Transport Phenomena in Nervous System PDF
Transport Phenomena in Nervous System PDF
Transport Phenomena in Nervous System PDF
Volume 65
DILEMMAS IN DIABETES
Edited by Stewart Wolf and Beatrice Bishop Berle - 1975
Volume 66
IMMUNE REACTIVITY OF LYMPHOCYTES: Development, Expression,
and Control
Edited by Michael Feldman and Amiela Globerson • 1976
Volume 67
ATHEROSCLEROSIS DRUG DISCOVERY
Edited by Charles E. Day. 1976
Volume 68
CURRENT TRENDS IN SPHINGOLIPIDOSES AND ALLIED DISORDERS
Edited by Bruno W. Volk and Larry Schneck. 1976
Volume 69
TRANSPORT PHENOMENA IN THE NERVOUS SYSTEM: Physiological and
Pathological Aspects
Edited by Giulio Levi, Leontino Battistin, and Abel Lajtha • 1976
Volume 70
KIN INS : J>harmacodynamics and Biological Roles
Edited by F. Sicuteri, Nathan Back, and G. L. Haberland -1976
Volume 71
GANGLIOSIDE FUNCTION: Biochemical and Pharmacological Implications
Edited by Giuseppe Porcellati, Bruno Ceccarelli, and Guido Tettamanti • 1976
TRANSPORT PHENOMENA
IN THE NERVOUS SYSTEM
Physiological and Pathological Aspects
Edited by
Giulio Levi
National Research Council
Rome, Italy
Leontino Battistin
University or Padua Medical School
Padua, Italy
and
Abel Lajtha
Research Institute of Neuroehemistry
New York, New York
I have no doubt that this meeting will belong among the most
pleasant memories and among the most productive and profitable
times of the participants.
The meeting would not have been possible without much help and
support. Special thanks are due to our host, Professor S. Rigotti
of the Department of Neurology and Psychiatry of the University of
Padova and also to Professor P. Pinelli of the Italian Society of
Neurology. My personal appreciation is added to those of the
participants, to myoid friends and colleagues, Dr. L. Battistin
and G. Levi, for their local arrangements and their arranging and
editing of the program. Special thanks are due also to the
University and to the Pharmaceutical firms, for their most generous
support, which made it possible for all of us to have such pleasant
surroundings for the exchange of information, such rich circumstances
for social exchange. These are hard days for people engaged in
medical research, but the three days of this symposium were a most
pleasant exception to such hardship. It was not the first or the
last symnosium on the subject, and if others will match its
cooperative and interested spirit, the field has a rosy future.
Abel Lajtha
February, 1976
Contents
SECTION I
METABOLITE TRANSPORT
METABOLITE TRANSPORT AT CELL MEMBRANES
H.N. Christensen 3
Mediation of apparently spontaneous metabolite migrations
Each active transport has a reverse phase
Meaning of duality or multiplicity of transport systems
Evidence that exodus occurs by reversal of a weaker pump
Increased importance of discriminating distinct transport
systems
Roles of receptor sites for amino acid transport in
neurotransmission?
The place of the hydrogen ion
The future: Generation versus interconversion of
energetic gradients
SECTI ON I I
BARRIERS IN THE LIVING BRAIN
TRANSPORT MECHANISMS IN THE CEREBROSPINAL FLUID SYSTEM
FOR REMOVAL OF ACID METABOLITES FROM DEVELOPING BRAIN
N. H. Bass and P. Lundbo rg 31
Maturation of bulk flow
Development of intracranial mechanisms for efflux of:
(a) para-aminohippuric acid (PAll)
(b) 5-hydroxyindoleacetic acid (5-HIAA)
Sink action of the cerebrospinal fluid (CSF) system
ix
x CONTENTS
hypertensive opening
Loci where there is no barrier
permeable vessels
SECTION I I I
TRANSPORT STUDIES IN VARIOUS NERVOUS TISSUE PREPARATIONS
THE CHARACTERISTICS OF GLUCOSE TRANSPORT ACROSS THE BLOOD
BRAIN BARRIER AND ITS RELATION TO CEREBRAL GLUCOSE
METABOLISM
A.L. Betz, D.D. Gi1boe, and L.R. Drewes 133
Glucose transport kinetics under physiological conditions
Glucose transport kinetics during hypoglycemia
Glucose transport kinetics during anoxia
Mechanism of glucose transport
Comparison with glucose transport in the erythrocyte
Proposed model for glucose transport at the blood brain
interface
SECTION IV
FACTORS INFLUENCING TRANSPORT
ENERGETICS OF LOW AFFINITY AMINO ACID TRANSPORT INTO
BRAIN SLICES
M. Banay-Schwartz, D.N. Teller, and A. Lajtha 349
scope
rationale for studying slices
Background
other reviews
brain slices
Does active transport of amino acids require glycolysis,
phosphorylation and ATP?
evidence from bacterial studies
comparison of properties of isolated vesicles, cells and slices
Experimental dissociation of transport from glycol¥sis
How does active transport of amino acids require K or Na+
the K+ requirement is not specific
the role of Na+ is more difficult to determine: a. changing
Na+ and K+ simultaneously
b. Na+ flux relationship to amino acid uptake
summary of Na+ relationship to uptake
Dissociation of ion pump activity from uptake
SECTION V
RELATIONSHIP OF IN VIVO AND IN VITRO STUDIES
THE USEFULNESS OF STUDIES IN VITRO FOR UNDERSTANDING
CEREBRAL METABOLITE TRANSPORT IN VIVO
A. Lajtha and M. Banay-Schwartz---- 415
Barriers --
in ----
vivo and --
in -vitro
----
Rates of cerebral protein synthesis in vivo and in vitro
Observations on -------
in vitro alterations
Substrate specificity of transport in vivo and in vitro
Aspects of transport that can be studied better in vitro
Function of the barriers
Metabolite compartmentation in vivo and in vitro
Conclusions
penicillin
gentamicin
SECTION VI
ALTERATIONS OF TRANSPORT IN PATHOLOGY
CEREBRAL PERMEABILITY PHENOMENA IN EPILEPSY
L. Battistin 465
Level and uptake in vivo
Uptake in vitro - - -
Regional uptake in vivo
Conclusions
We can say indeed that the Paduan School has always been the
heart of European Medicine, and that here was the origin of modern
medicine. In fact, during 1500, anatomical research on the corpse,
vivisection, and anatomo-comparative survey were systematically
carried out in this University. Moreover, here began the pharma-
ceutical teaching in the famous "Botanical Garden", as well as the
concept of "animal infection" and the clinical teaching at the bed
of the patient.
From such hopes is derived the need of meetings like this, where
non-medical researchers get closer to clinical science and share its
problems and anxieties; on the other hand, physicians can, by coming
into contact with. researchers, improve their technical knowledge with
better methodological and mental understanding.
Therefore I wish you good work, but I also hope that you
will find the time to visit our Padova, which with its old roads
and its various architecture shows some lovely contrasts and some
peculiar perspectives as well as some important monuments, that
incomparably testify to art and human creative genius.
Thank you.
Professor S. Rigotti
HALVOR N. CHRISTENSEN
INTRODUCTION
3
4 H.N. CHRISTENSEN
A B
A B
Fig. 3 may help you visualize how two pumps working together can
establish a cyclic flow. The stronger pump is able to maintain the
tank level at the higher point, Ii the weaker pump, only at the lower
point, II. At those points as many molecules of water are moving
downward as upward through the pump used. When both pumps work to-
gether, however, net entry occurs by the first, net exodus by the se-
cond, and an intermediate level (III) is maintained. Unfortunately
the analogy of Fig. 3 is defective in that it attributes the limit of
the level attainable by each pump to its inefficiency, whereas in the
biological context we should expect the energy available to each to
be an important factor. The model would be better if it showed the
second pump driven backward, so that it operated as an electric gen-
erator, when the water in the tank stands at the indicated level.
-I
-__..::.~- m
- IT
In the past the tendency has been to suppose that alanine and
other amino acids leave the epithelial cell by simple leakage - but
this tendency represents I believe the same fallacy that the mechani-
cal analog brought out: If the migration is down the gradient, we
tend to assume that it is just as spontaneous, just as energy wasting,
as it looks. Furthermore, when the spontaneity is in fact unknown,
we should avoid terminology that suggests high spontaneity. It is in
fact clear that amino acids are pumped into the intestinal epithelial
cell in the postabsorptive state and not merely during absorption.
Clearly the amino acid nutrition of the epithelial cell must be pro-
tected by having amino acids pumped in. In the same way the cellular
levels need to be protected during absorption by restraining exodus
by having it work against a pump. A high population of Na+-indepen-
dent pumps for amino acids and sugars appears to be characteristic
of the basolateral aspects of the epithelial cells of the proximal
tubule.
You may find it a little harder, for cells not engaged in absorp-
tion or secretion, to understand why a metabolite should be brought
in by one transport system and released by another. Consider, how-
ever, how often biochemical architecture of sorts other than gradients
is built up by one route and degraded by another. Such arrangements
no doubt permit superior control and greater energetic economy than
the alternatives.
the hydrogen ion rather than Na+. Co-transport of amino acids with
the hydrogen ion has now been seen in several species. The phenomenon
may follow logically from the ability of the membrane to recognize
the amino acid molecule first in one and then in another state of pro-
tonation 5 • Co-transport with H+ has, however, also emerged for sugars,
whose molecule seems unlikely to yield a hydrogen ion during the
process I6 ,17,13.
REFERENCES
Marian Orlowski
Department of Pharmacology
Mount Sinai School of Medicine of the
City University of New York
New York, New York
INTRODUCTION
13
14 M.ORLOWSKI
y-Glu-cysH
Add i t i o n s Activity
Specific Relative
None 20 100
Na+ (150 mMj 44 220
K+ (150 roM 46 230
CaZt- ~10 mM 150 750
Mg2+ 10 roM 140 700
Mg2+ 10 roM~
+ Na+ (150 roM) 80 400
Glycine 253 1300
L-Leucine 242 1200
L-Phenylalanine 253 1300
L-Aspartate 104 520
L-Asparagine 412 2100
L-Glutamate 205 1000
L-Glutamine 709 3500
L-Methionine 784 3900
L-Arginine 429 2100
L-Lvsine 366 1800
*The reaction mixtures contained GSH (0.005M), Tris-HCl buffer
(0.08 M; pH 8.8), dithiothreitol (0.005 M), enzyme and additions in-
dicated in the table, in a final volume of 0.5 ml.Activity is in
pmoles of cysteinylglycine released per mg enzyme per min. Relative
activities are relative to those obtained in the absence of any ac-
tivators. The activating effect of amino acids (20 roM) was measured
in the presence of Mg2+ (10 mM)
in amino acid transport came also from the study of hereditary hemo-
lytic anemia due to a deficiency of ¥-glutamylcysteine synthetase 64
In addition to hemolytic jaundice, the patients have signs of dege-
nerative disease of the CNS and signs of spinocerebellar degenera-
tion. Examination of the urine of the patients showed a marked ami-
noaciduria resulting from an increase in the excretion of both neu-
tral and basic amino acids.
CONCLUSIONS
REFERENCES
1. Albert, Z., Orlowska, J., Orlowski, M., and Szewczuk, A., Histo
chemical and biochemical investigations of gc;unma-glutamyl trans-
peptidase in the tissues of man and laboratory rodents, Acta His-
tochem. 18 (1964) 78-89.
2. Albert, Z., Orlowski, M., and Szewczuk, Z., Histochemical demons-
tration of y-glutamyl transpeptidase, Nature, 191 (1961) 767-
768.
3. Albert, Z., Orlowski, M., Rzucidlo, Z., and Orlowska, J., Stu-
dies on y-glutamyl transpeptidase activity and its histochemi-
cal localization in the central nervous system of man and d.iffe-
rent animal species, Acta Histochem. 25 (1966) 312-320.
4. Angielski, S., Niemiro, R., Makarewicz, W., and Rogulski, J.,
Aminoaciduria caused by maleic acid, Acta Biochim. Polon. 5
(1958) 396-402.
5. Ball, E.G., Cooper, 0., and Clarke, E.C., On the hydrolysis and
transpeptidation of glutathione in marine forms, Biol. Bull.
105 (1953) 369-370.
6. Ball, E.G., Revel, J.P., and Cooper, 0., The quantitative measu-
rement of y-glutamyl transpeptidase activity, J. Biol. Chern.
221 (1956) 895-908.
7. Banay-Schwartz, M., Teller, D.N., Gergely, A., and Lajtha, A.,
The effect of metabolic inhibitors on amino acid uptake and the
levels of ATP, Na+, and ~ in incubated slices of mouse brain,
Brain Res. 71 (1974) 117-131.
8. Binkley, F., Metabolism of glutathione, Nature, 167 (1951) 888-
889.
9. Binkley, F., Purification and properties of renal glutathionase,
J. Biol. Chern. 236 (1961) 1075-1082.
10. Binkley, F., and Nakamura, K., Metabolism of glutathione. 1.
Hydrolysis by tissues of the rat, J. Biol. Chern. 173 (1948)
411-421.
11. Bodnaryk, R.P., Membrane bound y-glutamyl transpeptidase. Evi-
dence that it is a component of the "amino acid site" of certain
neutral amino acids, Can. J. Biochem. 50 (1972) 524-528.
12. Bodnaryk, R.P., Kinetic aspects of the breakdown of y-glutamyl-
L-phenylalanine during sclerotization of the puparium of Musca
Domestica, Insect Biochem. 4 (1974) 439-454.
13. Bodnaryk, R.P., Bronskill, J.F., and Feterly, J.R., Membrane-
24 M. ORLOWSKI
30. Hanes, C.S., Hird, F.J.R., and Isherwood, F.A. Enzymic trans~e~
tidation reactions involving y-glutamyl ~e~tides and a-amino-acyl
~e~tides, Biochem. J. 51 (1952) 25-35.
31. Harrison, H., and Harrison, H., Ex~erimental ~roduction of renal
glycosuria, ~hos~haturia, and aminoaciduria by injection of ma-
leic acid, Science 120 (1954) 606-608 .
32. Hewitt, J., Pillion, D., and Leibach, F.H., Inhibition of amino
acid accumulation in slices of rat kidney cortex by diamide,
Biochim. Biophys. Acta 363 (1974) 267-276.
33. Hird, F.J.R., The y-glutamyl trans~eptidation reaction, Doctoral
dissertation, Cambridge University, England (1950)
34. Jellum, E., Kluge, T., Borresen, H.C., Stokke, 0., and Eldjarn,
L., Pyroglutamic aciduria - a. :1ew inborn error of metabolism,
Scand. J. clin. Lab. Invest. 26 (1970) 327-335.
35. Kamin, H., and Handler, P., Effect of infusion of single amino
acids u~on excretion of other amino acids, Amer. J. Physiol.
164 (1951) 654 - 561 • .
36. Kokot, F., Kuska, J. and Grzybek, H.,y~lutamyl trans~e~tidase
in the urine and intestinal contents, Arch. Immun. Therap. Exptl.
13 (1965) 549-556.
37. Kokot, F., and Sledzinski, Z., Die y-glutamyltransferase, Z.
Klin. Chem. Klin. Biochem. 12 (1974) 374-384. --
38. Kosower, N.S., Kosower, E.M., and Wertheim, B., Diamide, a new
reagent for the intracellular oxidation of glutathione to the
disulfide, Biochem. Biophys. Res. Commun. 37 (1969) 593-596.
39. Kosower, N.S., Song, K.R. and Kosower, E.M., Glutathione IV.
Intracellular oxidation and membrane injury, Biochim. Biophys.
Acta. 192 (1969) 23-28.
40. Lajtha, A., Berl, S., and Waelsch, H., Amino acid and ~rotein
metabolism of the brain- IV. The metabolism of glutamic acid,
J. Neurochem. 3 (1959) 322-332.
41. Marks, N., Pe~tide hydrolases, in Handbook of Neurochemistry
A. Lajtha (ed), Plenum Press, New York (1970) pp 131-171
42. ~1eister, A., On the enzymology of amino aCld tl'a.ns~6rf" Science
180 (1973) 33-39.
43. Meister, A., The y-glutamyl cycle, Ann. Intern. Med. 81 (1974)
247-253 •
44. Mohler, D.N., Majerus, P. W., ~1innich, V., Hess, C.E., and Gar·-
rick, M.D., Glutathione synthetase deficiency as a cause of here-
ditary hemolytic disease, N. Engl. J. Bed. 283 (1970) 1253-1257.
Okonkwo, P.O., Orlowski, M., and Green, J.P., Enzymes of the
y-glutamyl cycle in the choroid ~lexus and brain, J. Neurochem.
22 (1974) 1053-1058.
Oort, M., Loos, J.A., Prins, H.K., Hereditary absence of reduced
glutathione in the erythrocytes, Vox Sang. 6 (1961) 370-373.
Orlowski, M., The role of y-glutamyl trans~e~tidase in the inter-
nal disease clinic, Arch. Immun. Ther. Exptl. 11 (1963) 1-61.
48. Orlowski, M. and Meister, A., y-Glutamyl-~-nitroani1ide: A new
convenient substrate for determination and study of L- and D-y-
glutamyl trans~e~tidase activity, Biochim. Biophys. Acta 73
26 M. ORLOWSKI
(1963) 679-681 .
49. Orlowski, M., and Meister, A., Isolation of y-glutamyl transpep-
tidase from hog kidney, J. BioI. Chem. 240 (1965) 338-347.
50. Orlowski, M., and Meister, A., The y-glutamyl cycle: a possible
transport system for amino acids, Proc. Nat. Acad. Sci. U.S.A.
67 (1970) 1248-1255.
51. Orlowski, M., and Meister, A., Enzymology of pyrrolidone carboxy-
lic acid in P.D. Boyer ed., The Enzymes, Academic Press, Vol. 4
(1971) 123-151.
52. Orlowski, M., and Meister, A., Isolation of highly purified y-
glutamylcysteine synthetase from rat kidney, Biochemistry 10
(1971) 372-380.
53. Orlowski, M., and Meister, A., y-Glutamyl cyclotransferase: Dis-
tribution, Isozymic forms and specificity, J. BioI. Chem. 248
(1973) 2836-2844.
54. Orlowski, M., Okonkwo, P.O. and Green, J.P., Activation of y-
glutamyl transpeptidase by monovalent cations, FEES Letters
31 (1973) 237-240.
55. Orlowski, M., Sessa, G., and Green.J.P., y-Glutamyl transpepti-
dase in brain capillaries: Possible site of a blood-brain barrier
for amino acids, Science 184 (1974) 66-68
56. Orlowski, M., and Szewczuk, A., Colorimetric determination of y-
glutamyl transpeptidase activity in human serum and urine with
synthetic substrates, Acta Biochim. Polon. 8 (1961) 189-200.
57. Orlowski, M., and Szewczuk, A., Determination of y-glutamyl
trans:peptidase in human serum and urine, Clin. Chim. Acta
(1962) 755-760 •
58. Orlowski, M., and ~lilk, S., Intermediates of the y-glutamyl cycle
in mouse tissues, Eur. J. Biochem. 53 (1975) 581-590.
59. Orlowski, M., and Wilk, S., In vivo inhibition of y-glutamylcys-
teine synthetase by L-methionine-RS-sulfoximine, J. Neurochem.
1975, In press.
60. Ramakrishna,M., Krishnaswamy,P.R., and Rao, D.R. Metabolism of py-
rrolidone carboxylate in the rat, Biochem.J. 118 (1970) 895-897
61. Rathbun, W.B. and Wicker, K., Bovine lens y-glutamyl transpepti-
dase, Exp. Eye Res. 15 (1973) 161-171.
62. Reddy, V.N., Metabolism of glutathione in lens, Exp. Eye Res.
11 (1971) 310-328.
63. Reddy, V.N. and Unakar, N.J., Localization of y-glutamyl trans-
peptidase in rabbit lens, ciliary processes and cornea, Exp. Eye
Res. 17 (1973) 405-408.
64. Richards, F., Cooper, H.R., Pearce, L.A., Cowan, R.J., Spurr, C.
L., Familial spinocerebellar degeneration, hemolytic anemia and
glutathione deficiency, Arch. Int. Med. 143 (1974) 534-537.
65. Richter, R., Some properties of y-glutamyl trans:peptidase from
human kidney, Arch. Immun. Ther. Exptl. 17 (1969) 476-495.
66. Rosalki, s., y-Glutamyl transpeptidase p in: O. Bodansky and A. L.
Latner (ed.) Advances in Clinical Chemistry, Academic Press
New York (1975) pp. 53-107.
Rosenberg, L.E., and Segal, S., Maleic acid-induced inhibition
ROLE OF GLUTATHIONE IN TRANSPORT 27
31
32 N.H. BASS AND P. LUNDBORG
The central nervous system of the 5 day old rat contains: blood
vessels composed of primitive epithelial elements forming barely
patent channels surrounded by loosely apposed end-feet 7 ; extra-
cellular "lakes" formed by cellular processes of numerous poorly
differentiated neurons and few undifferentiated glia 7 ; and
relatively undifferentiated secretory epithelium in the choroid
plexus 19 At 30 days of age, extracellular spaces are reduced
to small intercellular clefts by growth of numerous neural processes,
and both brain capillaries and choroid plexus show an adult appear-
ance. Since intracranial sites for carrier-mediated transport of
organic acids contain poorly differentiated cells in the 5 day old
rat, we have taken an ontogenetic approach to the question: how
does the brain, which lacks a lymphatic system of the usual type,
remove its products of amine metabolism?
of this acid metabolite by the CSF system would require a bulk flow
rate of 33 pI/min. It is apparent that the normal circulatory rate
for CSF in the 5 day old rat can account for elimination of not more
than 6% of the 5-HlAA formed. Moreoever, the same condition holds
for the 30 day old rat, where its facilitated rate of bulk flow has
merely kept pace with an increased number of serotonergic synapses,
and is similarly capable of eliminating only 6% of the 5-HlAA formed
per minute. Clearly, if the CSF system is to function in the removal
of products of amine metabolism from the infant and adult central
nervous system, sites for carrier-mediated transport of such organic
acids must occur in proximity to the route of bulk flow.
100
:cZ 10
.. ........
0::
o'"
:II
5
'--
:u ..........
2
!
...Z
Q S Day Old Rat
..::;
...~ --,I I~ .....,..
oQ
'----.
e
'-----.
...... 50
!
...
.
o
.....
20
~
U
0. 10
Figure 1. Disappearance of 3H- PAH (.... ---.l) and 14C_inulin (A--l1) from
the CSF system of 5 and 30 day old rats following a five minute de-
livery of 40 and 90 ~l volumes respectively into the spinal subarach-
noid space (----1'"). (0-._.<» denotes 3H- PAH efflux after probenecid
(250 mg/kg, subcut); (.'.0') denotes combined intrathecal infusion
of 3H- PAH and 5-HlAA.
34 N.H. BASS AND P. LUNDBORG
Although the CSF system of the 5 day old rat has a markedly
sluggish rate of bulk flow, PAR was eliminated from the CSF at a
rate which was l7-fold greater than that found for inulin. This
efflux rate for PAR closely approached values which could theoret-
ically account for total removal of the 5-HIAA formed by the immature
brain. Moreover, addition of high doses of unlabeled 5-HIAA to the
infusion fluid, or systemic pretreatment with probenecid, decreased
the elimination rate by approximately 50% while having no effect
on inulin efflux suggesting that: (1) PAR is eliminated from the
CSF system by a carrier-mediated transport mechanism: and (2) 5-HIAA
and PAR probably share a common transport site. Previous in vitro
studies have shown that the choroid plexus of fetal and newborn
chicks, rats, rabbits, cats and dogs possesses an active transport
system for acid-sulfonated dyes, and radiolabeled sulphate, iodide,
and PAH 5,18 Hence, it may be concluded that poorly differen-
tiated choroid epithelial cells have a carrier-mediated transport
mechanism for organic acids, whose efflux rate approaches that
required for complete removal of metabolites of biogenic amines from
immature brain.
whole brain metabolism, the calculated e f flux rate for organic acids
from the CSF system of the 30 day old rat is too slow to constitute
the sale route for preventing their accumulation in brain. Hence,
further studies were performed to explore prior evidence suggesting
that the major route for removal of acid metabolites from adult brain
exists at transport sites presumably situated at the glia-capillary
interphase in the brain parenchyma.
40
20
10
..:c'"
z
...:z:
w
o
Z 5 Day Olel Rot
..
:c
t;
w
mnufes 60
(5
o
...
S
~
20
~
(;
...
Z
10
~
5
...~ '"
'"
2
"
30 Day Old Rat
mnutes
Figure 2. Disappearance of 3H- PAH (A- - ---A) from the brain of 5 and
30 day old rats following a five minute delivery of 40 and 90 ul
volumes, respectively into the spinal subarachnoid space (----t),
~.- . -o) denotes 3H- PAH efflux after probenecid (2~0 mg/kg, subcut);
(." ,.) denotes combined intrathecal infusion of H-PAH and 5- HIAA.
36 N.H. BASS AND P. LUNDBORG
**5 day old rat: Body wgt. 13.8 g; brain wgt. 0.43 g;
30 day old rat: Body wgt. 62.0 g; brain wgt 1.39 g.
By combining values for total CSF volume and bulk flow 2 with
rates of accumulation of 5-HlAA in brain and CSF after probenecid
treatment 4 , steady-state elimination rates for 5-HlAA from intra-
cranial compartments of 5 and 30 day old rats have been calculated
(Table). The total amount of 5-HlAA eliminated per minute from the
intracranial cavity of the 5 day old rat was 0.71 ng, a value in close
agreement with the reported rate of formation of this metabolite
in the brain of infant rats 20 No evidence for carrier-mediated
efflux at brain capillaries was found, as 5-HlAA did not accumulate
in immature brain after probenecid treatment. This lack of response
of the infant brain to inhibition of active transport was not caused
by a failure of 5-HlAA formation, since a rapid decrease of 5-HlAA
has been observed after monoamine oxidase inhibition 20 .
Additionally, bulk flow of CSF eliminated 0.04 ng of 5-HlAA/min,
amounting to only 6% of its formation rate. Hence, active transport
sites in the CSF system comprised the route for removal of 93% of
the 5-HlAA formed in the brain of the infant rat, eliminating the
metabolite at a rate of 0.67 ng/min. The total amount of 5-HlAA
Figure 3. Sink action of the CSF and its proposed role in the
removal of 5-HlAA from the brain of 5 day old (A) and 30 day old
(B) rats. Note developmental changes in major (~) and minor
(---~) efflux routes associated with reduction of intercelluar
clefts between neurons (N) and glia (G) and the appearance of
mediated transport sites at brain capillaries (~). Modified from
Davson and Bradbury (Symposium, Soc. Exp. BioI. 19:349, 1965).
38 N.H. BASS AND P. LUNDBORG
eliminated per minute from the intracranial cavity of the 30 day old
rat was 3.96 ng, a value in relative agreement with the endogenous
formation of this organic acid when corrected for decreased turnover
of serotonin to 60% of values reported for adult rat brain 20.
However, in contrast to the 5 day old animal, only 23% of adult brain
5-HIAA formed per minute (0.93 ng/min) was eliminated via CSF path-
ways by the combined mechanisms of active transport and bulk flow. In
fact, the maturation of active transport sites at the glia-capillary
interphase constituted the major route for efflux of 73% of the 5-HIAA
formed in the brain of the 30 day old rat, efficiently removing the
metabolite at a minimum rate of 3.03 ng/min.
ACKNOWLEDGMENTS
This research has been supported in part by the Swedish State Medical
Research Council (no. B73-l4P-3266-03 and B73-l4X-2464-06C),
Expressens Prenatal Forskning, and a Career Development Award to
N.H. Bass from the National Institutes of Health (NS 28-155)
References:
1 Ashcroft, G.W., Dow, R.D., and Moir, A.T.B., The active trans-
port of 5-hydroxyindolylacetic acid and 3-methoxyhydroxyphenyl
acetic acid from a recirculatory perfusion system of the cerebral
ventricles of the unanesthetized dog, {. Physiol. 19
(1961) 153-160.
2 Bass, N.H. and Lundborg, P., Postnatal development of bulk flow
in the cerebrospinal fluid system of the albino rat. Brain
Res.52 (1973) 323-332.
3 Bass, N.H., and Lundborg, P., Postnatal development of mechanisms
for the elimination of organic acids from the brain and cerebro-
spinal fluid system of the rat. Brain Res.56 (1973)285-298.
4 Bass, N.H., and Lundborg, P., Mechanisms for the elimination of
5-hydroxyindole-acetic acid from brain and cerebrospinal fluid
of the rat during postnatal development, Brain Res. 77 (1974)
111-120.
5 Bierer, D.W., and Heisey, S.R., Organic anion transport in the
maturing dog choroid plexus, ~rain Res b6 (1972) 113-119.
6 Butler, A.B. Mann, J.D., and Bass, N.H., Identification of the
major site for cerebrospinal fluid efflux in the albino rat.
Anat. Rev. 181 (1975) 323.
7 Caley, D.W. and Maxwell, D.W., Development of the blood vessels
and extracellular spaces during postnatal maturation of rat
cerebral cortex. J.Comp. Neurol. 138 (1970) 31-48.
8 Cserr, H.F., Physiology of the choroid plexus. Physiol. Rev.
51 (1971) 273-311.
9 Cserr, H.F., Relationship between cerebrospinal fluid and inter-
stitial fluid of brain, Fed. Proc. 33 (1974) 2075-2078.
10 Cserr, H.F., and Van Dyke, D.H., 5-Hydroxyindoleacetic acid
accumulation by isolated choroid plexus. Amer. J. Physiol.
220 (1971) 718-723. ~- -
11 Fenstermacher, J.D., Patlak, C.S. and Blasberg, R.G., Trans-
port of material between brain extracellular fluid, brain cells,
and blood. Fed. Proc. 33 (1974) 2070-2074.
12 Meek, J.L. and Neff, H.H., Is Cerebrospinal fluid the major
avenue for the removal of 5-hydroxyindoleacetic acid from the
brain? Neuropharmacology 12 (1973) 497-499.
13 Mi1horat, T.H., The third circulation revisited, J.Neurosurg.
42 (1975) 628-645.
40 N.H. BASS AND P. LUNDBORG
41
42 M.W. BRIGHTMAN AND R.D. BROADWELL
Tumors
Hyperosmotic Opening
Hypertensive Opening
.-
•
"",.
. ,,(
Permeable Vessels
outside of the eNS. Within the brain, the vessels of the choroid
plexus, as noted above, together with those supplying the circumven-
tricular organs (eVO) are fenestrated and contain more pits and
vesicles than the endothelium of the more common vessels in the eNS
20, 24. When HRP is injected intravenously, it rapidly crosses
vessles in the neurohypophysis median eminence, area postrema 20,
and other eva 6, 24. The extracellular spaces within these organs
become flooded with peroxidase. The neurohemal endings of the hypo-
thalamic neurosecretory cells avidly take up HRP from the spaces
around the fenestrated vessels in the posterior lobe of the pituitary
gland and median eminence. From these terminals, blood-borne
peroxidase is carried back to their cell bodies in the supraoptic,
paraventricular (fig. 3) and associated neurosecretory nuclei 6.
With time, the HRP flooding the extracellular clefts of the eva
spreads to adjacent areas. When high concentrations of 50 to 100 mg
of HRP (Sigma type VI or Worthington), tenfold greater than "probe"
amounts, are injected intravenously into adult mice, the areas around
the eva are completely inundated with protein within 2 hours 6. So
great is the spread from the median eminence, for example, that the
ventromedial nucleus and the dorsal and lateral areas of the hypo-
thalamus are also flooded. The inundation is intracellular as well
as extracellular. The cell bodies of neurons become homogeneously
brown, their cytoplasm having been infiltrated by HRP (fig. 5).
Protein that is freely dispersed in the cytoplasm connotes damage
to the cell membrane, since such a large molecule could only pass
directly across a membrane that is no longer semi-permeable 2.
If sufficient time (4 hours) is allowed, the protein is cleared from
the cell body by, presumably, orthograde cytoplasmic flow from soma
toward axon terminals. Between 8 and 12 hours, the HRP appears in
the cytoplasm of the neuronal soma as discrete particles: the
membrane-bounded organelles that are the recipients of protein
pinocytosed by the axon terminals (fig. 6).
Blood vessels outside of the eNS are the source of protein for
the cranial nerves. The capillaries of striated and cardiac muscle
are permeable to peroxidase 14. After intravascular injection, the
protein can then spread through the extracellular spaces of muscle to
synaptic clefts at myoneural junctions. From here, axonal terminals
of motor nerves can pinocytose the HRP 27 and transfer it back to
the centrally located parent cell body 15 In addition to motor
neurons, sensory and preganglionic autonomic neurons are also access-
ible to blood-borne protein 6. Thus, large molecules circulating
in peripheral, extracerebral blood can be carried intraneuronally
deep within the eNS even though these molecules, flowing in cerebral
capillaries, cannot reach the same neurons because of the capillary
barrier. Hematogenous viruses 1, proteins like nerve-growth factor
11, and presumably, some hormones and toxins may also gain entry to
the eNS from peripheral blood along these intracellular routes rather
than through the extracellular clefts within the brain. In this way,
the maintenance of certain neurons within the central n~rvous system
can be influenced by large and small substances in peripheral blood.
These substances, taken up by axon terminals, require a finite time
for retrograde transport and could trigger events in the cell body
long after they have been cleared from the blood.
MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 53
REFERENCES
1. Baringer, J.R., and Swoveland, P., Persistent herpre simplex
virus infection in rabbit trigemental ganglia, Laboratory
Investigation, 30 (1974) 230-240.
2. Brightman, M.W., The distribution with the brain of ferritin
injected into cerebrospinal fluid compartments. I. Ependymal
distribution, Journal of Cell Biology, 26 (1965) 99-123.
3. Brightman, M.W., and Reese, T.S., Junctions between intimately
apposed cell membranes in the vertebrate brain, Journal of Cell
Biology, 40 (1969) 648-677.
4. Brightman, M.W., Hori, M., Rapoport, S.I., Reese, T.S., and
Westergaard, E., Osmotic opening of tight junctions in cerebral
endothelium, J. Comparative Neurology, 152 (1973) 317-325.
5. Brightman, M.W., and Prescott, L., Blood vessels permeable to
peroxidase in virally-induced brain tumors, (in preparation).
6. Broadwell, R.D., and Brightman, M.W., Entry of peroxidase into
neurons of the central nervous system from extracerebral and
cerebral blood, J. Comparative Neurology (in press).
7. Castel, M., Sahar, A., and Erlij, D., The movement of lanthanum
across diffusion barriers in the choroid plexus of the cat,
Brain Research, 67 (1974) 178-184.
8. Claude, P., and Goodenough, D.A., Fracture faces of zonulae
occludentes from "tight" and "leaky" epithelia, Journal of Cell
Biology, 58 (1973) 390-400.
9. Dahlstrom, A., and Fuxe, K., Evidence for the existence of mono-
amine containing neurons in the central nervous system. I. Demon-
stration of monoamines in the cell bodies of brain stem neurons.
Acta Physiologica Scandinavia, 62, Suppl. 232 (1964) 1-55.
10. Feder, N., Reese, T.S., and Brightman, M.W., Microperoxidase, a
new tracer of low molecular weight. A study of the interstitial
compartments of the mouse brain, Journal of Cell Biology, 43
(1969) 35A-36A (Abstract).
11. Hendry, I.A., Stockel, K., Thoenen, H., and Iversen, L.L., The
retrograde axonal transport of nerve growth factor, Brain Research,
68 (1974) 103-121.
12. Hirano, A., and Zimerman, H.M., Fenestrated blood vessels in a
metastatic renal carcinoma in the brain, Laboratory Investigation,
26 (1972) 465-468.
13. Johansson, B., Li, C.L., Olsson, Y., and Klatzo, I., The effects
of acute arterial hypertension on the blood-brain barrier to
protein tracers, Acta Neuropatholgica (Berlin), 16 (1970) 117-124.
14. Karnovsky, M.J., The ultrastructural basis of capillary perme-
ability studied with peroxidase as a tracer, Journal of Cell
Biology, 35 (1967) 213-236.
15. Kristensson, K., Olsson, Y., and Sjostrand, J., Axonal uptake
and retrograde transport of exogenous proteins in the hypoglossal
nerve, Brain Research, 32 (1971) 399-406.
16. Long, D., Capillary ultrastructure and the blood-brain barrier in
human malignant brain tumors, Journal of Neurosurgery, 32 (1970)
127-144.
54 M.W. BRIGHTMAN AND R.D. BROADWELL
O.E. Pratt
Department of Neuropathology
Institute of Psychiatry
De Crespigny Park, London SE5 8AF, England
ss
56 O.E. PRATT
1. Glucose
30
PLASMA
GLUCOSE
.t' mol ml-1
10
o 25 50
MIN
• •
.....
A A •
INFLUX RATE
jJmol min-1 9-1 brain
50 100
GLUCOSE IN ARTERIAL PLASMA jJmol ml-1
2·0
GLUCOSE GAIN
BY BRAIN
jJmol min-1g-1brain
1-0
10 20 30
GLUCOSE IN ARTERIAL PLASMA mM
Fig. 3. The glucose gain by the brain at various blood glucose con-
centrations. • insulin-treated animals; • controls not given
insulin. Each point is mean of 3 to 12 experiments. The curved
line is taken from Fig. 2 (from Daniel, Love and Pratt 28).
METABOLITE TRANSPORT IN THE LIVING BRAIN 59
centration is raised above about 20 roM the influx does not increase
much more because the transport system is almost saturated. Despite
this, the cerebral glucose gain continues to rise until it catches
up with the influx, which means that, at this stage in the experi-
ments, the glucose efflux falls sharply (Fig. 3). Under these con-
ditions, especially if insulin is also given, more glucose enters
the brain than can be oxidised and an excess must be accumulating
within the brain cells. These experimental conditions correspond
roughly with what will happen shortly after a large glucose load
(as in an intravenous glucose tolerance test) when a combination of
hyperglycaemia and hyperinsulinaemia will not endure for long so
that glucose can only accumulate in the brain cells to a limited
extent. Efflux, which occurs under normal conditions, is likely to
be an important mechanism which protects brain cells from the osmotic
effects of an excess of glucose within them.
2. Ketone Bodies
Sources: (a) Banos, Daniel and pratt ll . (b) Daniel, Moorhouse and
pratt 3l ; (c) Daniel, Pratt and Wilson 32 (d) Crockett, Daniel and
Pratt l7 ; (e) Daniel, Love and Pratt 2S ; (f) Daniel, Love, Moorhouse,
Pratt and Wilson 25 ; (g) Daniel, Love, Moorhouse, Pratt and Wilson. 26
AMINO ACIDS
It is important to understand clearly how changes in the con-
centration of an amino acid in the blood plasma of a living animal
affect its rate of transport into the brain. When the concentration
of the amino acid is within, or not far above, the normal range the
cerebral influx is for all practical purposes directly proportional
to its concentration in the plasma, or in the case of L-tryptophan,
to the fraction that is not bound to albumin. 6 ,S However, at high
concentrations in the blood the amino acid saturates the transport
carrier (Table 2) and its influx eventually approaches a limiting
62 D.E. PRATT
the brain (Table 1) and although not used for cerebral protein
synthesis they may meet some other metabolic need.
Finally there is a third group, the amino acids which the
brain can readily synthesize, e.g. L-aspartate 35 and L-proline 57
and also amino acids which are not used for protein synthesis,
e.g. taurine. The neurotransmitter amines are not readily trans-
ported into the brain5l nor are the postulated neurotransmitter
amino acids. 59 Table 1 shows that the actual rates of transport
of aspartate, glutamate, glycine and taurine are slow. The slow
rates at which the amino acids in this third group enter the brain
are close to the entry. rates of those amino acids which are not
normally found in the body like 2-aminoisobutyrate and 2-amino-
adipate. 6 It may well be that all these amino.acids which are
not needed by the brain only cross the capillaries slowly by
diffusion even if some of them enter the brain cells by transport
systems. 13 In this group aspartate, glycine and glutamate may
be concerned in osmoregulation (Bradbury, this volume, p. 483 )
and proline and glycine have low entry rates even though present
at high concentration in the blood plasma.
On the other hand there are at least fourteen amino acids that
the cerebral cells either cannot or do not synthesize but which
they use in considerable quantities. The influx of these amino
acids (Table 1) is high enough to ensure entry at rates which are
related to the rates at which the brain uses them for protein syn-
thesis. It can be calculated from the mean rate of turnover of
cerebral protein of 0.7% hr- l , as given by Seta, Sansur and
Lajtha,54 that the rat brain uses amino acids at a rate of the
order of 75 nmol min-lg- l brain. Not all of these amino acids
are obtained directly from the blood. Some amino acids, that are
freed by the breakdown of cerebral protein, will be re-cycled,
bridging the gap between utilization and influx. Thus, the
fourteen amino acids with influx rates greater than about 1.0 nmol
min-lg- l brain provide a combined supply to the brain of 46 nmol
min-lg- l brain, i.e. less than two thirds of what the brain needs
(Table 1). These calculations suggest that the influx rates of
many of the amino acids may, on occasion, be barely adequate to
meet the needs of the brain for protein synthesis. It is signif-
icant that Lajtha44 has estimated that the half-life of many amino
acids free in the brain is less than 30 minutes.
a
2000 •• •
RECIPROCAL OF
ENTRY RATE OF
L-LEUCINE
,Pmo1-1. b'
min grain •
1000
20 40 60 80 100
-1
L-VALINE .... mol ml
Ratio of radioactivity:
-1
.uCi g brain
-1
f..lCi ml plasma
Min
Fig. 5. Exchange of radioactivity while constant specific activity
of l4C histidine is maintained in the circulating blood (from
Daniel, Love, Moorhouse and Pratt 24 ).
METABOLITE TRANSPORT IN THE LIVING BRAIN 67
1. Aminoacidurias
It has only recently become clear that insulin has an effect upon
the exchange of glucose between the brain and the blood.
The ability to control independently the insulin and glucose
levels in the blood has made it possible to separate the direct
effects of insulin upon cerebral transport or metabolism from
those due to changes in blood glucose. If the blood glucose is
raised to high levels the cerebral transport system becomes sat-
urated thus limiting the exchange of glucose between the blood
and brain. The increased influx in hyperglycaemia leads to
increased cerebral glucose gain. 28 This continues until a rise
in intracerebral glucose causes a comparable increase in efflux
which establishes a new equilibrium.
The response to hyperglycaemia is different when the blood
insulin is also raised to a high level. 28 The glucose influx
into the brain is not altered but the cerebral gain of glucose is
raised. Even at normal blood glucose levels insulin causes a
significant increase in gain which is associated with a reduction
in the efflux of glucose. Fig. 3 shows that as the blood glucose
is raised above normal in insulin-treated animals the cerebral
glucose gain rises faster than the influx until the efflux falls
to zero i.e. all the glucose transported into the brain is being
metabolized or retained. In these insulin-treated, hyperglycaemic
animals the rate of glucose gain is several times normal and is
sustained for 20 minutes or more. 28 This changed response to
hyperglycaemia as a result of insulin is likely to be due either
to a block of glucose efflux or to an increase in the rate of
cerebral glucose metabolism. Some of the extra glucose which is
gained by the brain when both insulin and glucose levels in the
blood are high may be oxidised to compensate for the lack of
ketone bodies which are removed from the bloodstream by the sys-
temic action of insulin. However, in hyperglycaemia (above about
20 mM plasma glucose) the cerebral oxygen uptake is insufficient
to oxidise all the glucose that is gained. In all probability
much of the excess glucose is being used to make glycogen, non-
essential amino acids or lipids. 29 This surprising effect of
insulin upon the brain merits further study.
TABLE 4. THE EFFECT OF THYROIDECTOMY UPON THE INFLUX OF SCME ESSENTIAL AMINO ACIDS INTO THE BRAIN
AND UPON THEIR INCORPORATION INTO CEREBRAL PROTEIN (Means ± S.E; No. of experiments in parentheses)
L-leucine 5.65±0.77(4)* 40.7 r S.5(4)* 1O.8±3.1(4)** 9.95 r O.75(3) 7S.6 r S.7(3) 21. ]±0.4(3)
L-valine 1.5a±O.22(S)* 10.4±1.5(S)* 3.1±0.9(S)** 2.94±0.44(2) 16.2±2.S(2) 1l.5±0.8(2)
L-lysine 2.26±0.41(4) 12.1±2.2(4) 2.9±0.2(4) 7.4G±1.4 (9) 24.8±4.6(9)
Significantly different from control *(P < O.OS, t-test), **(P < 0.01, t-test)
o
(From Daniel, Love and Pratt 30 ) m
"tI
::0
»
=I
METABOLITE TRANSPORT IN THE LIVING BRAIN 71
REFERENCES
4 Ba~os, G., Daniel, P.M., Moorhouse, S.R., and Pratt, O.E., The
passage of amino acids into the rat's brain. Journal of
Physiology, 210 (1970) 149P.
5 Ba~os, G., Daniel, P.M., Moorhouse, S.R., and Pratt, O.F.., The
entry of amino acids into the brain of the rat during the post-
natal period. Journal of Physiology, 213 (1971) 45-46P.
6 Banos, G., Daniel, P.M., Moorhouse, S.R., and Pratt, O.E., The
influx of amino acids into the brain of the rat in vivo: the
essential compared with some non-essential amino acids.
Proceedings of the Royal Society, London, B., 183 (1973) 59-70.
7 Banos, G., Daniel, P.M., Moorhouse, S.R. and Pratt, O.E.,
Inhibition of entry of some amino acids into the brain, with
observations on mental retardation in the aminoacidurias.
PSlchologica1 Medicine, 4 (1974) 262-269.
8 Banos, G., Daniel, P.M., Moorhouse, S.R., and Pratt, O.E.,
The requirements of the brain for some amino acids. Journal
of Physiology, 246 (1975) 539-548.
9 Ba«os, G., Daniel, P.M., Moorhouse, S.R., Pratt, O.E., and
Wilson, P., Inhibition of neutral amino acid entry into the
brain of the rat in vivo. Journal of Physiology, 237 (1974)
22-23P.
10 Ba~os, G., Daniel, P.M., and Pratt, O.E., Inhibition of entry
of L-arginine into the brain of the rat, in vivo, by L-lysine
or L-ornithine. Journal of Physiology, 214 (1971) 24-25P.
11 Banos, G., Daniel, P.M., and Pratt, O.E., Saturation of a
shared mechanism which transports L-arginine and L-lysine into
the brain of the living rat. Journal of Physiology, 236 (1974)
29-41.
12 Battistin, L., Grynbaum, A., and Lajtha, A., The uptake of
various amino acids by the mouse brain in vivo. Brain Research,
29 (1971) 85-99.
13 Blasberg, R., and Lajtha, A., Substrate specificity of steady
state amino acid transport in mouse brain slices. Archives of
biochemistry and biophysics, 112 (1965) 361-377.
14 Carlsson, A., The in vivo estimation of rates of tryptophan and
tyrosine hydroxylation: effects of alterations in enzyme environ-
ment and neuronal activity. In G.E.W. Wolstenholme and D.W.
Fitzsimons (Eds.) Aromatic amino acids in the brain, Ciba Found-
ation Symposium (new series), Elsevier, Amsterdam, 1974, pp.
117-125.
15 Christensen, H.N., de Cespedes, C., Handlogten, M.E., and
Ronquist, G., Energisation of amino acid transport, studied for
the Ehrlich ascites tumour cell. Biochimica biophysica Acta,
300 (1973) 487-522.
16 Cohen, S.R., and Lajtha, A., Amino acid transport. In A. Lajtha
(Ed.), Handbook of Neurochemistry, Plenum Press, New York,
London, 1972, vol. 7, pp. 543-572.
17 Crockett, M.E., Daniel, P.M., and Pratt, O.E., Saturation of
shared mechanisms transporting some neutral amino acids into the
brain. (1976) in preparation.
METABOLITE TRANSPORT IN THE LIVING BRAIN 73
INTRODUCTION
77
78 D.L. YUDILEVICH AND F.V. SEPULVEDA
r--l
"0
0
0
:a
E
'" 0.1
..
d
..........
on
0
0
c..
...
u
~
a.
L-...J
G.Ol
3 5 7 9
seconds
1.0
,--,
z
...
..
~
-......
J:;
<l.
:x:
""
I
-' 0.0
L-.-J
20 40 60 110 100
Percent total area un der 22Na
Ii I i , , , , If
1 2 3 4 5 6 769
seconds
Neutral
(14C)Glycine (0.041) -0.03 ± 0.04(3)
L-(14C)Alanine (0.059) -0.02 ± 0.04(5)
L-(14C)Serine
(14C)GABA
(0.053)
(4.357)
0.02 $
0.04(4)
-0.09 - 0.07(6)
L- (14C )Valine (0.033) 0.17 ± 0.05(6)
L-(14C)Glutamine (1.343 ) 0.01 ± 0.05(4)
£ (1.300) 0.33 ± 0.05(21)
!
L- 3H )Leucine
(1 C)Cyc10leucine 0.17 0.02(3)
L-(3H)Tyrosine (0.016) 0.47 - 0.04(5)
L-(3H)Phenylalanine (0.014) 0.39 ± 0.07(8)
L-(3H)Histidine (0.003 ) 0.26 ± 0.06(2)
Acidic
L-(14C)Aspartic acid (0.041) -0.08 +
- 0.08(4)
L-(3H)Glutamic acid (0.034) 0.03 ± 0.06(4)
Basic
L-(3H)Lysine
L-(14C)Arginine
(0.016)
(0.027)
0.03 !
0.07(5)
0.06 - 0.06(9)
SUGAR CARRIER
.1
1.0
0.5
~;~
L-Arab
5 5
l' , i;" , 0
... 0
0
~
... e
-..
N
Z
'E:::
N ci
..
N
.1
'"u0 0'" i
.= 0
<.!) 0. ~ c..
.....
O-Gal
...z u
.02
1M
ili'i" ,
1 5 Q.
1 1 5
G
I
0 ...
z
N
N
'0E=
0.5~
1 5
.1
.02
1
m
Phlor
5
NaCI
r"T'T"TTT'I
1 5
seconds seconds
I-
oa:
S!~
CI)
DOG 10 DOG 11 z
<t
a:
I-
w ::i
CI)
0 u.i
0 U (/j
0 0 :::J +1
0 C")
0 ....
0 0...1
0.... .c 0 II)
It)<!)
'0
0
8....
C") I/)
U ~ 0
~
0
....
0 0
c
I 0
....
0 ....
0 )(
>-
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aI 0
C")
C
II) II)
I/)
..J
0
i=
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(J
.cIII c::
I/)
.Q 0 C
I
ED
I
~
cu
(!) «
... ~
cu >-
)(
(J
~
u. ::I
0 ~
I I I I I I
*
C") C ..J C C C
0
FC-l -0.01
FC-2 -0.04
FC-3 0.03
FC-13 0.00 0.00 -0.03
FC-14 0.02 -0.02 -0.02
SPECIFICITY OF CAPILLARY TRANSPORT 85
REFERENCES
18. Park, C.R., Johnson, L.H., Wright, J.H. and Batsel, H. Effect
of insulin on transport of several hexoses and pentoses into
cells of muscle and brain. Am.J.Physiol. 191 (1957) 13-18.
19. Raichle, M.E., Larson, K.B., Phelps, M.E., Grubb, R.L., Welsh,
M.J. and Ter-Pogossian, M.M. In vivo measurement of brain
glucose transport and metabolism employing glucose-llC.
Am.J.Physiol. 228 (1975) 1936-1948.
The penetration of amino acids into the brain and into other
tissues is mediated by energy-requiring systems l - 3 , These systems
are not specific for each amino acid, but distinguish amino acids
into three groups: neutral, acidic, and basic 4 - 6 , The rate of
entry of anyone amino acid into the brain is controlled by the
ratio between its concentration in plasma and the concentration
of all other amino acids belonging to the same groupS-7.
On the other hand, the hypothesis that !2!!l, and not free,
89
90 A. TAGLIAMONTE ET AL.
The present report shows that insulin no~ only increases the
brain level of tryptophan but also increases the level of another
neutral amino acid, namely tyrosine, from blood to brain, though
decreasing its plasma concentration. This might suggest that
insulin directly controls the entry of amino acids into the brain.
Animals were stunned with a blow on the head, their blood was
rapidly collected from the abdominal aorta and allowed to clot at
+4oC, and the serum was frozen at -200C until analyzed.
RESULTS
TABLE I
Effect of Insulin Administration on the Concentrations of Tyrosine
and Tryptophan in Serum and in Brain.
TABLE 2
Effect of Oral Glucose Administration on the Concentrations of
Tyrosine and Tryptophan in Serum and in Brain.
DISCUSSION
REFERENCES
14. Curzon, G., Friedel, J., and Knott, P.J., The effect of fatty
acids on the binding of tryptophan to plasma protein, Nature
242 (1973) 198.
15. Tagliamonte, A., Biggio, G., Vargiu, L., and Gessa, G.L.,
Free tryptophan in serum controls brain tryptophan level and
serotonin synthesis, Life Sci., 12 (1973) 277.
16. Fernstrom, J.D., and Wurtman, R.J., Brain serotonin content:
increase following ingestion of carbohydrate diet, Sci., 174
(1971) 1023.
17. Wurtman, R.J., Effects of physiological variations in brain
amino acid concentrations on the synthesis of brain monoamines,
In, P. Seeman, G.M. Brown (Eds.), Frontiers in Neurology and
Neuroscience Research, First Int. Symposium of the Neuro-
science lnst., Toronto, 1974, p. 16.
18. Stefanini, E., and Biggio, G., Riv. Farmacol. Therap., Yr,
(1975) 46.
19. Costa, E., Spano, P.F., Groppetti, A., A1geri, S., and Neff,
N.H., Atti Ace. Med. Lomb., 23 (1968) 1100.
20. Snedecor, G.W., Statistical Methods, Iowa State College Press,
Ames, 1956.
21. Clark, A.J., Yamada, C., and Swendseid, M.E., Effect of L-
leucine on amino acid levels in plasma and tissue of normal
and diabetic rats, Am. J. Physiol., 215 (1968) 1324.
22. Manchester, K.L., The control by insulin of amino acid
accumulation in muscle, Biochem. J., 117 (1970) 457.
23. Biggio, G., Fadda, F., Fanni, P., Tagliamonte, A., and Gessa,
G.L., Rapid depletion of serum tryptophan, brain tryptophan,
serotonin and 5-hydroxyindoleacetic acid by a tryptophan-free
diet, Life Sci., 14 (1974) 1321.
24. Wurtman, R.J., In, P. Seeman, G.M. Brown (Eds.), Frontiers in
Neurology and Neurosciences Research, First Int. Symposium
of the Neuroscience lnst., Toronto, 1974.
25. Tagliamonte, A., Tag1iamonte, P., Corsini, G.U., Mereu, G.P.,
and Gessa, G.L., Decreased conversion of tyrosine to catechol-
amines in the brain of rats treated with p-ch1oropheny1a1anine,
.]. Pharm. Pharmac., 25 (1973) 101.
26. Wurtman, R.I., Larin, F., Mostafapour, S., and Fernstrom,
Brain catechol synthesis: control by brain tyrOSine concentrat-
ion, Science, 185 (1974) 183.
THE INFLUENCE OF LIVER-BYPASS ON TRANSPORT
Jill E. Cremer
95
96 J.E. CREMER
CH3.
1
*co. COOH
l,+c~
CH3. CH2. : CH2. *COSCoA
+CO~CH3 " .. ~
*COSCoA ~-----.....
~
*Aspartic ~*GI utamic-tGlutamine
acid ~ cycle j" acid
~
Figure 1. Schematic outline of the labelling of
metabolites during the oxidative metabolism of substrates
specifically labelled with l4c.
,
"
......
.......... GLN
,:
E S-O '
~ Q.
.2 ~ )( 4·0
:t:1C)
U
CD '0 2-0
Q. -
Cf)
Substrate injected
d.p.m.!g of brain
Time after Total % of total
injection radioactivity Glutamate Glutamine counts in
(seconds) GLU + GLN
10 -3 x d.p.m./g of brain
14 ~1_14C2But~rate
(1- C2Acetate
Control 2 26 7 7 67 19 30
Operated 2 4 1 0.7 33 10 11
Control rats p)
or rats 3 weeks after a portocaval-anastomosis (e)
120 • •
100 #Oe
0
0 0 0 0
0
80 0
00
0
60 0
0
40
•• • •
20 •
0
Minutes after intravenous injection
Figure 3
100 J.E. CREMER
Acetate 14
Butyrate 46
Octanoate 95
Acknowledgements
References
11. Veech, R.L., Harris, R.L., Veloso, D., and Veech, E.H.,
Freeze-blowing: a new technique for the study of brain in
vivo, Journal of Neurochemistry, 20 (1973) 183-188.
CERTAIN ASPECTS OF DRUG DISTRIBUTION TO BRAIN
William H. Oldendorf
INTRODUCTION
In brain, the cells making up the capillary wall are fused to-
gether, thus closing the intercellular cleft and obliterating this
non-specific route of exchange. Exchanges between plasma and brain
ECF must take place through the brain capillary and are trans-cell-
ular. Undoubtedly, such trans-cellular exchanges take place through
the general capillary wall but the extracellular route is so effici-
ent that it obscures any transcellular exchanges.
INTER-CELWLAR TRANS-CELWLAR
EXCHANGE EXCHANGE
GENERAL BRAIN
CAPILLARY CAPILLARY
•
INTERFACE
tV
ENZYMATICALLY
PLASMA DRUG CARRER
PROTEIN....... MOL~ PROTEIN
~,
---. SUBSTRATE
-
MOLECULE
CARRIER
PLASMA7'f'" " •• "PROTEIN
WATER
o
ENom.~JAL
PLASMA
CYTOPLASM
t
INNER
t
OUTER
MEMBRANE t.£t.tlRANE
LIPID LIPID
\, 1.-____________ _
:--
~ ~N NICOTIE (BRAW) PCO.4 -------------------E
~
~ OHMCL_
E
LSD (BRAIN)
124 8 16 64
TIME IN (MIN) AFTER I.V. IN.JECTION
••
NlCO-.• IM"UMINt
"HANOL
".eMU
N~~~N'~____________________
e __ •
100 •
•
..
.0 .~
O,FFI4NI
e.8_NlETMYL_
.
HEROIN
•
METHADONE
.
2 30
•
'HENOIAlIITAl
•
DUNnN
ASCOIIiC ACD
• MOI.'HIHI
MnHO~.EJlA!~nL5AUC'uc • ______ ~T~OD_ '.!.C!!I~~ ~E!.. ___________ _
2 ----l"iCm- - - - - - - - - - - -
.-
(noSH: • • IENZYLPINICLUN
AI.IWOSIDE $-IODO·2·DfOXYUIIDINE
10 100
'"OLIVE
.001 .10 1.0
Oil
....RTITION COfFfICENT
H 20
where they may reside for very long intervals. This rapid washout
and redistribution of very lipid-soluble barbiturates (such as thio- 6
pental) has been shown to be the basis of their brief central action.
REFERENCES
INTRODUCTION
111
112 E. LEVIN AND C.E. TRADATTI
Assuming that all CSF proteins were derived from blood, their
plasma liquor ratio would be approximately 200, showing on the one
hand the low permeability for these compounds, and on the other
hand the presence of regulatory mechanisms capable of maintaining
a steady protein concentration in CSF. We know very little about
these mechanisms apart from a presumably passive efflux by bulk
flow of the CSF.
Proteins with smaller size enter the eNS more easily than
bigger ones, again with selective differences. Serous-transferrin,
type II (mol.wt.88,000) is found in normal eSF, but transferrin
type I of the same size is not - it has been found only in abnormal
eSF when the total protein content rises above 100 mg percent 19.
Albumin,~;-glycoprotein, transferrin and some lipoproteins, all
wi th mol. wt. below 200,000 are present normally in eSF. In contras t,
fibrinogen,~glycoprotein, and certain lipoproteins (mol.wts.above
200,000) are excluded or only traces are present.
Routes of penetration:
parenchyma
The profile of penetration was different for total and for TCA
insoluble radioactivity, and thus, it was possible to determine the
diffusion coefficient (D) for Fab and for RISA in the tissue.
.7 I
I
I
RISA ,, Fab
E ,,
I I
,,
I I
:)
.4 I
"0
I
,,
I
,
CI>
I
E I
I
...
I \
,
u
I
C
0 I ' ...
u I
.......... .1 I
CI>
u
-;;
I
,, tissue
I
tissue
u
C
.....
0
u
5 10 5
distance from medium surface in mm
Fig.2. Diffusion patterns for Fab and for RISA in brain tissue
and in agar. -1
Diffusion coefficients in cm- 2 .sec .10- 7 Fab:Dtissue = 1.27
Dagar = 6.48 - RISA:Dtissue = 1.20; Dagar = 18.8; Dtissue for
inulin is 12.0 and for sulfate is 52.1.
Dotted line = total radioactivity (protein bound + TCA soluble),
deviating from diffusional patterns.
INCREASED PENETRATION
changes the protein attachment. Or, perhaps after edema and the
appearance of new protein species, there may be new interactions
and binding with the entering molecule. It is known that during
brain edema new proteins appear from proteolysis, metabolism,
and from removal of the macromolecules 27 • When new fractions
are formed, even nonspecific binding of proteins could be very
strong and might be fundamental to the state of the molecules in
a biological medium.
The main differences between this and other studies that utilized
hypertonic solutions, are: a) The combination of an "osmotic" solute
with another agent active in transport processes; b) blood supply
to the brain was maintained during the infusion; c) quantitative
measurement of the protein penetration; d) the addition of a lipid
soluble substance (DMSO) favored the protein penetration; and e) we
obtained frankly positive results, i.e., up to 20 times control
values, without macro- or micro-evidence of tissue damage.
TIM
N Agents Conc.* Osmol. more than Tissue
6 percent damage
(Positive
penetration)
Cortex 39 60 23 58 62 75 20 80
White matter 42 55 31 70 81 83 12 86
Thalamus 40 53 52 71 35 65 27 82
REFERENCES
18. Lorenzo, A.V., Shirahige, I., Liang, M., and Barlow, C.F.,
Temporary alterations of cerebrovascular permeability to plasma
proteins during drug induced seizures, Am.J.Physiol., 223
(1972) 268-277.
l8a.Lowenthal, A., Chemical physiopathology of the cerebrospinal
fluid. In A.Lajtha (Ed.). Handbook of Neurochemistry, Plenum
Press, New York, 1972, vol.VII, pp.429-464.
19. Lumsden, C.E., The proteins of cerebrospinal fluid in multiple
sclerosis. In D.McAlpine, C.E.Lumsden and E.D.Acheson (Eds.),
Multiple Sclerosis. A reappraisal. Livingstone Ltd., Edinburgh
and London, 1965, pp.252-299.
20.Matsen, F.A.III, and West, C.R., Supracortical fluid: a monitor
of albumin exchange in normal and injured brain, Am.J.Physiol.,
222 (1972) 532-539.
2l.Nauta, J.W., Kaiserman-Abramof, I.R., and Lasek, R.J.,
Electronmicroscopic observations of horseradish peroxidase
transported from the caudoputamen to the substantia nigra in the
rat: possible involvement of the agranular reticulum, Brain
Research, 85 (1975) 373-384.
22.0chs, S., Systems of material transport in nerve fibers (axoplas-
mic transport) related to nerve function and trophic control,
Ann.N.Y.Acad.Sci.,228 (1974) 202-223.
23.Paravicini, U., Stoekel, K., and Thoenen, H., Biological import-
ance of retrograde axonal transport of nerve growth factor in
adrenergic neurons. Brain Research, 84 (1975) 279-291.
24. Rapoport , S.I., Hori, M., and Klatzo, I., Testing of a hy~othes
is for osmotic opening of the blood-brain barrier, Am.J: Physiol.
223 (1972) 323-331.
25.Rapoport, S.I., and Thompson, H.K., Osmotic opening of the blood-
brain barrier in the monkey without associated neurological
deficit, Science, 180 (1973) 971-9
26. Rapoport, S.I., Thompson, H.K., and Bidinger, J.M., Equi-
osmolal opening of the blood-brain barrier in the rabbit by
different contrast media, Acta Radiol. ,15 (1974) 21-32.
27.Rasmussen,L.E., and Klatzo, I., Protein and enzyme changes in
cold injury edema, Acta Neuropath. 13 (1969) 12-28.
28.Reperant, J., The orthograde transport of horseradish peroxidase
in the visual system, Brain Research, 85 (1975) 307-312.
29.Schuller, E., Aspects actuels de la recherche sur les proteines
du liquide cephalo-rachidien, Ann.Biol.Clin. (Paris), 30
(1972) 297-300.
30.Sterrett, P.R., Thompson, A.M., Chapman, A.L., and Matzke,H.A.,
The effects of hyperosmolarity on the blood-brain barrier. A
morphological and physiological correlation,Brain Research, 77
(1974) 281-295.
3l.Westergaard, E., and Brightman, M.W., Transport of proteins
across normal cerebral arterioles, J.Comp.Neurol. 152 (1973)
17-44.
Transport Studies in
Various Nervous Tissue
Preparations
THE CHARACTERISTICS OF GLUCOSE TRANSPORT ACROSS THE BLOOD BRAIN
INTRODUCTION
133
134 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES
cells. The many studies using brain slices are probably more
relevant to transport across brain cell membranes than to trans-
port across the BBB.
Quantification of the rate of transport requires knowledge of
the blood flow rate and solute concentration. With few exceptions,
these values are unknown when whole animals are used; therefore,
studies made with the entire animal are generally qualitative
rather than quantitative. Interpretation of data obtained in
studies using intact animals is further complicated by the hormon-
al and metabolic influences of extracerebral tissues.
The ability to measure and control the perfusate composition
and flow rate in the isolated perfused canine brain combined with
the ability to study unidirectional solute extraction using the
indirttor dilution technique has allowed us to quantify the
rate of unidirectional glucose transport across the BBB. Thus,
the effects of both physiologic conditions and pharmacologic
agents on the kinetics of glucose transport across the BBB can be
studied.
1.60 • •
•
c • •
E 120
"e •
•
"..
0>
'"
'0
e~ 0.80
~
0.40
0.10 0.20 0.30 0.40 0.50 0.60
-!,.(mM'-'
A
10 20 30 40 50
A (mM)
"2
·e....
E
!?
,,
.,
II>
0.40
,
~
~
0.30
'y.-- --t- -i---i
? 0.20
0.10
o 5 10 15 20 25 30 35 40 45 50 55 60
Minutes of Anoxia
123456 10 15 20 25 30
Minutes of Anoxia
than 24% of normal a~§er 10 min of anoxia and to near zero follow-
ing 30 min of anoxia . These data suggest that the brain could
survive longer periods of anoxia if the blood glucose concentra-
tion were elevated. Prolonged post-decapitation EEG activity and
maintenance of a more normal metabolic state ~tS been observed in
mice made hyperglycemic prior to decapitation .
The entry of glucose into the brain during anoxia becomes
g
even more inadequate as the result of subsequen ~?anges in
properties of the BBB glucose transport system ' . After 2
minutes of anoxia, the rate of unidirectional glucose transport
begins to decline and after 15 minutes it levels off at a rate
that is approximately half that of the control value (Fig. 2).
These data indicate that the BBB glucose transport mechanism is
altered as a consequence of cerebral anoxia. That this is not due
to a general change in BBB permsability is indicated by t?5 fact
that both diffusion of fructose and transport of leucine are
unchanged by anoxia. There are several possible explanations for
this phenomenon: a) an inhibitor of glucose transport may be
produced during anoxia, b) cerebral blood flow may be substantia-
ly redistributed during anoxia, c) the facilitated diffusion --
transport system may be modified as a result of the decrease in
brain glucose levels, d) glucose transport across the BBB may be
directly coupled to metabolic energy, or e) glucose transport
across the BBB may be indirectly ~ouple$ to metabolic energy
through cotransport with ions (Na or H). Each of these possibi-
lities is discussed.
43.9±0.6mM
2.0
C 26.3±0.2mM
·E 16.8.:tO.3mM
......
E 1.5 6.11±0.!7mM
~
~'"
0
E 1.0
<.
~
0.5
20 40 60 80 100
A (mM)
Table 1
Effectiveness of Various Monosaccharides as Inhibitors
of Glucose Transport in Brain and Erythrocyte
hypothet ico l
cytocho losin 8
binding si te
SUMMARY
ACKNOWLEDGEMENT
REFERENCES
Department of Physiology
King's College, Strand, London WC2R 2LS, U.K.
and
*Laboratoire de Neurobiologie Cellulaire
CNRS, 91190 Gif-Sur-Yvette, France
INTRODUCTION: K+ HOMEOSTASIS
151
152 N.J. ABBOTT AND Y. PICHON
INVERTEBRATE STUDIES
CRUSTACEAN STUDIES
TABLE 1
_____________________________
Preparation Ion T,0.5 ______________
(mins) n __
a 13
mM --control
.... .... ethlcrynlc acid
- - - strophanthldln
It
_.- 3 M urea
2 8 10 14 18
min
b entry efflux
0 0
In ( K,.!C..)
\ ",
' .,,,,,".,,".".
1Io·!C..
\ " ';;''.;,; ''
-(}02 \
\
-1 , "
' ', .' .....
"'.
\ " "-,
"............
\
i
i -2
"
,'. ' "- "-
,.
"'-.,
-()-Q4
\ "-
"-
"
"'i \
-(}08
\
-3
"'\,
\
\
\
0 2 2 e 10
min '"
Figure 2a. Effect of 2 min 100 mM K Ringer pulse (horizontal
bar) on K concentration at the axon surface (K)a' in control and
after treatment with inhibitors or urea. S04 replaced Cl in the
K-Ringer. Results with KCl-Ringer were comparable.
Medium
(a)
(b)
Tris
CI
60
mV
40
20
O+---f-"""T""__l""t'"rT'I'r---r-""""'"
20
CONCLUSION
REFERENCES
27. Sugaya, E., Takato, M., and Noda, Y., Neuronal and glial
activity during spreading depression in cerebral cortex of
cat, J. Neurophysiol., 38 (1975) 822-841.
28. Sypert, C.W., and Ward, A.A., Unidentified neuroglia potentials
during propagated seizures in neocortex, Exp. Neurol., 33
(1971) 239-255.
29. Trachtenberg, M.C., and Pollen, D.A., Neuroglia: biophysical
properties and physiologic function, Scienc~ 167 (1970)
1248-1251.
30. ... v, "
Vyskoc~l, F., Kr~z, N., an
d Bures,
.. J., Potassium selective
microelectrodes used for measuring the extracellular potassium
during spreading depression and anoxic depolarization in rats,
Brain Res., 39 (1972) 255-259.
31. Walker, J .L., Ion specific liquid ion exchanger microelectrodes ,.
Analyt. Chern., 43 (1971) 89 A-93 A••
AMINO ACID TRANSPORT IN SPINAL AND SYMPATHETIC GANGLIA
Peter J. Roberts
I. INTRODUCTION
It has been suggested 16 that the glial cell package which envelops
synaptic regions in the brain and spinal cord, and also the cell
bodies of ganglia, is present to regulate the substances reaching
the neuronal membrane; such substances could include neurotrans-
mitters, K+, and blood-borne, neuroactive materials. The release
AMINO ACID TRANSPORT IN GANGLIA 167
TABLE 1
UPTAKE OF AMINO ACIDS INTO RAT DORSAL ROOT GANGLIA
Amino Acid Tissue: Medium Ratio
0.5 hr 4 hr
activity over the satellite glial cells. With GABA, the presence
of AOAA in the medium greatly enhanced the silver grain density.
Similar 3results were obtained with desheathed superior cervical
ganglia 6; there was heavy labelling over the satellite cells and
especially over the Schwann cells around unmyelinated fibres.
Incubation of dorsal root ganglia 34 with a high (300 ~) concentrat-
ion of 3H-GABA, i.e. with the low affinity uptake system predominat-
ing, a large number of silver grains was observed over the neuronal
cell bodies and connective tissue sheath in addition to over the
glia. When ganglia were incubated with 3H-GABA in the absence of
sodium, the extent of labelling was minimal, with no selective
localization. Investigations with tritiated glycine, a-alanine,and
leucine under the same conditions as for GABA, revealed an equal
localization over the neuronal cell bodies and satellite glial cells.
TABLE III
DISTRIBUTION OF AMINO ACID RADIOACTIVITY DERIVED FROM l4C-GLUTAMATE
(5 ~), l4C-PYRUVATE (450 ~), AND l4C-ACETATE (80 ~).
l4 C_glu l4 C_pyr 14C-ac
Amino Acid Endogenous
conc. -1
(IJIIlO 1. g ) S.A. R.S.A. S.A. R.S.A. S.A. R.S.A.
TABLE IV
IC sO VALUES FOR THE INHIBITION OF GABA UPTAKE INTO CORTICAL SLICES
AND GANGLIA.
Compound IC501__________~_
Cortex Ganglia
700-J~
L-2,4-diaminobutyric acid 50~'~
l20-J~
!3-alanine
-J~ from Schon and Kelly34 19
** from Iversen and Johnston
IC sO value is the concn. of inhibitor required to produce a 50%
inhibition of the uptake of 0.5 ~ 3H-GABA after a 10 min incubation .
Tissues pre incubated with the inhibitor for 10 min.
Recent evidence (see Levi et al., this book) has indicated that
homoexchange is a major mechanism by which radioactive GABA is trans-
ported into synaptosomes. This process is strongly sodium-dependent,
and occurs even at very low concentrations of GABA in the external
medium, and hence might merely simulate a high-affinity uptake system.
To test whether homoexchange occurs in dorsal root ganglia, ganglia
were finely sliced and incubated with 3H-GABA (0.8 ~) followed by
superfusion for 30 min (unpublished). When varying concentrations
of unlabelled GABA were added to the superfusion medium, homoexchange
occurred, with a detectable increase in release of 3H-GABA occurring
at a concentration of 5 ~ unlabelled GABA, and a maximal release at
approximately l500~. The heteroexchange of 3H-GABA with DAB and
~-alanine (both 100 ~) was also investigated: ~-alanine was approx-
imately three times as effective as DAB as substrate for the carrier
system. Several other substances including O-aminovalerate and 3-
hydroxy GABA were also able to heteroexchange.
AMINO ACID TRANSPORT IN GANGLIA 175
IX CONCLUSIONS
The glial cells of both dorsal root ganglia and superior cervical
ganglia behave very similarly to isolated glia, cultured glial tumor
cells, and nerve endings in their ability to accumulate neuroactive
amino acids by sodium-dependent, high-affinity systems. Hence, it
can be proposed that the function of the glial cells in the dorsal
root ganglion and superior cervical ganglion is to regulate the levels
of neurotransmitters and other neuroactive materials in the extra-
cellular space and to form a protective barrier against substances
entering from the blood supply; however, it has never been suggested
that either glutamate or GABA playa transmitter role in ganglia,
and their neurones are insensitive to glutamate. GABA will depolarize
neurones of both sensory12 and superior cervical ganglia ll , and this
can be antagonized by the GABA - receptor antagonists, bicuculline
and picrotoxin13 • Close intra-arterial injection of GABA readily
depolarizes ganglionic neurones; hence the glial cells would not seem
to be particularly efficient at these sites in a 'protective' role.
What then are the functions of the uptake systems found in the satelli-
te glia and Schwann cells? It seems quite feasible that the uptake
sites are mere vestiges of a property common to all glial cells, but
which are active only in the central nervous system in areas where
GABA or glutamate are neurotransmitters. Such a concept is supported
by the finding that Sc~wann cells of both dorsal and ventral spinal
roots will accumulate 4C-glutamate in a similar manner28. An
alternative proposal 4 for the function of the glial uptake systems
is that the glial cells are the site of the 'GABA-shunt' in nervous
tissue.
REFERENCES
INTRODUCTION
179
180 B. HABER AND H.T. HUTCHISON
v
=
S
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 181
v +D
s
Where Km and Vmax are Michaelis constants and maximum velocities,
respectively, and D is a diffusion constant. The velocities, V,
have units of picomoles of substrate taken up per milligram of
cell protein per minute of incubation; the substrate concentra-
tions, S are expressed in micromolar units. The data are plotted
as vIs vs V, a modified Hofstee 28 plot. A transport system obey-
ing Michaelis-Menton kinetics (a "carrier mediated" transport sys-
tem) yields a straight line on a plot of VIS versus V, whereas a
two-carrier mediated system for the same substrate, or a single
carrier mediated system and diffusion, yield hyperbolas. Using
the aforementioned method, kinetic data from our laboratory are
obtained using statistically weighted fits 21 and are presented with
standard errors.
10 20
V (pmoles/mg-min)
A B
0.04
0.04
-
0.03 0.03
3-
E
f
'ii
'ii
~
0.02 i
,g.
0.02
,g.
>
>
0.01 0.01
10 20 5 10 15 20
minutes minutes
TRANSPORT OF CHOLINE
30 Km Vmax D
Ai4.2 303 10.8
. t3D tao !.6
Kml Vmax Kmz Vmax
B.
"ii5 201 2,605 34,733
t5.1 !60 !I,022 !12,744
20
V
S
10
- ---------+-
B
A
5000 10000
V
Fig. 3:
A. Graphic description of a singZe high-affinity
transport system pZus a diffusionaZ component.
B. Graphic description of a carrier-mediated trans-
port system, consisting of two components of
high and ZOW affinities.
Inset gives numericaZ vaZues for the kinetic parameters des-
cribed by curves A and B. Each point represents the mean
of five determinations; th~ bar, the standard error of the
mean (from Ref. 49).
parameters obtained from these fits are shown in the inset to Fig.
3. Either fit yields an estimated Km for the high-affinity cho-
line transport system in the range of 10-14 ~M. The low-affinity
component is less well characterized by these data,but if it is a
saturable system, it has a Km greater than 1 roM and is not satu-
rated at 10 roM, the highest concentration used in this series of
experiments. These data are included to graphically illustrate the
possibility of multiple possible fits for double-affinity trans-
port systems; that even rigorous statistical evaluation of data
yields Km values which should be assigned a range rather than an
absolute numerical value. Furthermore, it is difficult, if not
impossible, to unequivocally decide whether a low-affinity system
is in fact carrier-mediated or represents diffusion. These con-
siderations apply equally to transport studies of other neuro-
transmitters and precursors.
A B
15
I(tI'II Vrncu _ C_
. No° Il7!131 7B!n7 4~! 0 4
~ 10
- No" 1t1 4,! 1.0
S 26~24
,,
\
'---------------------------------------
TABLE I
% INHIBITION
INHIBITOR Cone. (M) NE 5HT
ACKNOWLEDGMENTS
REFERENCES
10. Schrier, B., and Thompson, E.J., Uptake, excretion, and meta-
bolism of putative neurotransmitters by cultured glial tumor
cells, J. Biol. Chem., 249 (1974) 1769-1780.
14. Schubert, D., The uptake of GABA by clonal nerve and glia,
Brain Research, 84 (1974) 87-98.
16. Lanks, K., Somers, L., Papermeister, B., and Yamamura, H.,
Choline transport by neuroblastoma cells in tissue culture,
Nature, 252 (1974) 476-478.
19. Benda, R., Lightbody, J., Sato, G., Levine, L., and Sweet,
W.H., Differentiated rat glial cell strain in tissue culture,
Science, 161 (1968) 370-371.
21. Harris, A.J., Heinemann, S., Schubert, D., and Tarakis, H.,
Trophic interaction between clonal tissue culture lines of
nerve and muscle, Nature, 231 (1970) 296-301.
22. Schubert, D., Humphreys, S., de Vitry, F., and Jacob, F.,
Induced differentiation of a neuroblastoma, Devel. BioI.,
25 (1971) 514-546.
23a. Kukes, G., deVellis, J., and Elul, R., A linked active trans-
port system for Na+ and ~ in a glial cell line. Brain
Research, 1975 (in press).
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 195
23b. Kukes, G., Elul, R., and deVellis, J., The ionic basis of
the membrane potential in a rat glial cell line, Brain
Research, 1975 (in press).
24. Varon, S., Neurons and glia in neural cultures, Exp. Neuro-
~, 48 (1975) 93-134.
34. Schon, F., Beart, P.M., Chapman, D., and Kelly, J.S., On
GABA metabolism in the gliocyte cells of the rat pineal gland,
Brain Research, 85 (1975) 479-490.
196 B. HABER AND H.T. HUTCHISON
43. Kuhar, M.J., Sethy, V.H., Roth, R.H., and Aghajanian, G.K.,
Choline: Selective accumulation by central cholinergic
neurons, J. Neurochem., 20 (1973) 581-593.
44. Liang, C.C. and Quaste1, J.H., Effects of drugs on the uptake
of acetylcholine in rat brain cortex slices, Biochem. Pharma-
col., 18 (1969) 1187-1194.
46. Haga, T., and Noda, H., Choline uptake systems of rat brain
synaptosomes, Biochern. Biophys. Acta, 291 (1973) 564-575.
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 197
49. Hutchison, H.T., Suddith, R.L., Risk, M. and Haber, B., Uptake
of neurotransmitters and precursors by clonal lines of astro-
cytoma and neuroblastoma. III. Transport of choline, Neuro-
chemical Research, 1975 (submitted).
55. Haber, B., Werrbach, K., Risk, M. and Hutchison, H.T., Uptake
of serotonin by clonal lines of astrocytoma and neuroblastoma
in culture. Fifth ISN Meeting, 1975, p. 169.
60. Wong, D.T., Horng, J.F., Bymaster, F.P., Hauser, K.L., and
Molloy, B.B., A selective inhibitor of serotonin uptake:
Lilly 110140, 3-(P-trifluoromethylphenoxy)-N-methyl-3-
phenolpropylamine, Life Sciences, 15 (1974) 471-479.
CULTURES
INTRODUCTION
199
200 R. MASSARELLI AND P. MANDEL
Cell Cultures
Neuroblastoma clones: Cholinergic clones 821 and 820
and inactive clone NI8 were a generous gift of Dr. M. Nirenberg;
astroblasts from hamster (clone NN) were obtained from North Ame-
rican Biological Inc. and glioblastoma clone C6 and neuroblastoma
clone CCL 131 neuro 2a from American Type Culture Collection. The
cells were stored in liquid nitrogen and thawed when needed. The
cultivation was performed in Falcon plastic flasks (75 cnf) with
Dulbecco's medium (Gibco) containing 10 % foetal calf serum in a
100 % humidified atmosphere of 5 % C02/95 % Air at 37 0 C. The
cultures were replicated in Falcon plastic Petri dishes (28 cm2 )
and used at confluency.
Chick embryo cultures : The cerebral hemispheres of em-
bryos at various ages were dissociated through a nylon sieve (48~)
and seeded in Falcon plastic Petri dishes. The cultures were incu-
bated in Eagle's modified Dulbecco's medium containing 20 % fetal
calf serum in a 100 % humidified atmosphere of 5 % C02/95 % Air
at 37 0 C. Cultures from 8 day old embryos in culture for 7-10 days
show a uniform2~ayer of glial-like cells on top of which neurons
are dispersed -)0. The presence of synaptic junctions has been
shown in cultures of this age. Cultures from 14 day old embryos in
culture for 7 days present the same morphological aspect 29-30;
however, when these cultures are kept for longer periods in cultu-
re (14-21 days) the neurons will degenerate and only differentia-
ting glial cells will be left 30.
50 100
MIN
CN DNP Refe-
Ouabain 4°C
rences
Cultures
S21 (ImM) 83% --- (O.lmM) 65% 10% (32)
NI8 88% --- 75% 8% (32)
E8c7
EI4c7
--- ( ImM) 61%
--- ---
81%
90%
13%
12%
(33)
(33)
S~naE-
tosomes
(4mM) 98% (0.2mM)96% (0.2mM) 74% --- (23)
( ImM) 27% (0. 2mM) 40% (O.lmM) 55% 22% (22)
(0.lmM)73%
Homo~enates
corpus
striatum (0.5-100) I. 43 9.3 (22)
cerebral
cortex (0.5-100) 3.0 3.3
cerebellum (0.5-100) --- 4. I
8ynapto-
somes
--
(0.5-100) 2.0 2.S (29)
(2.0- SO) 4.B 4.0 (23)
(2. 0-100) 3.5 --- (24)
Table 2: High affinity (HA) and low affinity (LA) Km's of choli-
ne uptake in nerve cell cultures and synaptosomes. NIS: neuroblas-
toma inactive clone; 821, 826: neuroblastoma cholinergic clones;
NIE 115: neuroblastoma adrenergic clone; NN: normal astroblasts
from hamster; C6: rat glioblastoma; BB2: fibroblasts 37 ; EBCIO:
B day-old chick embryos in culture for 10 days; E14C7: 14 day-old
embryos in culture for 7 days; E14C21: 14 day-old embryos in cul-
ture for 21 days. x = not specified.
204 R. MASSARELLI AND P. MANDEL
e e
I
..1
y " .1.
y "
.1 .5 1.0 .1 I
.-1..
S
DISCUSSION
171
J
BW284 C51
-6 .,.,P
10 M
s
y
-30 -10 0 10 30 50
s
Fig. 5 Effect of a selective inhibitor of acetylcholinesterase
on the kinetics of choline uptake in clone N18.
REFERENCES
10. Freeman, J.J., Choi, R.L., and Jenden, D.J., Plasma choline:
its turnover and exchange with brain choline. J. Neurochem.
24 (1975) 729-734.
II. Choi, R.L., Freeman, J.J., and Jenden, D.J., Kinetics of plas-
ma choline in relation to turnover of brain choline and forma-
tion of acetylcholine. J. Neurochem. 24 (1975) 735-742.
12. Spanner, S., Hall, R.C., and Ansell, G.B., Arteriovenous dif-
ferences of choline and choline lipids across the brain of rat
and rabbit. Biochem. Soc. Trans., 3 (1975) 120.
13. Martin, K., Concentrative accumulation of choline by human ery-
throcytes. J. Gen. Physiol., 51 (1968) 497-516.
14. Green, A.R., Boullin, D.J., Massarelli, R., and Hanin, I.,
Can the human blood platelet be used as a model for choliner-
gic nerve ending? Life Sci., II (1972) 1049-1058.
15. Schuberth, J., Sundwall, A., Sorbo, B., and Lindell J.O., up-
take of choline by mouse brain slices. J. Neurochem.,13 (1965)
347-352.
16. Potter, L.T., The uptake of choline by nerve endings isolated
from the rat cerebral cortex. In P.N. Campbell (Ed.), The In-
teraction of Drugs and Subcellular Components on animal Cells,
Churchill, London, 1968, pp. 293-304.
17. Marchbanks, R.M., The uptake of [I4C]choline into synaptosomes
in vitro. Biochem. J. 110 (1968) 533-541.
18. Diamond, I., and Kennedy, E.P., Carrier mediated transport of
choline into synaptic nerve endings. J. BioI. Chem., 244
(1969) 3258-3263. ---
19. Diamond, I., and Milfay, D., Uptake of [3H]methyl choline by
microsomal, synaptosomal,mitochondrial and synaptic vesicles
fractions of rat brain. J. Neurochem., 19 (1972) 1899-1909.
20. Hemsworth, B.A., Darmer, K.I., and Bosmann, H.B., The incor-
poration of choline into isolated synaptosomal and synaptic
vesicles fractions in the presence of quaternary ammonium
compounds. Neuropharmacol., 10 (1971) 109-120.
21. Yamamura, H., and Snyder, S.H., Choline: high affinity uptake
by rat brain synaptosomes. Science, 178 (1972) 626-628.
22. ----- High affinity transport of choline into synaptosomes of
rat brain. J. Neurochem., (1973) 1355-1374.
23. Haga, T., and Noda, H., Choline uptake system of rat brain sy-
naptosomes. Biochim. Biophys. Acta, 291 (1973) 564-575.
24. Guyenet, P., Lefresne, P., Rossier, J., Beaujouan, J.C., and
Glowinski, J., Effect of sOdium, hemicholinium-3 and antipar-
kinson drugs on [l4C] acetylcholine synthesis and [3H] choline
uptake in rat striatal synaptosomes. Brain Res., 62 (1973)
523-529.
25. ----- Inhibition by hemicholinium-3 of [14C] acetylcholine
synthesis and [3H] choline high affinity uptake in the rat
striatal synaptosomes. Molec. Pharmacol., 9 (1973) 630-639.
26. Dowdall, M.J., and Simon, E.J., Comparative studies on synap-
tosomes: uptake of [Me- 3H]-choline by synaptosomes from squid
208 R. MASSARELLI AND P. MANDEL
41. Kuhar, M.J., Sethy, V.H., Roth, R.H., and Aghajanian, G.K.,
Choline: selective accumulation of central cholinergic neu-
rons. J. Neurochem., 20 (1973) 581-593.
42. Sorimachi, M., and Kataoka, K., Choline uptake by nerve ter-
minal: a sensitive and a specific marker of cholinergic In-
nervation. Brain Res., 72 (1974) 350-353.
43. Massarelli, R., Stefanovic, V., and Mandel,P., Effect of ChE
inhibitors on choline high affinity uptake and ectocholines-
terase activity in nerve cell cultures. Vth International
Meeting of the International Society for Neurochemistry,
a b s t r ac t I 53 (I 975) .
44. Stefanovic, V., Massarelli, R., 11andel, P., and Rosenberg, A.,
Effect of cellular desialylation on choline high affinity up-
take and ecto-cholinesterase activity of cholinergic neuro-
blasts. Biochem. Pharmacol., 24 (1975) 1923-1928.
THE UPTAKE AND RELEASE OF y-AMINOBUTYRIC ACID (GABA) BY THE RETINA
M.J. Neal
INTRODUCTION
The present study concerns the uptake and release of the putative
synaptic transmitter substance y-aminobutyric acid (GABA) by the
retina. It is reasonable to assume that the retina utilizes the
same transmitter substances as the rest of the CNS and so the study
of retinal transmitters, in addition to providing information on
visual physiology, may also provide clues to understanding central
synaptic mechanisms in general.
211
212 M.J. NEAL
24
22
(S)
20 (4)
18
16
.2 14
C;
a:: 12 0-<> [I4 C]-GABA
E (4) lO-8 M)
.~ 10
"0 .--e L3 H]-Mannitol
-''""
~
:J
III
8
6
(lO-7 M)
III
;:: 4
2 (8) (8) (8)
__ ..... ____ .... _____ - - - - - I
5 10 15 20 25 30 35 40 45 50
Incubation Time (min)
80
60
40
.!!
800 ~ 20
S
....
600 .
~
c
c
0
-20
..1 x 10-6 ""
u
V 400 -40
-60
-80
I
-100
400 600 800 1000
..1.10"3 I nl1 10nl1 100nl1 111" 10,,11 100,.,.. ImH IOml1
5 AOAA concentration
Fig. 2 Fig. 3
ACKNOWLEDGEMENTS
REFERENCES
Institute of Neurobiology
University of Goteborg
Fack, S-400 33
Goteborg, Sweden
INTRODUCTION
221
222 A. HAMBERGER ET AL.
Most amino acid uptake systems are active, i.e. against a con-
centration gradient. Accordingly, uptake of either transmitter or
226 A. HAMBERGER ET AL.
120
T
~ 80
Q.
U
,.."
..
. ............ ;
I \
"..... ..........
\
40
~ .
,/
5 15 min
those leading to high efflux rates and vice versa. Thus, ouabain and
low sodium stimulate GABA efflux from pre-loaded fractions 34 , where-
as they inhibit uptake. Of the greatest interest is that K+-stimul-
ated release of amino acids from glial cells could imply a physi-
ologically important mechanism of modulation at the synaptic site;
i.e. neuroactive amino acids released from the glial compartment in
response to high K+ during pre-synaptic activity could compete with
synaptically released amino acid for occupation of post-synaptic
receptors.
In our hands, K+-stimulated glial release of GABA failed to
show the "classical" Ca++-dependence shown for synaptosomes;
failure to exhibit calcium-dependence 55wou ld be expected of a non-
vesicle-mediated phenomenon. Neuronal perikarya, on the other hand,
showed calcium-dependence for release, like synaptosomes, and here
we are not dealing with vesicles.
80000
60000
40000
20000
GLIAL CElLS
SYNAPTOSOMES
o+---~-----r----~--~-----r----'
o 10 15 20 25 30
MIN
GLUTAMATE-GLUTAMINE COMPARTMENTATION
300
o Ring.r
2 ••
.c 150
'e.·
.........
'"
-Z..
·
~
E
10 •
U
•
·
'0
E
50
SUMMARY
ACKNOWLEDGEMENTS
REFERENCES
31 Katz, R.I., Chase, T.N., and Kopin, I.J., Effects of ions on sti-
mulus-induced release of amino acids from mammalian brain slices,
J.Neurochem.,16 (1969) 961-967.
32 Kuriyama,K., Weinstein, M., and Roberts, E., Uptake of y-amino-
butyric acid by mitochondrial and synaptosomal fractions from
mouse brain, Brain Res.,16 (1969) 479-492.
33 Kvamme,E., and Torgner, I. Aa., The effect of acetyl-coenzyme A
on phosphate-activated glutaminase from pig kidney and brain,
Biochem.J., 137 (1974) 525-530.
34 Levi,G., and Raiteri,M., Exchange of neurotransmitter amino acid
of nerve-endings can stimulate high affinity uptake, Nature,250
(1974) 735-737.
35 Logan,W.I. and Snyder, S.M., Glycine, glutamic and aspartic acids:
Unique high-affinity uptake systems in central nervous tissue of
the rat, Nature ,234(1971) 297-299.
36 Logan, W.J. and Snyder, S.M., High-affinity uptake systems for
glycine, glutamate and aspartic acids in synaptosomes of rat cen-
tral nervous tissue, Brain Res.,42 (1972) 413-431.
37 Lowry, O.H., The quantitative histochemistry of the brain: Histo-
logical sampling, J.Histochem.Cytochem.,l (1953) 420-428.
38 Medzihradsky, F., Sellinger, O.Z., Maudhasri,P.S., and Santiago,
J.C., ATPase activity in glial cells and in neuronal perikarya
of rat cerebral cortex during early postnatal development,
J.Neurochemistry 19 (1972) 543-545. 3
39 Minchin, M.C.W. and Iversen, L.L., Release of ( H) gamma-amino-
butyric acid from glial cells in rat dorsal root ganglia, J.
Neurochem. , 21 (1974) 533-541.
40 Nagata, Y., Mikoshiba, K., and Tsukada, Y., Neuronal cell body
enriched and glial cell enriched fractions from young and adult
rat brains: preparation and morphological and biochemical pro-
perties, J.Neurochem. 22 (1974) 493-503.
41 Orkand, P.M., Bracho·, M., and Orkand, R.K., Glial metabolism:
alteration by potassium levels comparable to those during neural
activity, Brain Res., 55 (1973) 467-471.
42 Pamiljans, V., Krishnaswamy, P.R., Dumville, G. and Meisler, A.,
Studies on the mechanism of glutamine synthesis: Isolation and
properties of the enzyme from sheep brain, J.Biochem., 1 (1962)
153-158.
43 Poduslo, S.E., and Norton, W.T., Isolation and some chemical
properties of oligodendroglia from calf brain, J.Neurochem.,19
(1972) 727-736.
14
44 Roberts, P.J., and Keen, P., C-glutamate uptake and compartmen-
tation in glia of rat dorsal sensory ganglion, J.Neurochem., 23
(1974) 201-209.
45 Roberts, P.J., and Keen, P., High-affinity uptake system for gluta-
mine in rat dorsal roots but not in nerve-endings, Brain Res.,
67 (1974) 352-357.
236 A. HAMBERGER ET AL.
INTRODUCTION
237
238 S.S. OJA. P. KONTRO. AND P. LAHDESMAKI
Brain 1 2
Femoral muscle 3 5
Heart 10 80
Liver no 130
"0
Q. A B
~ •
c:
!300
"0
E
c:
..............
".. 4
.>t.
a.
:l .........
------
---....................
"c: 2
'§
t!!!
2 4 6 8 10 12 20 40 60 80 100
Taurine concentration mmol/l Taurine concentration I'molll
- means that the authors have not detected this component in the
saturable taurine influx
Amidosulphonic acid 88
Glycine 66 62 125 87 72
Hypotaurine 9 58 50 11
S-Alanine 14 21 42 49 17
L-Alanine 94 82 70
L-Cysteine 54 86 68
L-Cysteic acid 86 75
L-Cysteine sulphonic acid 83 71
y-Aminobutyric acid 38 39 93 65 31
S-Aminoisobutyric acid 62
a-Aminoisobutyric acid 87
N-Methyltaurine 47 117 77 55
S-Guanidinopropionic acid 74
6-Aminovaleric acid 37
£-Aminocaproic acid 80
Isethionic acid 85
~t 0.15/
E~
"0
E
c
0.10
c
:8
1!
C
~ "C
C ;,
8 .8 0.05
1 2 3 4 5 1 2 3 4 5
Taurine concentration mM Taurine concentration mM
iii
£
40
'0 Elution order
111.
30 )
~I)
::J
iii 20
05
I)
10
C
0;c:
::J
~
.
0
ill
z
0
~
it
""Z'"
I»
'"""
Z
I»
0
g
Z
0
g
Z .g
0 0
;;
~
::a
CD
III
c:
C
I» Q.
Q Q J: Z ~ C; !!!.
Q ~ '"VI I»
0 C;
::J
::J
3
I» ..0 J:
>:< It
~ >:< 00 3
g-
is
o I»
::J
CD
III
CONCLUSIONS
REFERENCES
32. MINATO, A., HIROSE, S., OGISO, T., UDA, K., TAKIGAWA, Y., and
FUJIHIRA, E., Distribution of radioactivity after adminis-
tration of taurine _ 35 S in rats. Chern. Pharm. Bull. 17 (1969)
1498-1504.
33. MIRAGLIA, R.J., MARTIN, W.G., SPAETH, D.G., and PATRICK, H.,
On the synthesis of taurine from sulfate by the chick: I. Inf-
luential dietary factors, Proc. Soc. expo BioI. Med. 123
(1966) 725-730.
34. NEAL, M.J., PEACOCK, D.G., and WHITE, R.D., Kinetic analysis
of amino acid uptake by rat retina in vitro, Brit. J. Pharma-
col. 47 (1973) P656-P657. •
35. OJA, S.S., Exchange of taurine in brain slices of adult and
7-day-old rats, J. Neurochem. 18 (1971) 1847-1852.
36. OJA, S.S., KARVONEN, M.-L., and LAHDESMAKI, P., Biosynthesis
of taurine and enhancement of decarboxylation of cysteine sul-
phinate and glutamate by the electrical stimulation of rat
brain slices, Brain Res. 55 (1973) 173-178.
37. OJA, S.S., and PIHA, R.S., Changes in the concentration of
free amino acids in the rat brain during postnatal develop-
ment, Life Sci. 5 (1966) 865-870.
38. OJA, S.S., UUSITALO, A.J., VAHVELAINEN, M.-L., and PIHA, R.S.,
Changes in cerebral and hepatic amino acids in the rat and
guinea pig during development, Brain Res. 11 (1968) 655-661.
39. PASANTES-MORALES, R., KLETRI, J., LEDIG, M., and MANDEL, P.,
Influence of light and dark on the free amino acid pattern of
the. developing chick retina, Brain Res. 57 (1973) 59-65.
40. PASANTES-MORALES, H., KLETRI, J., URBAN, P.F., and MANDEL, P.,
The physiological role of taurine in retina: uptake and effect
on electroretinogram (ERG), Physio1. Chern. Phys. 4 (1972)
339-348.
41. PASANTES-MORALES, R., KLETRI, J., URBAN, P.F., and MANDEL, P.,
The effect of electrical stimulation, light and amino acids on
the efflux of 35S- taur ine from the retina of domestic fowl,
Exp. Brain Res. 19 (1974) 131-141.
42. PASANTES-MORALES, H., URBAN, P.F., KLETRI, J., and MANDEL, P.,
Light stimulated release of J 35 SJtaurine from chicken retina,
Brain Res. 51 (1973) 375-378.
43. PECK, E.J., and AWAPARA, J., Formation of taurine and isethi-
onic acid in rat brain, Biochim. Biophys. Acta 141 (1967)
499-506.
44. PIHA' R.S., OJA, S.S., and UUSITALO, A.J., The effect of
chlorpromazine on free amino acids in the rat brain, Ann. Med.
expo BioI. Fenn. 40, Supp1. 5 (1962) 1-28.
45. RASSIN, D.K., Amino acids as putative transmitters: failure to
bind to synaptic vesicles of guinea pig cerebral cortex, J.
Neurochem. 19 (1972) 139-149. -
46. READ, W.O., and WELTY, J.D., Effect of taurine on epinephrine-
and digoxin-induced irregularities of dog heart, 1. Pharmacol.
expo Therap. 139 (1963) 283-289.
252 5.5. OJA, P. KONTRO, AND P. LAHDESMAKI
Henry McIlwain
Institute of Psychiatry
The Maudsley Hospital
Denmark Hill, London, England (U.K.)
INTRODUCTION
253
254 H. MciLWAIN
140
.!:
.. 120
~Q.
0
rlOO
...
Q
Q.
C
..,
E
~
U 60
£
....
'0
.
> 40
0
u
a:
u 20
!
Pentobarbital
66lJM ; 60lJmoles kg Decreased Post-mortem increase
opposed
CONCLUSION
...
Transport from extracerebral regions:
as PURINES and PURINE PRECURSORS
Extracerebral
.:
Extracellular transport:
as NUCLEOSIDES or PURINES
Brain,
ext [a-
cellular Release and
Receptor sites
metabol ism
Brain"
cellular
Cellular transport
as NUCLEOfIDES
ACKNOWLEDGEMENT
REFERENCES
INTRODUCTION
k1
kO
k2 = t· k,
H ....
Vt V2
Table I
~MANNITOL
5 10 20 30 40 50 60
TIME OF EXPOSURE TO ISOTOPES,MIN.
Table I I
Ischemic k1
0.015 0.18 3.0
brain (min- 1 )
The model (Fig. 1) was fitted to the uptake (Fig. 2). The
table shows the values of ko and k1 , which gave the best
fits. The k1-va1ues given in the lower row were obtained
on slices from ischemic brain.
...J
0I- ~
-I-
ZJ:
Z(!)
«-
::::EILI
~
60
0
ZI-
«ILl
If)~
a:...J
~~
:::J-
40
If)IL
IL DI
o E
ILl 8
... -
:0::-
:e- 20
Q.:::L
:::J~
5 10 20 30 40 50 60
TIME OF EXPOSURE TO ISOTOPES, MIN.
This study was supported by grant no. 512-5210 from the Danish
medical research council.
272 H. LUND·ANDERSEN AND C.S. KJELDSEN
REFERENCES
INTRODUCTION
273
274 G. LEVI, U. POCE, AND M. RAITERI
8
111M GIU
100 ,M
-....
c 7 50 ,M
-....
10 ~M
5 ,M
6
1 pM
co.
Cantlll
.
.~
co
5
W
~
.
c:
3
..
.... 2
o~~~~ __~__~__~__~
2 4 6 8 10 12
Fraeli .. number (1 fracllon : lmin · O.47ml J
10
_ Contro l
9
..- ..... 10~M Glu
c:
0 . 'I, " l l - i > 20 ~M
..., 8 .. / \ o - 0 5 0~ M
'" ,/ \ '
t . ·~ '\
• .200 ~M
;;; 7
Co 1/ \ \
> : \ b
.~ 6 ,6 '\ \
~ 1/ \ \
'" f, \ '\ .
\ \'
0
5
"C
~
I \ '\ .
I .................. \,. ~
'"
;:!
4 I / I -.-.. , ' . , 6
I •, .
~
c: 3 I ,' ....
...,
'" ,
<;; {,,
a.. 2
~-
A'..
2 4 6 8 10 12
fraction number Cl fract io n= lmin = O.6mf)
Number of expts. 4 4 4 4 7 7
take. The experiments reported above also showed that the unlabeled
glutamate added to the superfusion fluid stimulated the release of
the l4C-aspartate derived from l4C-glucose (data not presented). In
conclusion, it appears that exogenous glutamate can enter the synap-
tosomesby exchanging with a pool of endogenous glutamate and with a
pool of endogenous aspartate. A comparison between total exchange
rates and initial rates of uptake would be possible only if the size
of the endogenous glutamate and aspartate pools exchanging with ex-
ogenous glutamate were known.
.-. Control
-. .......•..
0··-0 Sucrose
'"
.!!
5 Sucrose +
-.-....
1>-1>.
500 pM GABA
::!
•...•. ConI 101 +
50 pM GABA
--..
"~
4 ..•.
:E
ow
D
:;;
".
3
---
::!
;;
D
-..
'"
2
.-
a.
o~~~ __~__~__~~~~~__
2 4 & 8 10 12
Fraction number ( 1 fraction =1min U 7ml )
100
........
90
80
c 70
:!>'
c;
0
u
60 ...- exchange
-;; w····" uptake
-
~
u
50
~
c...
40
30
20
10
10 25 50 75 100 138
Sodium concentration ImMI
tyric acid, was also a strong stimulator of 3H-GABA release from sy-
naptosomes 30 , probably through a heteroexchange process. Another
strong, and, according to Schon and Kelly33,34, specific inhibitor
of neuronal GABA uptake, 2,4-L-diaminobutyric acid (DAB), was also
able to stimulate 3H-GABA release (Fig. Sa).
u
co 6 6
f
i
t
.::
:; 4 4
o
"'0
co
. / •. - .. -.l ......
f .4_.4 _ ..
"'"'"
u
~
0..
®t
4 6 10 4 8 10
traction number (1 fractlon= 1 min= 0.6 mt)
Beside the cases that have been already mentioned, the uptake
and exchange of GABA behaved in a parallel way also in other circum-
stances. In two series of experiments V we obtained a partial de-
pletion of the endogenous synaptosomal GABA content by two different
methods: 1) by washing the prelabeled synaptosomes on Millipore fil-
ters with incubation medium at 0°. This procedure led to a 60-70%
loss of intracellular GABA; 2) by superfusing the prelabeled synap-
tosomes with a depolarizing concentration of KCl, a condition cau-
sing an increased release of GABA and a depletion of synaptosomal
GABA of about 30%. After an equilibration period of 5 or 10 min with
Concentration at the
prestimulation time 16.4 28.4 12.4 70.0
(nmoles/mg protein)
Exchange rate (nmoles/ 0.96 33.3 0.56 67.8
mg protein/min)
Uptake of 20 pM 14C-GABA 5.6 31. 9 4.3 81.8
(nmoles/mg.protein)
:; 8
u
10 pM GABA •
'" 7- 1001lM ouabain
'"
0. 1O,IMGABA'
:= 6- 19pM A 23187
'"
o
.
! '.
-c
'" 4- /_ ..........., 4· ../ '. .........10p\1GARA
{
/ " 10 pM GABA
I
f I
/ ,0' _0 100 pM ouballl
0: I
'"u 191'M A23187
__ 0 •• 0--0"0-'0 o·
'"
0..
Coni rol
Ii;l;lt-O>-'-._~~_ Con lllli
a I iJ I
4 6 8 4
fraCtIOn numher 11 frac1101l=11l1l1l=O.611l1 ,
c:
0
'" 6 6
~ lO~MGlu.
0-
19 ~M A23187 10 ~M Glu •
~ 5 5
100~M ouabain
.~
u
4 4 \
'"
!:!
"0
• IO~M Glu
~
3 3
'"
0
CONCLUDING RE~ARKS
Acknowledgments: 1-Ie thank Mr. Alberto Coletti for the excellent tech-
nical assistance, and Dr. Peter J. Roberts for collaborating in some
of the more recent experiments. The ionophore A23l87 was a generous
gift of Eli Lilly and Company (Indianapolis). This investigation
was partly supported by Research Grant No. 922 of North Atlantic
Treaty Organization.
REFERENCES
Donald F. Bogdanski
INTRODUCTION
291
292 D.F. BOGDANSKI
MECHANISMS OF TRANSPORT
OUTSIDE INSIDE
i:
.
x
Co n
:II
o
m
" Z
""
::;
:s
:II
., o
Z
c: i:
m
'" ~
......
OJ
~ ..
M
low
INEI
II:
(0 ..
rejected and two others were adopted for amine transport 54. In
their original form, the two models provided for an effect of the Na+
gradient.
Another report rejected the ion-gradient hypothesis on the basis
that Na+, ~-ATPase was not involved 53. Reports on the effects of
ouabain generally suggest that amine transport persists after enzyme
activity or ion gradients have been inhibited or diminished but not
blocked or abolished. Amine transport and enzyme activity are both
abolished in the presence of high 'concentrations of inhibitor, how-
ever. Also, the indirect linkage between electrolyte and amine trans-
port renders proportionate inhibition unlikely. In any event, the
magnitude of the electrolyte gradient may be greater than indicated
by the incorrect assumption that the electrolyte is evenly distributed
in the cytosol. Moreover, the data on inhibition of transport by low
concentrations of ouabain 53 are quantitatively at variance
with data reported elsewhere 1,25,28,45.
It has been reported that the transport of metaraminol in rat
uteri was energized by Na+, ~-ATPase activity and not by the Na+
gradient 38. Thus, in the presence of ouabain, an imposed Na+ gra-
dient did not energize the accumulation of metaraminol. These data
are consistent with data for NE reported eqrlier by us 47. However,
an imposed Na+ gradient energizes initial NE as well as 5-HT trans-
port in synaptosomes in which Na+, ~-ATPase activity was inhibited
by a ~-free medium 45, and by the ~-free medium or ouabain, re-
spectively 9,47. Moreover, the effectiveness of either inhibitor
was decreased by a low concentration of extracellular Na+ in accord
with the hypothesis. Sustained transport, unlike initial transport,
required Na+, ~-ATPase activity 9,45,47 in order to maintain ion
gradients.
The facts are consistent with a possible role for outward transport
in transmitting nerve impulses. Transport is rapid and complete 4,33.
Also, the proposed shuttle of transmitter out of and into vesicles
attached to the presynaptic membrane has some merit for nerve endings
in which the theory of exocytosis raises some problem of transmitter
supply, large molecules are not secreted in the proportion in which
they exist within the cell, and in which a single impulse may not
produce a response by the effector organ. In any event, there is now
TABLE 1
Rats were injected with 12.5 ~Ci of dl- 3H-Norepinephrine (3H- NE ) and
killed 18 hrs later. Ventricle slices were incubated in Krebs HC03
medium at 37°, then desipramine or cocaine was added to a concentration
of 3 ~M. Ouabain was added to a concentration of 0.5 roM after 20 min.
Preincubation was continued to 60 min for all. Slices were then trans-
ferred to modified Krebs HC03 containing 12.5 roM Na+; choline chloride
replaced deficient Na+. Other slices were transferred to fresh Krebs
HC03 medium. As indicated on the table, 3H- NE , and potassium sulfate
(K2S04) were added to concentrations of 0.1 ~M or 50 roM, respectively.
The medium was sampled every 20 min. to 140 min. The figures represent
the half time of tissue 3H- NE .
302 D.F. BOGDANSKI
REFERENCES
24. Green, R.D. III, and Miller, J.W., Evidence for the active
transport of epinephrine and norepinephrine by the uterus
of the rat. J. Pharmaco1. Exp. Ther. 152 (1966) 42-50.
25. Harris, J.E., and Ba1dessarini, R.J., The uptake of 3H-dopamine
by homogenates of rat corpus striatum: effects of cations.
Life Sci. 13 (1973) 303-312.
26. Heinz, E. (Ed.) Na-1inked transport of organic solutes. Springer-
Verlag, Berlin, 1972.
27. Hitzemann, B.A., Hitzemann, R.J., and Loh, H.H., On the speci-
ficity of trypsin (EC 3.4.4.4) of nerve ending particles to in-
hibit norepinephrine transport. J. Neurochem. 24 (1975) 323-330.
28. Ho1z, R.W., and Coyle, J.T., The effects of various salts,
temperature and the alkaloids veratridine and batrachotoxin
on the uptake of [3H]-dopamine into synaptosomes from rat striatum.
Mol. Pharmaco1. 10 (1974) 746-758.
29. Iversen, L.L., The uptake of noradrenaline by the isolated per-
fused rat heart. Br. J. Pharmaco1. 21 (1963) 523-537.
30. Iversen, L.L., The uptake and storage of noradrenaline in sym-
pathetic nerves. Cambridge University Press, New York, 1967.
31. Iversen, L.L., Neuronal uptake processes for amines and amino
acids. In E. Costa and E. Giacobini (Eds.), Advances in Bio-
chemical Psychopharmacology 2 (1970) 109-132.
32. Iversen, L.L., and Kravitz, E.A., Sodium dependence of trans-
mitter uptake at adrenergic nerve terminals. Mol. Pharmaco1.
2 (1966) 360-362.
33. Keen, P., and Bogdanski, D.F., Sodium and calcium ions in uptake
and release of norepinephrine by nerve endings. Am. J. Physiol.
219 (1970) 677-682.
34. Lee, C.O., and Armstrong, W.McD., Activities of sodium and po-
tassium ions in epithelial cells of small intestine. Science
175 (1972) 1261-1264.
35. Ling, C.M., and Abdel-Latif, A.A., Studies on sodium transport in
rat brain nerve ending particles. J. Neurochem. 15 (1968) 721-
729.
36. Ling, G.N., and Ochsenfe1d, M.M., Mobility of potassium ion in
frog muscle cells, both living and dead. Science 181 (1973)78-81.
37. Maxwell, R.A., Keenan, P.D., Chaplin, E., Roth, B., Batmang1idje,
S., Eckhardt, S., Molecular features affecting the potency of tri-
cyclic antidepressants and structurally related compounds as in-
hibitors of the uptake of tritiated norepinephrine by rabbit.
aortic strips. J. Pharmaco1. Exp. Therap. 166 (1969) 320-329.
38. Paton, D.M., Cation and metabolic requirements for retention of
metaraminol by rat uterine horns. Br. J. Pharmaco1. Chemotherap.
33 (1968) 277-286.
39. Pietryzk, C., and Heinz, E., The sequestration of Na+, K+ and
C1- in the cellular nucleus and its energetic consequences for
the gradient hypotheses of amino acid transport in Ehrlich cells.
Biochim. Biophys. Acta. 352 (1974) 397-411.
SYNAPTOSOMAL AMINE TRANSPORT 305
G. Takagaki
Department of Neurochemistry
Tokyo Metropolitan Institute for Neurosciences
Fuchu-shi, Tokyo, Japan
307
308 G. TAKAGAKI
To study the release from slices, the pre loaded slices with
labelled glutamic acid (10 ~) for 15 min were transferred to a
superfusion chamber and the slices were super fused with oxygenated
medium for 10 min. Thereafter 2-min fractions (4 ml) were collected"
The high-potassium stimulation was effected by switching the super-
fusion solution to that containing 50 mM K+, over a 10-min period.
Amino acids in each fraction were isolated and counted as described
above.
RESULTS
Table 1
Metabolism of Glutamic Acid in Guinea-pig Cerebral Cortex Slices
L-Glutamate(lO ~)
48.75 4.17 43.95 0.79
Slices 97.66
± 2.66 ± 0.46 ± 3.64 ± 0.13
D-Glutamate(lO ~M)
96.32 0.16 0.99 0.06
Slices 97.53
± 1.92 ± 0.05 ± 0.31 ± 0.01
A B •
OA OA
D.2
JDI
......
40
-a
5. 12 C 12 0 •
8 8
2 2
4 • • 4
•
1~ __---JD.5
~~-40~-6~0 0 0 10 20 40
Time(min)
Fig. 1
Uptake of L- and D-Glutamic Acids into Guinea-pig Cerebral Cortex
Slices. Effects of Ouabain(0.05 mM) and of omitting Sodium Ions
from Medium.
The radioactivity in glutamic acid fractl.on which was isolated
from the TCA extract of incubated slices was expressed as pmol of
glutamic acid per g initial fresh weight of slices. To examine the
effects of ouabain and of omitting sodium ions from medium, the
former was included in and the latter was left ~ut from the medium,
.:
from the beginning of the preincubation.
A: L-Glutami c Acid, 10 pM Control(left-side scale)
B: D-Glutamic Acid, 10 pM A: Ouabain, 0.05 mM(right-
C: L-Glutamic
D: D-Glutami c
Acid,
Acid,
0.5 mM
0.5 mM .: side scale)
Sodium-free(right-side
scale)
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 311
2000
A
~ 50
"'E'
~1000 40
30ii
...'"
1:!!
e
.!!. 20;;
10
0 0
0 10 20 30 40 50
Time (min)
B
0.75
Fig. 3
Double Reciprocal Plots of Uptake of Glutamic Acid.
Each plotted point represents the mean of 4-5 independent determi-
nations. . , L-Glutamic Acid; 0, D-Glutamic Acid
A: Uptake into guinea-pig cerebral cortex slices. Velocity(V) is
expressed as ;mol of glutamic acid accumulated in 5 min per ml
of non-inulin space of the slices.
B: Uptake into P2 fraction from rat cerebral cortex. Velocity(V)
is expressed as nmol of glutamic acid accumulated in 2 min per
mg protein.
314 G. TAKAGAKI
B
:;
\\ 0.5
i····....... I
~
.
'. ' .•........... D-
••••<;>0..
r
.......'0
"
10.25
o~ ___~___~___~___~
o 5 10 15 20 5 10
Time(min)
15 20
Time(min)
Fig. 4
Uptake and Release of Glutamic Acid by Synaptosomes(P2 fraction)
from Rat Cerebral Cortex. Effects of Ouabain and of omitting
Sodium Ions from Medium.
0, Control; e, Ouabain(0.05 mM); ~,Na+-free.
A: L-Glutamic Acid(lO pM); B: D-Glutamic Acid(lO pM)
Each plotted point represents the mean with SD of 4-5 independent
determinations. At arrows, 50 mM KCl was added.
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 315
...
~a.
r4
.....
"'0
e...
C
2
0~__~__~~-7.~~~
o 5 10 15 20
Time(min)
Fig. 5
Uptake and Release of L-Glutamic Acid(lO pM) by Subfractions from
Crude Synaptosomal Fraction(P2) from Rat Cerebral Cortex.
See the legend for Fig. 4. Uptake of L-glutamic acid(lO pM) into
the P2 fraction subjected to hypo-osmotic shock is also shown( • ).
A: O. Subfraction A(myelin)
B: 0, Subfraction B( synaptosomes)
C: ~, Subfraction C(mitochondria)
316 G. TAKAGAKI
DISCUSSION
The results may indicate that the high affinity uptake system
for glutamic acid is specific to L-isomer and localized in nerve
terminals. When L-glutamic acid is added to the medium at 10 pM,
the concentration per mg protein reached in the synaptosomal
fraction(subfraction B) is more than threefold higher than in the
slices. D-Glutamic acid may be taken up only by the low affinity
system. In the cerebral cortical slices the high affinity component
is probably masked by the predominant uptake due to the low affinity
system. It is highly likely that the bulk of the low affinity up-
take is due to non-terminal structures which are included in the
slice preparations? It is known that L-glutamic acid is accumu-
lated at a markedly higher rate and reaches a higher level in the
glial fraction than in the neuronal fraction, and the release
by high potassium stimulation was also observed from the glial
fraction by Hamberger 2 • The high affinity uptake system for
glutamic acid seems to be peculiar in view of its high resistance
to ouabain. Or, it may easily be substituted by a transport system
which is resistant to ouabain. The uptake by the low affinity
system is greatly inhibited by ouabain. Either high or low affinity
system is dependent on the presence of sodium ions in the medium.
REFERENCES
1 Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H., Metabolic
compartments in vivo. Ammonia and glutamic acid metabolism in
prain and liver. J.biol.Chem., 237(1962) 2562-2569.
2 Hamberger, A., Amino acid uptake in neuronal and glial cell frac-
tions from rabbit cerebral cortex. Brain Research, 31(1971) 169-
178.
3 Hopkin, J., and Neal, M.J., Effect of electrical stimulation and
high potassium concentrations on the efflux of [14c]glycine from
slices of spinal cord. Br.J.Pharmacol., 42(1971) 215-223.
4 Lajtha, A., Amino acid transport in the brain in vivo and in vitro.
In G.E.W. Wolstenholme, and D.W. Fitzsimons(Eds.), Aromatic Amino
Acids in the Brain, Elsevier, Amsterdam, 1974, pp. 25-49.
5 Levi, G., and Rai teri, M., Detectabili ty of high and low affinity
uptake systems for GABA and glutamate in rat brain slices and
synaptosomes. Life Sci., 12(1973) 81-88.
6 Matsui, S., and Yamamoto, C., Release of radioactive glutamic
acid from thin sections of guinea-pig olfactory cortex in vitro.
J.Neurochem., 24(1975) 245-250.
7 Nicklas, W.J., Puszkin, S., and Berl, S., Effect of vinblastine
and colchicine on uptake and release of putative transmitters by
synaptosomes and on brain actomyosin-like protein. J.Neurochem.,
20(1973) 109-121.
8 Quastel, J.H., Amino acids and the brain. Biochem.Soc.Trans.,
2(1974) 765-780.
9 Snyder, S.H., Young, A.B., Bennett, J.P."and Mulder, A.H.,
Synaptic biochemistry of amino acids. Federation Proc., 32(1973)
2039-2047.
10 Takagaki, G., Developmental changes in glycolysis in rat cerebral
cortex. J.Neurochem., 23(l974) 479-487.
11 Takagaki, G., Uptake and release of glutamic acid in vitro in the
mammalian CNS. Proc.Vth International Meeting of the Inter-
national Society for Neurochemistry, Barcelona, 1975, p. 178.
12 Whittaker, V. P., and Barker, L.A., The subcellular fractionation
of brain tissue with special reference to the preparation of
synaptosomes and their component organelles. In R. Fried(ed.),
Methods of Neurochemistry, Vol. 2, Marcel Dekker, New York, 1972,
pp. 1-52.
RELEASE OF BIOGENIC AMINES FROM ISOLATED NERVE ENDINGS
INTRODUCTION
319
320 M. RAITERI ET AL.
SUPERFUSION OF SYNAPTOSOHES
,
, I
,
I
THERMOSTATIC
BAT H
' ••7""
:::J PUMP
perfusion, utilizing the upper reservoirs for adding the new medium.
The main advantage of this superfusion technique, however, is that
the transmitter is removed by the stream of the superfusion fluid
as soon as it is released, so that its reuptake is prevented. This
is demonstrated by the fact that drugs or experimental conditions
known to inhibit reuptake of a given neurotransmitter do not cause
any increase of its spontaneous release (see ref. 27 and 28, and
following paragraphs).
12 -
~€
'"o
.
:;;;
~ 6-
~
'0
.. . '
..
co
....
a..
2-
-- --~
6_
nal incubation technique 26 . This is due to the fact that the spon-
taneously released 3H-NE can not be recaptured by the nerve endings
in the superfusion conditions~
When synaptosomes prelabeled with 3H-NE, in the absence of cal-
cium ions, were superfused with a medium containing 56 mM potassium
chloride, a calcium-dependent stimulation of the radioactivity re-
leased was observed. Fig. 2 shows a sharp increase in the total
tritium released when calcium ions were added in conditions of mem-
brane depolarization. A similar finding has been reported by seve-
ral authors with various neurotransmitters and in different brain
tissue preparations 3 ,6,15. In our experiment the increased release
of radioactivity following stimulation was totally accounted for by
unmetabolized 3H-NE; the release of deaminated metabolites was al-
most unchanged. This indicates that the calcium-dependent release
involves a pool of norepinephrine which is protected from monoamino-
oxidases both during the storage and the "release step and may sug-
gest the involvement of an exocytotic mechanism I6 ,35.
Most of the NE in nerve terminals seems to be localized in the
vesicles and very little in the cytoplasm I9 ,25. Any increase in the
13
12
..,
~ JD
A.\\
~"~,~
" 4..
""l
.......... --6~---A
c:
COIPU~ Slft.tum
.~ HYPOll lil-l!II l,IS
u
-= 1
Conllol
ConUCl I
d - Amptil!:lfll'l lflt IO-$, M 6
(OI1IUII d · A mp~!llm l ne lO -5 M
CI · Al'IIllheUmlflf: IO-" M
.. 3
o
c:
~
u
~
0.. I 2 3 4 5 7 8 I II
1 1 3 4 I 6 7 8 9 1 11
1 1 1 4 ~ 6 J J 11
fraction number
.
u
..
m
COAIfIllI • • CelII"ClI . . - COIloI'",'
' -Amphtl. 'O~ l M o d - Amphllurllne 10 - 5 M
c. -- 10- " M
-0
0.-0 d- AmphEII'IUlllll0- )M
~ 10-~M S
:~
u
m
o
~ "
§
'0
"..
~ I
C>.. ®
~'~1~]~'~S7.'1·8·9~IO"1I-- 1 2 10 11 11l"56J8~1[I11
fracl illn nllmher
9 9
C!lonl rOI
" CDIll 1101
." ~
Ir - it.
0/,11 10"/,1
d -Am"heUtnmt 10· S M
d ·Alf'lptltllmln,.IlM I
d-J\mphtt.mln. 10- 5 M
"- Aflllphll,mlftt·
81"ltfO,'ft, ( ltIoth IO-5 MI
...
..
I bo" 10' 5/,1 1 0--0 811UlloplnllQa 5 M
6
~
...."
;; 4 4
;;
;;
~
.
"-
@
2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 a 9 10 II 12 13
fraction number
,
durlng the 'lncu b '
atlon 'h t h e ra d'loactlve
Wlt , ,21,32 .
amlnes
It is also important to exclude that the releasing effects ob-
served are due to inhibition of reuptake of the spontaneously re-
leased amines. That an effect on reuptake is not involved is demon-
strated by the following observations: desmethylimipramine, a strong
inhibitor of NE and 5-HT uptake, did not stimulate the release of
the two amines (ref. 10 and Fig. 7); benz tropine , an inhibitor of
DA uptake stronger than d-amphetamine 22 , was only a weak stimulator
of 3H-DA release from striatal synaptosomes (Fig. 6b).
In summary, d-amphetamine was unable to stimulate 3H- NE release
from the hypothalamus 3 an area rich in noradrenergic nerve endings,
but it could release H-NE from striatal and cortical synaptosomes.
As dopaminergic terminals are particularly abundant in the striatum
and are also present in good amount in the cortex 33 , and since do-
paminergic terminals are lik~ly to accumulate 3H- NE in the labeling
step32, it is possible that H-NE is released by d-amphetamine only
after being artificially stored in dopaminergic nerve endings. This
hypothesis is supported by the fact that the amphetamine-stimulated
release of 3H-NE from striatal synaptosomes was only slightly dimi-
nished by the presence of desmethylimipramine (Fig 8), thus showing
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 327
Control
~
0 ~
8 Cont I 01
~ OMI 10- 5 M OMI 10- 5 M
u
'" ........ d - Amphetamine 10- sM "0 d· Amphetamine IO-5M
~ d· Amphetamine + OMI 6--6 d- Amphelamlne+DMI
0
~
c. I bOl h 1O- 5 M I I both 1O- 5 M I
> 6 6
>
u
'"0
"0
'"
-;;; 4 4
0 "' ...8.- _.6,
o ",
C 3
0 '6
o ......~
~
-'6--.
t
~
t
u
~
c..
® (6)
1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 1011 12
fr act ion number
10
ConfJol
DMllO 'M
"o Jf-----i( d Amphelamlne 10 5 M
d Amphetamine +DMI
I both 10 5 MI
6·
1 2 3 4 5 6 1 8 9 10 11 12 13
fraction number
nergic nerve terminals; from these in vitro results one could de-
duce that the reported increased availability of NE in the synaptic
cleft caused by the drug is essentially due to inhibition of reup-
take of the released amine. On the other hand, d-amphetamine seems
to influence the dopaminergic transp~rt system both by inhibiting
DA uptake and by a direct releasing effect.
Control
""'
. KCI 56 mM
KCI 56 mM + d -Amphetamine 10. 5 M
.
.
"
~
1 2 3 4 5 6 7 8 9 10 11 12 13 14
fraction number
BENZYL AMINE
- CH2"NH 2
@
p - TYRAMINE
H O - @ -CH2" CH i NH 2
y H3 p - O~-AMPHETAMINE
H O - @ -CHiCH-NH2
Cont rol
<=
0
Benzylamine 10- 5 M
0--0 p-OH-d-Amphetamlne
u
.....----..
'"
.;:
~-Phenylethylamine
p- Tyramine
'"
Co
>
,.
u 4
'"
0
-c
'"
'"
;:
~
<=
'"u
t
'"
Cl.
1 2 3 4 5 6 7 8 9 10 11 12 13
fractIOn number
............ Control
<=
0
............ ~·Phenvlethvlamine 5 ·10-7 M
0--0 p.OH·d·Amphetamine
" p-Tyramine
.::'" ~ d·Amphetamine
Q;
c.
>-
.:!
"'"0 4
~
'"
e
(;
t
<=
"'"Q;
0-
1 2 3 4 5 6 7 8 9 10 11 12 13
fraction number
>p-tyramine> f'-phenylethylamine.
®
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
fraction number
Cont TO\
"
0
o-<:J ~·Phenylethyl'm,"e 5.10- 7 M
u ~ d-Amphetamine
ro
0---0 P-OH-d-Amphetamme
~ p-Tyramme
=
~
,.
ro 4
0
"0
ro
ro
0
-;
"
~
"-
t
1 2 3 4 5 6 7 8 9 10 11 12 13
fractIOn number
REFERENCES
I - INTRODUCTION
337
338 J.P. TASSIN ET AL.
a General characteristics
As already mentioned, dopaminergic and noradrenergic terminals
can be differentiated by their reuptake processes. In earlier
studies of peripheral noradrenergic neurons, it was found that the
estimation of 3H-NA uptake provided a good index of the noradrener-
gic innervation12 • A direct relationship was observed between the
NA content and the intensity of 3H-NA uptake in various structures
innervated by the sympathetic system. The elimination of this
innervation by 6-0HDA resulted in a decline of both NA levels and
3H-NA uptake in most organs 4 ,13,14. The uptake process of catechol-
amines in central catecholaminergic neurons was also used to
specifically label these neurons IO • In addition to these studies
Snyder and COIle examined more precisely the regio~rl differences
in 3H-NA and H-DA uptake in rat brain homogenates • They describ-
ed differences between the uptake affinities of 3H-NA and 3H-DA and
found that in most structures 3H- DA could be taken up by a high
(Km = 8.l0-8M) and a low (1.4 lO-6M) affinity uptake process. If
both 3H-DA and 3H-NA can be taken up by noradrenergic or dopaminergic
neurons, there exists nevertheless, a specific uptake process in
each type of terminal. Indeed, specific competitive inhibitors
can block one carrier or the ~ther: desipramine (DMI) acts specifical-
lyon noradrenergic terminals whereas benz tropine interferes more
markedly with the high affinity transport of 3H-DA in dopaminergic
terminals 5 • In the striatum, the 3H-dopamine uptake decreased in
direct relation to endogenous DA levels after the selective
destruction of nigrostriatal dopaminergic neurons induced by the
injection of 6-hydroxydopamine into the substantia nigra 14 • During
ontogenesis, the development of 3H-catecholamine upta~e in various
structures parallels that of endogenous amine content. Jonsson
et al. 15 estimated the uptake of 3H-NA to confirm the sprouting
of noradrenergic neurons in the brain stem following a 6-0HDA induced
lesion of this region: no change could be seen in the affinity of
the transport process, but a two fold increase of the Vmax was
obtained. These few examples illustrate the use of catecholamine
uptake estimations as a tool for the determination of the respective
importance of dopaminergic and noradrenergic innervation in the CNS.
Using the method described by Tassin et al.,23 the uptake was linear
for at least 2 minutes, unaffected by desipramine (Table 1) and
inhibited by benztropine (-75%). Moreover, no loss of uptake
activity was seen when compared to that obtained with unfrozen
tissue when the uptake activity was expressed per mg of tissue.
Differences were observed when the results were expressed in nCi
of 3H-DA per min per mg of protein; this is due to a two fold
purification of synaptosomes occurring during the centrifugation
when the activity of homogenates of fresh tissue is measured (Table 1).
TABLE 1
The striatum was cut on the frontal plane in 9 slices each 500 ~
thick. Three punches were made in each slice in the dorsal, medio
latera~ and ventral positions except for the 1st, 2nd, and 9th slice
in which only 1, 2 and 1 microdiscs respectively were punched out.
The 3H-DA uptake activity was rather homogene~ in the dorso-ventral
plane but very heterogeneous in the rostro-caudal plane. The uptake
activity decreased progressively from the rostral to the caudal part
of the striatum23 • Similar distributions were obtained when the DA
endogenous content 23 and the DA sensitive adenylate cyclase 3 from
identical micro discs were measured.
DOPAMINE TRANSPORT IN CORTEX AND STRIATUM 341
_ _ C4;mtrol,
____ '+ ___ PC s. 60HOA l es ion
- - .- - A,O e LectrolytlC.• L IH lon
ROSTRAL CAUDAL
IJOCO
Fig . 1 Fig . 2
REFERENCES
1. Berger, B., Tassin, J.P., Blanc, G., Moyne, M.A., and Thierry,
A.M., Histochemical confirmation for dopaminergic innervation of
the rat cerebral cortex after destruction of the noradrenergic
ascending pathways, Brain Research, 81 (1974) 332-337.
2. Berger, B., Thierry, A.M., TaSSin, J.P., and Moyne, M.A., Dopa-
minergic innervation of the rat prefrontal cortex: fluorescence
histochemical study, Brain Research (1975) in press.
3. Bockaert, J., Premont, J., Glowinski, J., Thierry, A.M., and
Tassin, J.P., Topographical distribution of dopaminergic inner-
vation and of dopaminergic receptors in the rat striatum: II.
Distribution and characteristics of dopamine adenylate cyclase.
Interaction of d-LSD with dopaminergic receptors, Brain Research,
in press.
344 J.P. TASSIN ET AL.
18. Lindvall, 0., Bj8rklund, A., Moore, R.Y., and Stenevi, V.,
Mesencephalic dopamine neurons projecting to neocortex,
Brain Research, 59 (1974) 332-337.
19. Palkovits, M., Isolated removal of hypothalamic or other brain
nuclei of the rat, Brain Research, 59 (1973) 449-450.
20. Routtenberg, A., and Sloan, M., Self-stimulation in the frontal
cortex of Rattus norvegicus, Behav. BioI., 7 (1972) 567-572.
21. Snyder, S.H., and Coyle, J.T., Regional differences in 3H-norepi-
nephrine and 3H-dopamine uptake into rat brain homogenates,
J. Pharmacol. expo Ther. 165 (1969) 78-86.
22. Tassin, J.P., Blanc G., Stinus, L., Berger, B., Glowinski, J. and
Thierry, A.M., Distribution of dopaminergic terminals in the
cerebral cortex, in preparation.
23. Tassin, J.P., Cheramy, A., Blanc, G., Thierry, A.M., and
Glowinski, J., Topographical distribution of dopaminergic
innervation and of dopaminergic receptors in the rat striatum:
I. Micro-estimation of 3H-dopamine uptake and dopamine content
in micro discs, Brain Research, in press.
24. Tassin, J.P., Thierry, A.M., Blanc, G., and Glowinski, J.,
Evidence for a specific uptake of dopamine by dopaminergic
terminals of the rat cerebral cortex, Naunyn-Sch. Arch. Pharmacol.
282 (1974) 239-244.
25. Thierry, A.M" Blanc, G., Sobel, A., Stinus, L. and Glowinski,
J., Dopaminergic terminals in the rat cortex, Science, 182
(1973) 499-501.
26. Thierry, A.M., Stinus, L., Blanc, G., and Glowinski, J.,
Some evidence for the existence of dopaminergic neurons in the
rat cortex, Brain Research, 50 (1973) 230-234.
Factors Influencing Transport
ENERGETICS OF LOW AFFINITY AMINO ACID TRANSPORT
INTRODUCTION
349
350 M. BANAY-SCHWARTZ, D.N. TELLER, AND A. LAJTHA
lL.. 100 r - - - - - , . - - - - - - r - - - - - - - - - r - - - - - - ,
o
/0' UPTAKE AT 37°
40
20
~-GLUTAMIC
O~------L-------L---------------~--~
o 15 30 60
o~
DURATION OF INCUBATION WITHOUT GLUCOSE
BEFORE AMINO ACID ADDITION - MINUTES
Fig. 1. Brain slices were incubated at 37 0 in HEPES-G medium
i.e., HEPES-2 medium 3 ,4, minus glucose, for 15, 30 or 60 min
then l4C-amino acids were added for additional 10 min periods.
Initial concentration of l4C-amino acids in the medium: D-glu,
1 mM; a-AlB and L-1ys, 2 mM. Control levels are those reached
30 min after incubation in HEPES-2, followed by 10 min incubation
with l4C-amino acid. C.U. (concentrative uptake) = ~oles of amino
acid m1- 1 intracellular H2 0 (T), above the medium concentration (M),
at the end of incubation; C.U. = T- [(MxE) / l-E] -M, where E =
(extracellular space), m1 inulin/m1 tissue H2 04. Thirty min
incubation at 37 0 in the absence of glucose is sufficient to block
amino acid uptake. For HEPES-2 medium composition see ref. 44.
60
40
L~
20
Depletion and
restoration with
glucose-(b) 0.45 134 43 11.6 7.6 86 77
Depletion and
restoration with
SNP (c) 0.36 177 44 66 16.1 8.8 119 89
Table I. (a) Includes 30 min. temperature equilibration at 37 0 before amino acid addition.
(b) Includes 30 min incubation in medium lacking glucose (HEPES-G), 30 min in control
medium, + 10 min in presence of 14C- amino acid. (c) Includes 30 min in medium lacking
glucose, 30 min in presence of SNP (HEPES-G, + 20 mM succinate, 5 mM malate, and 5 mM
RYruvate) + 10 min in presence of l4C-amino acid. id) Na+ and ~ are expressed as
)lequiv. ml- l tissue H20. (e) Per cent swelling. [H20 in slices after incubation
(ml gm-l frozen weight)/H20 in unincubated slices~x 100 (-) 100. Swelling measurements
were performed only with D-glu. Thel~swe1ling of slices 30 min after restoration in the
SNP medium, but before addition of C-amino acid for uptake, was 53. (f) Concentrative
uptake, C.U. (see legend Fig. 1). Initial amino acid concentrations in the medium:
D-glu, lmM; a-AlB, 2 mM.
lAc 3x10- 4 23 52 22 56 57 43 56 32 99 46 1 4
Ouabain 2x10- 6 73 42 47 35 50 26 40 18 58 20 2 4
01igom. 9.2xlO- 7 86 97 71 87 33 25 14 17 33 45 8* 7*
Table II. a) Inhibitor added at t(incubation) = 0 for 30 min before amino acid was added.
b) All other columns of data were obtained after incubations that depleted the endogenous energy
stores of the slices for 30 min in HEPES-G, followed by addition of inhibitor and energy source
for 30 min (restoration period) prior to addition of l~C-amino acid for 10 or 20 min uptake.
FAA = f1uoroacetate; lAc = iodoacetate; 2,4-DNP = 2,4-dinitropheno1; SMP = succinate, 20 mM;
malate, 5 mM and pyruvate, 5 mM. a-G1y-P = a-glycerophosphate, 20 mM; glucose or lactate, or
pyruvate, 10 mM. *Rotenone, 0.04 ~M; antimycin; 0.2 ~M and oligomycin, 0.46 ~M.
c) 100~values = uptake by undep1eted slices in HEPES-2, without inhibitor; for 1 mM D-g1u
control, 100~ of 10 min C.U. = 13.5; 100~ of 20 min C.U. = 22.9 ; for 2 mM a-AlB, 100~
of 10 min C.U. = 9.9; 100~ of 20 min C.U . • 18.1. The following procedure will give the
concentrative uptake values with any of the substances used for "restoration", or after treatment
with the inhibitor: [(~ of control x control C.U.)/100J.
Control
C.D. +gluc (b) -gluc +gluc -gluc -gluc +gluc -gluc
+K -K -K +K -K -K -K
+Rb +Rb +Rb
+SHP
D-g1u 28.6 86 4 11 16 24 100 99
a-AlB 22.1 82 14 20 33 52 73 93
GABA 28.3 103 18 36 50 55 101 98
L-his 18.0 94 32 22 20 38 91 95
gly 20.7 98 12 27 30 44 87 100
L-leu 3.57 73 24 39 41 81 81 89
L-val 7.63 88 28 22 24 46 61 93
L-orn 5.50 110 57 65 70 91 91 91
L-1ys 5.44 97 54 76 97 85 104 89
Table III. a) Brain slices were incubated for three successive 10 min intervals in 5 ml
of oxygenated media lacking both glucose and K+. This was followed by a 30 min incubation
in media containing the substances listed <It the column heading (b) for "restoration" of
energy, and thereafter 30 min uptake of l4C-amino acids was measured after incubation
under the same conditions as the restoration. Inhibitors were added for both the 30 min
restoration period as well as for the 20 min uptake period. The initial concentration of
the amino acids in the uptake media waS-l mH, except for a-AlB and L-lys, which were 2 mH.
When present, glucose = 10 roM; ~ or Rb+ = 5 mH; SMP = succinate, malate, pyruvate
(20, 5, 5 mH).
e,e_
e-e-e
K~--
o 10
a 70 27 -43 85 73
b 70 40 -30 97 76
c 5 57 +52 105
d 5 44 +39 80 92
e 5 2 -3 97
f 5 25 +20 90
g 70 16 -54 103 84
Table IV. Most of the uptake of D-Glu or a-AlB was maintained whether K+ went into
the tissue, or came out, from high or low initial ~ levels in the tissue. In all
cases, the brain slices were depleted of their endogenous ~ levels by 3 transfers
at 10 min intervals, in HEPES-G (-~), followed by 30 min preloading, either in
HEPES media containing glucose and: 50 mH K+ (a,b,g); 50 mH Rb+ (e,f); or no K+
(c,d). Then the slices were washed and transferred to media containing the amino
acid, 10 mH glucose, and the following combinations of ions: (a) 0 roM ~;
(b,f) 5 mH KT; (c) 15 mH t+; (d) 10 mH~; or (e,g) 5 mH Rb+. The duration
of uptake was 10 min. in: (a), (b), (e), (f), (g); and 30 min. in (c), and (d).
Control values of D-glu uptake from initial 1 mH medium concentration for 10 min •
13.5; from 2 mH AlB = 9.90; for 30 min uptake, the control C.U. values were 26.6
and 22.1, respectively.
24 120~
::i c
u .;::
"20
~
<t
~/6
:::)
lIJ
">12
i=
c;(
~8
~
~4
c
u
to the sixth minute, the rates of uptake are maximal while the rate
of Na+ change is nearly zero. Only with valine does the uptake rate
fall continuously in this case. From these results it is also
obvious that the sodium level in the tissue does not have to be
set at some arbitrarily high (i.e., 100 meq/l) level for maximum
rates of transport, 20-40 meq/l appear to keep the tissue uptake
system quite 'happy'.
362 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA
2·0 • 25
1.5
20 IJ
Na+
f1
c.u. 0
15 MIN.
MIN. 1.0
GABA
/0
/
I.~£-·-£
0.5
iK. e,<;AIB 5
6. [J ....
"'[J=- VAL
o - ~- Ih I~ 21 0
TRANSFER
Fig. 5. The maximal rate of GABA, AlB and glu uptake occurs after the
flux of Na+. Slices were depleted by two 15 min incubations in
HEPES-G (-) Na+ (+) 5 roM K+ (+) 100 roM sucrose, Tris-adjusted to
pH 7.35. They were transferred into this medium (+) Tris-SMP for
30 min, and were then transferred into this medium (+) 80 roM Na+,
containing l4C-amino acid. The ordinate is the change (delta) per
min of tissue Na+ or C.U., for 3 min intervals after the last transfer.
Na+ flux (in) is essentially complete in 2 min (cf. Fig. 3). Uptake
rate is maximal for three "sodium-dependent" amino acids after the
Na+ flux, but decreases steadily for L-valine.
DISCUSSION
K+ 23.7 78 60 41 23.2 62 16 14
Rb+ 23.7 77 57 34 22.6 43 9 10
Cs+ 15.9 35 16 15.6 8 10
is maintained with sucrose and Tris, the uptake is much better yet
Na+ levels are not so closely related (Fig. 4 b). As reported by
Weiss and Hertz 4l , K+ levels of 50 mM also exert a maximal effect
on slices, causing swelling and increased 02 uptake, and independent-
ly increase the efflux of glutamate (thereby decreasing the apparent
uptake at equilibrium).
very closely with our finding of the initiation of the most rapid
D-glu and AlB transport during the second 3-minute period (after
K+ levels remained relatively constant as shown in Fig. 3 a and
~). If the rate of transport is indeed closely coupled with
NADPH reduction, our finding of pronounced inhibition of transport
by ouabain in SMP medium is in agreement with their observation that
the glycoside also blocks the NADPH reduction.
ACKNOWLEDGEMENTS
The authors thank Mr. Henry Sershen for the suggestion to try
Rb+ as a K+ substitute; Ms. Teresita De Guzman, Ms. Denise McHale,
and Mr. Stanley Reid for their skilled technical assistance. We
are also indebted to Dr. Amos Neidle for help with amino acid
analyses and to Dr. M.A. Verity, whose publications on synaptosomal
energy requirements for ion accumulation encouraged us to attempt
this work. The experiments, summarized here, were supported in part
by NIH grant No. NS-03226, and in the main by the New York State
Department of Mental Hygiene, Research Division, G.C.Salmoiraghi, ,M.D.,
Ph.D., Associate Commissioner.
REFERENCES
Leif Hertz
Department of Anatomy
University of Saskatchewan
Saskatoon, S7N own, Canada
INTRODUCTION
371
372 L.HERTZ
Release
min. a/a
-...
...:
.s::.
37.5 - 75 205
~
-¢
~
.s::.
-...
01)
25.0 - 50 155 III
01)
III
"0
0.0 - 55 E
00 20 40 60 80 100 120 ::1-
-25
-20
-15
f
I
c
"e -10
~
.= -5
c
•
CI
C
0
"
.s=
U
5
5 10 15 20 25 30 A
Age (days)
a major part of the release in brain slices occurs from sources other
than synaptosomes. Evidence for a potassium-induced release of GABA
from satellite cells has been obtained in dorsal root ganglia of
the rat 32 , 33 and it has been suggested that the potassium-induced
release of GABA which develops after the age of 21 days, at least
to some extent, may occur in glia ce11s45. A potassium-induced
release of GABA has been observed both in neuronal-enriched and
glia cell-enriched fractions obtained by gradient centrifugation
(A.Hamberger and A.Sel1strom, personal communication). Attempts
to demonstrate a potassium-induced release of glutamate and GABA
in either all-glial or mixed neuronal-glial primary cultures from
100 I
0
•~. 50
CD 0
-
::J
II)
.!! 0
25
0
c
0
C)
c 0
·c 10 0
.-
.iii 00
E 00
...
CD
5
00
0
>. 0 ....
~
>
;; 0000
u 2.5 000
.~
"C
ca
0:::
10 15 30 45 60 75 90
Time from start of washout, min.
Fig. 3. An example of washout curves showing, as a function of
time, release of radioactivity from a mixed neuronal-glial culture
(dissociated brain cells from a 7-day-old chick embryo cultured for
7 days in a modified Eagle's minimum essential medium as originally
described by M.Sensenbrenner and coworkers I9 ,48). The culture had
been loaded with U-(14cJ- labelled glutamate and the washout was into
a 'physiological' (0) medium (0-45 min+ and 69-90 min.) and (45-69
min.) into a medium containing 80 mM K (e). From such curves, the
halftimes (~) can, after an initial deviation (0-20 min.), be direct-
ly read, and the changes in ~ shown in Figs. 1-2 are the differences
between the ~ before and during, the exposure to excess potassium.
376 L. HERTZ
Uptake
Energy Metabolism
Table 1
Specific K+
PreEaration SEecies activity conc.
CONCLUSION
ACKNOWLEDGEMENTS
REFERENCES
15. Henn, F.A., Haljamae, H., and Hamberger, A., Glial cell
function: Active control of extracellular K+ concentration,
Brain Res., 43 (1972) 437-443.
16. Hertz, L., Possible role of neuroglia: A potassium-mediated
neuronal-neuroglial-neuronal impulse transmission system,
Nature, 206 (1965) 1091-1094.
17. Hertz, L., Neuroglial localization of potassium and sodium
effects on respiration in brain, J.Neurochem., 13 (1966)
l373-l387.
18. Hertz, L., Potassium effects on ion transport in brain slices,
J.Neurochem. 15 (1968) 1-16.
19. Hertz, L., Dittman, L., and Mandel, P., K+-induced stimulation of
oxygen uptake in cultured cerebral cells, Brain Research,
60 (1973) 517-520.
20. Hertz, L., and Schou, M., Univalent cations and the respiration
of brain-cortex slices, Biochem.J., 85 (1962) 93-104.
21. Hillman, H.H., and McIlwain, H., Membrane potentials in mamma-
lian cerebral tissues in vitro: Dependence on ionic environ-
ment, J.Physiol.Lond. 157 (1961) 263-278.
22. Himwich, H.E., Bernstein, A.O., Fazekas, J.F., Herrlich, H.C.,
and Rich, E., The metabolic effects of potassium, temperature,
methylene blue and paraphenylene diamine of infant and adult
brain, Am.J.Physiol.137 (1942) 327-330.
23. Horstmann, E., and Meves, H., Die Feinstruktur des molekularen
Rindengraues und ihre physiologische Bedeutung, Z.Zellforsch.
mikrosk. Anat., 49 (1959) 569-604.
24. Hopkin, J., and Neal, M.J., Effect of electrical stimulation
and high potassium concentrations on the efflux of [14c] glycine
from slices of spinal cord, Brit.J.Pharmacol., 42 (1971) 215-
223.
25. Hultborn, R., and Hyden, H., Microspectrophotometric determina-
tion of nerve cell respiration at high potassium concentration
Exp.Cell Res., 87 (1974) 346-350.
26. Katz, R.I., Chase, T.N., and Kopin, I.J., Effect of ions on
stimulus induced release of amino acids from mammalian brain
slices, J.Neurochem., 16 (1969) 961-967.
27. Kimelberg, H.K., Active potassium transport and Na+ + K+ ATP-
ase activity in cultured glioma and neuroblastoma cells, J.
Neurochem. , 22 (1974) 971-976. --
28. Lund-Andersen, H., and Hertz, L., Effects of potassium and glut-
amate on swelling and on sodium and potassium content in brain-
cortex slices from adult rats, Exp.Brain Res. , 11 (1970)
199-212.
29. Lux, H.D., Neher, E., and Prince, D., K+-activity determinations
in cat cortex, Pflugers Arch. 332 (1972) R89.
30. Machiyama, Y., Balazs, R., Hammond, B.J., Julian, T., and Richter,
D., The metabolism of r-aminobutyrate and glucose in potassium
ion-stimulated brain tissue in vitro, Biochem.J.116 (1970) 469-481.
382 L. HERTZ
Ricardo Tapia
INTRODUCTION
385
386 R. TAPIA
GABA-DEPENDENT INHIBITION
GLUTAMATE
GAD
GABA~STORAGE
---
GABA
-
Figure 1. Synthesis-dependent release of GABA from
inhibitory nerve endings •• It is postulated that a certain
proportion of GAD is bound to the presynaptic membrane in
the presence of Ca 2 +, and that the pool of GABA synthesized
by the membrane-bound enzyme is released into the synaptic
space (®). A second pool of GABA, synthes ized by GAD
soluble in the synaptoplasm, would be stored for its
release stimulated by depolarization <@).
*
t Cutnulative radioactivity released in the 40 tnin superfusion period.
Labeled atnino acid released in 40 tnin superfusion as percentage of the total radioactivity in
the atnino acid.
Values significantly different frotn the control values (p":0.02 or p<0.05.l test). All other
differences were not significant (p"0.05).
Our data also suggest that the equilibrium between the newly
synthesized pool and the storage pool of GABA is very slow, or,
alternatively, that they do not mix. In contrast, the pool of
recently synthesized catecholamines or acetylcholine, although also
preferentially released upon stimulation, seems to be in a relatively
rapid equilibrium with the storage pool, which apparently can supply
neurotransmitter for release and thus maintain the synaptic function.
REFERENCES
38. Tews, J.K., Carter, S.H., Roa, P.D., and Stone, W.E., Free
amino acids and related compounds in dog brain: Post-mortem
and anoxic changes, effects of ammonium chloride infusion, and
levels during seizures induced by picrotoxin and by pentyl-
enetetrazol, J. Neurochem., 10 (1963) 641-653.
39. Thierry, A.M., Blanc, G., and Glowinski, J., Effect of stress
on the disposition of catecholamines localized in various
intraneuronal storage forms in the brain stem of the rat,
J. Neurochem., 18 (1971) 449-461.
40. Urquhart, N., Perry, T.L., Hansen, S., and Kennedy, J., GABA
content and glutamic acid decarboxylase activity in brain of
Huntington's chorea patients and control subjects, J. Neurochem.
24 (1975) 1071-1075.
41. Von Euler, C., Skoglund, S., and S8derberg, U. (Eds.),
Structure and Function of Inhibitory Neuronal Mechanisms,
Pergamon Press, Oxford, 1968, 563 p.
42. Wood, J.D., The role of y-aminobutyric acid in the mechanism
of seizures, Progr. Neurobiol., 5 (1975) 77-95.
43. Wood, J.D., and Peesker, S.J., The effect on GABA metabolism
in brain of isonicotonic acid hydrazide and pyridoxine as a
function of time after administration, J. Neurochem., 19 (1972)
1527-1537.
44. Wood, J.D., and Peesker, S.J., The role of GABA metabolism in
the convulsant and anticonvulsant actions of aminooxyacetic
acid, J. Neurochem., 20 (1973) 379-387.
TRANSPORT OF AMINO ACIDS AND CATECROLAMINES IN RELATION
Department of Biochemistry
Imperial College of Science and Technology
Prince Consort Road
London SW7 2AZ, Great Britain
I INTRODUCTION
395
396 J.S. de BELLEROCHE AND H.F. BRADFORD
active amino acids are the most affected indicates the importance
of this cycle for neurotransmitters.
TABLE 1
Amino acid release from rat cerebral cortex synaptosomes and the
effect of ouabain,~-aminobutyric acid (BABA),2,4-diaminobutyric
(DABA) and N-acetylglutamic acid (NAG).
Incubate Wash
40' trans'er
~ synaptosomes
14C Dopamine
,..
.. . 3H Dopamine uptake
.. . ., ..
"
a) Synaptosome total n mo n mol "
per 100 mg protein per
100 mg
b) Per cent released protein
,. L:.!-,
II !
••
--'-____ . . ,'.. . .- - ----1 DA external !JM
,. . '0 •
• .1 n It
DA external !JM
Fig. 2. - Synaptosome beds were incubated in Krebs-bicarbonate med-
ium (10 mM glucose) containing 3.9 !JM DL-3(3,4-dihydroxyphenyl)ala-
nine-[2- l4 C]at 37 0 for 40 min. The beds were then rinsed twice in
medium containing no radioisotope and placed in fresh-b carbonate
medium (10 mM glucose) containing C3Badopamine (10 ~Ci) present at
the concentrations of dopamine indicated. After 3 min the incubation
was terminated and the (3H1and[14CJcontents of the final incubation
medium and synaptosomes were determined by double isotope scintillat-
ion spectrometry, Individual values are indicated by filled circles
for control incubations, those carried out in the presence of d-
amphetamine (0.12 mM) are indicated by open triangles. The total
synaptosome content of~14CJdopamine is shown in (a) expressed as nmol/
100 mg protein, with the proportion (%) released to the medium being
shown in (b). The synaptosome content of(3H)is shown in (c).
Figure 3
400 J.S. de BELLEROCHE AND H.F. BRADFORD
.
DOPAMINE RELEASE
DOPAMINE RELEASE
a' CONTROL )
n mol
"
40' I
40 'I
b)
n Ci
I
100me
100
20' I
Figure 4
ACKNOWLEDGEMENTS
REFERENCES
Federico Piccoli
Department of Neurology
University of Palermo
Palermo, Italy
INTRODUCTION
405
406 F. PICCOLI
The level of ATP and the specific activities of Mg++ and Na+-
K+ ATPase have been investigated in the developing rat brain 11 . The
level of ATP decreases during post-natal development, while Mg++
and Na+ - K+ ATPase increase. The exposure to nitrogen of the fetus
at the 21st day of intrauterine life slightly depresses Mg++ ATPase
activity, while the Na+ - K+ ATPase activity remains unchanged, and
a significant fall in ATP level occurs. Conversely, the exposure
of the fetus to 169'ooxygen increases the Na+ - K+ activity, and
mainly the level of ATP.
TABLE I
WATER CONTENT AND SPACES IN INCUBATED SLICES OF RAT BRAIN
Age Total Water (ml/g dry wt.) Swelling (percentage Inulin space
(days) before after increase of water (percent of
incubation incubation after incubation) total water)
The water content per unit dry wt of the intact brain decreases
by more than 50 per cent from fetus to adult. There is no swelling
in incubated slices from fetal brain, and the extent of swelling
increases with age; consequently the changes in fluid content at
the end of incubation are less than those in unincubated slices as
a function of development. The non-inulin space of incubated brain
slices decreases from fetus to adult by about 50 per cent; this
decrease probably does not imply that as development proceeds the
cellular space shrinks.
When uptake of amino acids by brain slices from newborn and adult
is compared, most compounds appear to be taken up by slices from
adult brain to a greater extent, although some compounds such as
ethanolamine or tyrosine are taken up more by newborn slices 7 .
z
o
8
•
I-
oct
Ill:
I-
Z
w 6
U
Z
o
()
Ill: 4
oct
...J
~
...J
...J
W
U
oct 2
Ill:
I- ____~________~~/rAU
Z
12 18 24 30 36 42
AGE (days)
The maximal flux also does not occur at the time the levels
reach a peak (glycine levels reach peaks at birth and after 32 and
90 days, for example).
60
w GLY
:..:
<I: f-+-IltLU
~J-o
~
D..
::> 40
w
>
I- f---I ;"'AU
<I:
a:
I-
Z
w 20
()
Z
0
()
12 18 24 30 36 42
LLJuJ
60 Ad.
AGE (days)
CONCLUSIONS
REFERENCES
415
416 A. LAJTHA AND M. BANAY·SCHWARTZ
2 9.9 7.2 73
7 9.8 5.6 57
9 9.9 5.3 54
14 7.0 3.4 49
brain in vivo and in vitro. The rates shown in vivo in Table 1 are
comparable to those reported recently from other laboratories 19 ,24,52.
Although the incorporation rates il, slices shown are the highest we
can obtain, still they are below the levels in vivo. The difference
between slices and in vivo is not constant; it is smaller in the
young brain and much larger in the adult. The greater stability of
the system for protein synthesis in young brain has been observed
previously59. Table 1 illustrates how studies of changes in rates
of protein synthesis during decelopment must take into account
changes that are due to such factors as this stability before meas-
urements in vitro can be related to those in vivo. It also is of
interest that most studies of mechanisms of protein synthesis used
systems in vitro in which activity is only a very small fraction
of that in vivo ..
o Lysine uptake
• AI B uptake
Sliced bulb
.•
20
,--.-
Intact bul b
•
.•"'-
E
"
" 12
'E"
4 ..1---..A_--'
. - ___-:.:!e=--=---e---e---..a
'" _.-::r
~-
30 60 90 120 I~O
Tissue K+ pmol/ml 70 73
Uptake units are given in vivo as per cent taken up from tracer
i~fusion55, 62, uptake in slices as tissue medium concentration
ratio after 30 min incubation. The inhibition in vivo is not
significant, the small uptake possibly being via diffusion;
the concentrative uptake in slices (with tissue levels 4-5 fold
higher than medium) is significantly inhibited by analogs 35 at
low or high concentrations and in short and long time experi-
ments.
transferase activity. Although this may not cause any net utiliza-
tion of glutamate, it will cause the appearance of label from glut-
amate in a variety of compounds. The rate of net glutamate in brain
is unknown, and we still cannot exclude the possibility that the
major replacement for the glutamate that is utilized in the brain
comes from plasma glutamate. In turn, the final distribution and
regional level (perhaps even the regional metabolism) of glutamate
depends on cellular transport processes 30 •
TABLE VI. Levels of amino acids in vivo and uptake in brain slices
Concentration Uptake
Adult Newborn Adult Newborn
Glutamate ---g:g- 4.4 ~ 50
Taurine 8.3 19 44 17
Glycine 0.91 2.30 53 34
Lysine 0.19 0.23 8.8 10
Leucine 0.04 0.08 5.1 6.5
Phenylalanine 0.05 0.11 3.3 4.7
CONCLUSIONS
REFERENCES
50. Neidle, A., Kandera, J., and Lajtha, A., The uptake of amino
acids by the intact olfactory bulb of the mouse: A comparison
with tissue slice preparations, J.Neurochem. 20 (1973) 1181-
1193.
51. Neidle, A., Kandera, J., and Lajtha, A., Compartmentation and
exchangeability of brain amino acids: Evidence from studies of
transport into tissue slices, Arch.Biochem.Biophys., 169 (1975)
397-405.
52. Oja, S.S., Incorpolation of phenylalanine, tyrosine and trypto-
phan into protein of homogenates from developing rat brain:
kinetics of incorporation and reciprocal inhibition, J.Neuro-
chern., 19 (1972) 2057-2069.
53. Oja, S.S., and Vahvelainen, M.L., Transport of amino acids in
brain slices, In N.Marks and R.Rodnight (Eds.), Research Meth-
ods in Neurochemistry, Vol.3, Plenum Press, New York, 1975,
pp.67-l37.
54. Okamoto, K., and Quastel, J.H., Uptake and release of glutamate
in cerebral-cortex slices from the rat, Biochem.J., 128 (1972)
1117-1124.
55. Oldendorf, W.H., Brain uptake of radiolabeled amino acids,
amines, and hexoses after arterial injection, Amer.J.Physiol.,
221 (1972) 1629-1639.
56. Oldendorf, W.H., and Szabo, J., Amino acid assignment to one of
three blood brain barrier amino acid carriers, Amer.J.Physiol.,
1975, in press
57. Piccoli, F., Grynbaum, A., and Lajtha, A., Developmental changes
in Na+, K+ and ATP and in the levels and transport of amino
acids in incubated slices of rat brain, J.Neurochem., 18 (1971)
1135-1148.
58. Pull, I., Jones, D.A., and McIlwain, H., Superfused cerebral
tissues in hypoxia: neurotransmitter and amino acid retention;
labile constituents and response to excitation, J.Neurobiol.,
3 (1972) 311-323.
59. Roberts, S., Effects of amino acid imbalance on amino acid
utilization, protein synthesis and polyribosome function in
cerebral cortex.ln Aromatic Amino Acids in the Brain, CIBA
Foundation Symposium 22, American Elsevier, New York, 1974,
pp.299-324.
60. Rose, S.P.R., and Sinha, A.K., Some properties of isolated
neuronal cell fractions, J.Neurochem., 16 (1969) 1319-1329.
61. Sershen, H., and Lajtha, A.,The distribution of amino acids,
Na+, and K+ from surface to centre in incubated slices of
mouse brain, J.Neurochem., 22 (1974) 977-985.
62. Sershen, H., and Lajtha, A., in preparation.
63. Seta, K., Sansur, M., and Lajtha, A., The rate of incorporation
of amino acids into brain proteins during infusion in the rat,
Biochim.Biophys.Acta, 294 (1973) 472-480.
64. Snyder, S.H., Young, A.B., Bennett, J,P., and Mulder, A.H.,
Synaptic biochemistry of amino acids, Fed.Proc., 32 (1973) 2039-
2047.
434 A. LAJTHA AND M. BANAY-SCHWARTZ
65. Teller, D.N., Banay-Schwartz, M., De Guzman, T., and Lajtha, A.,
EnergeLics of amino acid transport into brain slices. Effects
of glucose depletion and substitution of Krebs cycle inter-
mediates, Brain Research, 1976, in press.
66. Wade, L.A., and Katzman, -R., Transport of L-DOPA and related
amino acids across cerebral capillaries: evidence for the
presence of the L transport system, Abstracts 5th Meeting
Internatl.Soc.Neurochem., Barcelona, 1975, p.462.
RELEASE OF AMINO ACIDS FROM THE SPINAL CORD
Robert W. P. Cutler
INTRODUCTION
435
436 RoWoP oCUTLER
100
0
0
50
<!>
• <D 56% k ' 002 min·'
~
z 20 ® 2 1 % k ' .040min- '
<i
:;;
w
® 2.3% ~ '. 115 min- '
a:
w
z 10
U
>-
...J
<!>
}'
-=.... 5
~
0
2
,
60
,
90
.
120
.
150
TIME . mi n
Figure 1. Efflux of [14C] glycine from the intact rat spinal cord.
[14C] glycine, 10 ~Ci, was injected into the spinal fluid. After
incubation for 30 min , cisternal-lumbar perfusion with artificial
CSF was carried out (see reference 11). Rate constants and com-
partment sizes were calculated from the desaturation curve.
z 200
0
~
a:
....z
w 15 0
U
z
0
U
w
a.
....
0
0
100
~
w
>
~
...J
50
W
a:
0 ,
0 5 10 15 20
SAMPLE NUMBER
80 0
~ I
o 5 10 15 20
SAMPLE NUMBER
However, when the sciatic nerve was stimulated during the time of
action of p-CMBA, an increased rate of release of glycine was in-
duced. Because the glycine was not labeled, it originated in the
spinal cord and was released into the spinal fluid under conditions
which presumably blocked its reuptake. An attempt was then made
to induce the release of glycine into the fluid by blocking its
binding to postsynaptic receptor sites with strychnine. This drug
is not known to affect the uptake of glycine by the tissues. Strych-
nine alone, after intravenous injection, did not alter the resting
flux of glycine, but when strychnine was administered in combina-
tion with simultaneous sciatic nerve stimulation, there was a sig-
nificant release of glycine into the fluid (Figure 4). The enhanced
rate of release continued for at least 40 minutes after nerve stimu-
lation ceased. There was no increase in the rate of release of any
442 R.w.P. CUTLER
180
SAMPLE NUMBER
I
300
C
"e i
01lio ,5
.. E
"0 ~
E a.
8 u
~ ~- 200
II> ...
<t Z
w w
--' w
~ ~
a
u \
\
~ 'b---'\,
u
~ u
,
100 ....
,
''b''_...D_-"o .......
"'!!
!
II
"'0
!
~ .o IT''----.....,,'''~---....,,
f . .o
5
If
20
o 20 40 60 eo 100 120
PERfUSION Ti ME, min
60mM K+
60
o 2 • 10
SAMPLE NUMBER
CONCLUSION
REFERENCES
I. INTRODUCTION
447
448 A.V. LORENZO AND R. SPECTOR
1. Salicylic Acid
(6)
12 EXPERIMENTAL 15MIN " OF
It......... ..........
CONDITION UPTAKE CONTROL
10 I
(41 CONTROL
(O.OO/mll)
11.0tO.7 -
I ~, SALICYLATe
{5mMI
3.0!0.3 21.3
I " ......Salicylate
8 -<
95 "No'"
3.1 :!:.0.3 2B.3
I 5" COZ
d'c
5.7tO.4
2.8:t0.2
52.0
25.0
6
l
I
'" , (6)
DIODRAST
{ImNI
1.5:!:'0.2 13.0
I ' PAH
~
3.3±0.3 30.0
4 I {fOmMl
I MEDIUM
60MIN
C. PLEXUS
HOMOGENATE
SUPERNATE
I ACTIVIlY
(CPll/mll
ACTIVITY ACTIVITY
2 ~ (6)
(CPM/Qm I (CPM/mll
o 10 20 30 40 50 60 TIM£(minlJtes)
.0
0.8
• • 125I _ HSA
14
C- Sucrose
0.6 • 3H-Salicylate
TIME (minules)
that both drugs are transported by the same system. However, during
ventriculocisternal perfusions in rabbits the non-saturable clearance
of PAS was found to be less than that of salicylate. This was some-
what unexpected since the dissociation constant for PAS (pKa= 4.0)
isgreater than for salicylate. Therefore in CSF, PAS should be less
ionized than salicylate and presumably be able to penetrate from
CSF to blood more readily. This disparity becomes m!)!'e "'pparent
when the clearance from blood to CSF (Ki) (Table 3) is compared for
both drugs. The clearance value for salicylate is some 6 times
greater than for PAS. Since both drugs are bound to plasma protein
to approximately the same extent, the relatively higher influx rate
for salicylate must be attributable to some other factors. Pos-
sibly, the intramolecular hydrogen bonding which converts salicylate
to a chelate (not observed with the para and meta hydroxylated
derivatives of benzoic acid), and/or the interaction with lipids
such as lecithin which markedly enhance the solubility of salicylate
in organic solvents may be the basis for the higher influx rate39.
Also, the classical analysis of Brodie 7 and Mayer et al. 27
which assumes that only undissociated drug can pass through lipid
membranes is known not to be correct l6 ,33, since in the presence of
high concentrations of ionized moeity the relatively small amount of
ionized drug that penetrates across membranes constitutes a signifi-
cant fraction of the total amount (ionized and unionized) that passes
across the membrane. Whatever the explanation, in the same unanes-
DISTRIBUTION OF DRUGS 453
Table 3. Clearance of drugs from and into the CSF during ventricu-
locisternal 2erfusions.
Ko Ko s Ki
K~os Ko/Ki
Drug ~ml/min} ~ml/min}
Salicylic .026 .022 .024 1.2 1.1
.025 .017 1.5
.022 .009 .004 2.4 5.5
.028 .004 .002 7.0 14.0
.011 .0009 12.0
.690 040 17.5
et al. avson and Pollay
3. Penicillin
suggest that with meningitis the weak acid efflux transport system
is inhibited. The finding that the uptake (T/M) of penicillin by
the choroid plexuses obtained from H. Flu inoculated rabbits was
also inhibited, lends support to this suggestion. The degree of
inhibition does not appear to be related to the severity of the
disease, since the inhibition was similar whether the meningitis
was induced with H. Flu, which produced a mild type of meningitis
that resolved within 5 days, or with S. Aureus, which produced a
severe meningitis that proved fatal within 24-48 hours. Moreover,
the inhibition produced during meningitis was only partial, since
administration of probenecid led to further reduction in the clear-
ance of penicillin from the CSF. As yet we have not resolved the
factor(s) which act to depress the carrier-mediated transport
process of the choroid plexus. The fact that no inhibition was
apparent during ventriculocisternal perfusions (penicillin clearance
(Ke) controls = 0.014 ± .002 ml/min; H. Flu = 0.011 ± .003 ml/min)
could be due to anesthesia as discussed above.
4. Gentamicin
Table 4. Penicillin clearance from the CSF and uptake into choroid
plexus in control and H. Flu inoculated rabbits l
CSF Retention! Choroid Plexus 2
Penicillin Inulin 5 min uptake
Group % of in1. dose Day dpm/ml x 104 T/M ratio
Control 4.1 ± 0.2 1 0.85 + 0.19 10.8 + 1.1 (14)
H. Flu 8.9 ± 2.1 1 0.81 ± 0.14 7.5 + 0.7 (16)
3 9.6±1.3 (8)
5 15.8 ± 1.2 (6)
lAnimals were inoculated intracisternally with 5 x 10 7 Hemophilus
influenza type b (H. Flu) in 0.2 ml phosphate buffer. The follow-
ing day, l4c-penicillin and 3H-inulin were injected intraventricu-
larly. The animals were killed 2 hours later and cisternal CSF was
drawn. The amount of penicillin retained in CSF was expressed as
l4C/3H activity in CSF 100
14C/3H activity in injectate x
21n the choroid plexus experiments, choroid plexuses were obtained
from rabbits inoculated with 0.2 ml buffer or H. Flu in buffer
intraCisternally 1, 3 and 5 days after inoculation. The choroid
plexuses were then incubated in artificial CSF containing l4C-peni-
cillin, according to the procedures described in Figure 1.
456 A.V. LORENZO AND R. SPECTOR
20 1 ) - - - - - - - - - - 0 14C ·Penicill in
....
u
c:
0 18
()
E
.2 16
"0
G,)
~
"- 14
u
c:
0
() 12
...RI
::;,
10 14C-p-Amino
G,)
U Salicylic Acid
....c:...
RI
8
.....
UJ
~
«
....el. 14C· Salicylic
:J Acid
~_ _ _ _ _ _ _ 14C-Gentamycin
3H-lnulin
15 30 45 60
TIME (minutes)
III. DISCUSSION
References
Leontino Battistin
.465
466 L. BATTISTIN
even though they have the great advantage of being much more similar
to human pathology.
Control Metrazol
Level and uptake in vivo. The levels of free amino acids in the
mouse brain under physiological conditions and after generalized
convulsions are given in Table I. In the normal brain the levels of
the various compounds are fairly similar to those described elsewhere
5, 16; after convulsions, we found that some amino acids (alanine,
AMINO ACID UPTAKE IN CONVULSIONS 467
L-aspartic acid
0 (Control) 0.10 3.27 32.7
0 (Metrazol) 3.43
10 (Control) 4.99 3.77 0.76
10 (A) 9.87 3.33 0.34
10 (B) 8.21 3.29 0.40
L-methionine
o (Control) 0.042 0.061 1.45
o (Metrazol) 0.054 0.073 1.35
10 (Control) 0.41 0.11 0.27
10 (A) 0.55 0.17 0.31
10 (B) 0.52 0.22 0.42
Control 47 46 40 39
L-Aspartic acid 34 35 40 38
L-Glutamic acid 35 36 39 39
L-Lysine 48 45 48 44
Glycine 43 42 43 39
L-Leucine 43 42
Concentrative uptake
5 min 60 min
control experiment control experiment
L-Aspartic acid 10.03 8.47 46.3 44.7
L-Glutamic acid 9.41 10.48 45.7 44.9
Glycine 8.97 8.27 42.5 44.6
L-Alanine 6.68 6.09 32.1 29.8
L-Leucine 2.93 2.65 3.43 3.49
L-Valine 5.09 5.27 7.59 6.89
L-Lysine 2.79 3.54 8.37 9.44
L-Methionine 3.15 2.49 6.28 5.88
L-Histidine 6.16 6.89 38.7 38.9
Control experiment
L-Aspartic acid 75 71
L-Glutamic acid 48 47
Glycine 56 54
L-Valine 73 68
L-Lysine 35 61
L-Methionine 36 45
CONCLUSIONS
ACKNOWLEDGEMENTS
REFERENCES
8. Berl, S., Takagaki, G., and Purpura, D.P., Metabolic and pharma-
cological effects of injected amino acids and ammonia on cortical
epileptogenic lesions,J.Neurochem., 7 (1961) 198-209.
9. Cohen, S.R., The estimation of extracellular space of brain
tissue in vitro. In N.Marks and R.Rodnight (Eds.),Research
Methods in Neurochemistry, Vo 1. I, Plenum Press, New York, 1972,
pp.179-2l9.
10. Cohen, S.R., Blasberg, R., Levi, G., and Lajtha, A., Compart-
mentation of the inulin space in mouse brain slices, J.Neurochem.,
15 (1968) 707-720.
11. Cutler, R.W.P., Lorenzo, A.V., and Barlow, C.F., Changes in blood-
brain permeability during pharmacologically induced convulsions,
In A.Lajtha and D.H.Ford (Eds.), Brain Barrier System, Progress in
Brain Research, Vol.29, Elsevier, Amsterdam, 1968, pp.367-378.
12. Guidotti, A., Badiani, G., and Pepeu, G., Taurine distribution
in cat brain,J.Neurochem., 19 (1972) pp.43l-435.
13. Kandera, J., Levi, G., and Lajtha, A., Control of cerebral meta-
bolites levels. II. Amino acid uptake and levels in various areas
of the rat brain, Arch.Biochem, 126 (1968)249-260.
14. Lajtha, A., Transport as control mechanism of cerebral metabolite
levels. In A.Lajtha and D.H.Ford (Eds.), Brain Barrier Systems,
Progress in Brain Research, Vol.29, Elsevier, Amsterdam, 1968,
pp.20l-2l8.
15. Lee, J.C., Evolution in the concept of the blood-brain barrier
phenomenon. In H.M. Zimmerman (Ed.), Progress in Neuropathology,
Grune and Stratton, New York, 1971,pp.84-l45.
16. Levi, G., Kandera, J.,Lajtha, A.,Control of cerebral metabolite
levels. I.Amino acid uptake and levels in various species,Arch.
Biochem., 119 (1967) 303-311. --
17. Levin, E., Wolosiuk, R., Scilli, G., and Glanczepigel, R., In
vitro incubation of brain hemispheres. Fluid spaces and amino
acid uptake, Neurol. (Minneapolis) 20 (1970) 584-593.
18. Lorenzo, A.V., Shirahige, J., Liang, M., and Barlow, C.F.,
Temporary alteration of cerebrovascular permeability to plasma
protein during drug-induced seizures, Amer.J.Physiol.,223 (1972)
268-277 •
19. Lovell, R.A., Some neurochemical aspects of convulSions, In
A.Lajtha (Ed.), Handbook of Neurochemistry, Vol.6, Plenum Press,
New York, 1971, 63-102.
20. Neame, K.D., Transport, metabolism, and pharmacology of amino
acids in brain. In A.N.Davison and J.Dobbing (Eds.), Applied
Neurochemistry, Blackwell , Oxford, 1968, pp.1l9-l77'.
21. Oja, S.S., and Lahdesmaki, P., Is taurine an inhibitory neuro-
transmitter? Med.Biol., 52 (1974) 138-143.
22. Perry, T.L., Sanders, H.D., Hansen, S., Lesk, D., Kloster, M.,
and Gravlin, L., Free amino acids and related compounds in five
regions of biopsied cat braln,J.Neurochem., 19 (1972) 2651-2656.
23. Purpura, D.P., Girado, M., Smith, T.G., Gomez, J.A., Synaptic
effects of systemic aminobutyric acid in cortical regions of
AMINO ACID UPTAKE IN CONVULSIONS 477
479
480 M. SPATZ AND I. KLATZO
(1) ischemia, (a) the effect on BBB, (b) the effect on synaptosomes;
(2) the effect of oxygen saturation and pC0 2 tension on the trans-
port from blood to brain, (a) in vivo studies, and (b) in vitro
studies. - -- -- - - -
Ischemia
Table II. Brain Uptake of l4C Glucose Analogues and Amino Acids
The injected mixture contained 1.5 ~C of each 3H20 and 14C test
substance in concentrations of .01 - .02 mM except for 3 ~C of
glutamic acid (.04 mM) and serine (.06 mM). Percent of control
Brain Uptake Index values are means ± S.E.M. of 3-9 experiments.
* Increased percent of control BUI. ** No effect.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 485
In Vivo Studies
14
of C glucose analogues and most of the amino acids was observed
in severe hypoxia SA02 of 15-18% (values derived from rabbit hemo-
globin dissociation curve and corrected for variation in pH4).
The most affected substances appeared to be L-Tyrosine, L-Methionine
and D-Leucine (but the quantitative differences may not be signifi-
cant since precise assessment is not possible by this method) while
little or no effect was seen in the BUI of L-Serine and L-Glutamic
acid, respectively. A similar BUI reduction of these substances
was also found in severe hypercapnia (pCO Z 98-lS0 mmHg) with the
exception of L-Alanine and L-Serine, show~ng only a slight decrease
of BUI, while the BUI of glutamic acid was found normal in this
severe respiratory acidosis (pH 7.1). However, in hypocapnia
(pC02 lS-l8 mmHg) the BUI of L-Tyrosine and L-Methionine appeared
little altered while the other tested compounds revealed the BUI
to be increased or normal. Although a variety of differently
classified amino acids have been screened in this experiment, the
changes observed cannot be attributed to any group39. Only the
glutamic acid, which was unaffected, belongs to the acidic amino
acids. In hypocapnic animals with respiratory alkalosis (pH 7.7S
and pC02 about IS mmHg) the BUI of 3H 3-MG was 3S% higher than the
BUI of controls. Inhibition studies revealed that the hypocapnic
rabbits were more sensitive than the control animals; that is, the
Km or inhibition concentration (cold 3-MG) necessary to decrease
the BUr by SO% was .S mM and S mM, respectively. The control uptake
of all the substances were similar to the BUI reported by Oldendorf 4l
and by different indicator techniques by other investigators 37 ,6S.
(3) There is some other evidence to support the contention that the
BUI do not simply reflect blood flow changes: (a) hypercapnia (pC0 2
= 100 mmHg) had been shown in dogs to have a much stronger dilating
effect on cerebral than severe hypoxia 23 , causing possibly a greater
blood flow in hypercapnia. The BUI values in hypercapnia are similar
with one exception, to those observed in hypoxia, Table I. (b)
On the other hand, hypoxia (SA02 less 60%) has been shown in dogs
to abolish autoregulation and results in a passive pressure-flow
relationship23. However, the BUI of 2-DG in severe hypoxia was
found to be independent of a wide range of arterial blood pressure,
which in the absence of autoregulation, would expect to result
in a correspondingly wide range of cerebral blood flow. (c)
Single-step hyperoxia to levels above 300 mmHg has been shown to
decrease cerebral blood flow by at least 20% in man l • In the
rabbit experiments, there were no significant differences in the
BUI found in a similar single-step hyperoxia, and the control
animals 5 • (4) If the BUI of the tested compounds would entirely
reflect the results of uptake caused by increase or decrease of
blood flow, then there should be a more uniform BUI response to
the altered p02 saturation and pC02 tension (Table II).
In Vitro Studies
The aim of this paper has been to review and discuss the past
and the recent investigations concerned with the study of cerebral
transport phenomena in pathological conditions which have been
divided into two main parts: (1) the effects of experimentally
induced blood brain barrier (BBB) injury by (a) HgClZ or (b) hyper-
osmolar intracarotic perfusate; and (Z) the effects of ischemia or
of an altered oxygen saturation and pCOZ tension on glucose and/or
amino acids and/or protein transport across the BBB, in the syanpto-
somes and cerebral capillaries. The most important observations
were as follows: (1) HgClZ or hyperosmolar perfusates produced an
increased BBB permeability to protein tracers but the brain up-
take of glucose analogues was found decreased following the former,
and increased (except for lactamide) after the latter treatment.
(Z) (a) In ischemia, the noted increased vesicular transport of
peroxidase, as well as the increased saturable and non-saturable
passage of glucose analogues across the BBB depended on the dura-
tion of cerebral deprivation of blood supply which never resulted
in degeneration of endothelial cells of the brain vessels. (b)
The progressively decreased specific Z-deoxy-D-glucose uptake in
the synaptosomes seen during cerebral ischemia of 30-180 minutes
returned to the level of controls 1 hour after reestablishment
of cerebral circulation. (c) A decrease in brain uptake of glu-
cose analogues and amino acids (with few exceptions) was
observed in severe hypoxia and hypercapnia while an increase
or no change in the brain uptakes was seen in hypocapnia. (d)
Preliminary investigations of the Z-DG uptake by the cerebral
capillaries obtained by fractionation of the brain from animals
subjected to normal or altered oxygen saturation and pC02 tension
suggested that cerebral glucose uptake may be directly related to
its capillary function.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 491
REFERENCES
(1970) 233-243.
15. Crone, C., Facilitated transfer of glucose from blood into
brain tissue, Journal of Physiology, 181 (1965) 103-113.
16. Cutler, R. W. P., and Barlow, C. F., The effect of hypercapnia
on brain permeability to protein, Archives of Neurology, 14
(1966) 54-63.
17. Cutler, R. W. P., Lorenzo, A. U., and Barlow, Ch. F., Changes
in blood brain barrier permeability during pharmacologically
induced convulsions, Progress in Brain Research, 29 (1968)
367-377 .
18. DeRobertis, E., and Rodriguez de Lores Arniaz, G., Structural
components of the synaptic region. In A. Lajtha (Ed.), Hand-
book of Neurochemistry, Vol. II, Structural Neurochemistry,
Plenum Publishing Co., New York, 1969, pp. 365-392.
19. Diamond, I., and Fishman, R. A., High-affinity transport and
phosphorylation of 2-deoxy-D-g1ucose in synaptosomes, Journal
of Neurochemistry, 20 (1973) 1533-1542.
20. Eklof, B., Lassen, N. A., Nilsson, L., Norberg, K., Siesjo,
B. K., and Tor1of, P., Regional cerebral blood flow in the rat
measured by the tissue sampling technique: a critical evalu-
ation using four indicators C14-Antipyrine, C14-Ethanol, H3-
water and Xenon, Acta Physiologica Scandinavica, 91 (1974) 1-10.
21. Fujimoto, T., Berson, F., Spatz, M., and Klatzo, I., The effect
of oxygen saturation and C02 tension on amino acid brain uptake
in the rabbit, unpublished observation (1975).
22. Goldstein, G. W., Wolinsky, J. S., and Diamond, I., Isolation
of metabolically active capillaries from rat brain, Transactions
of the American Society for Neurochemistry, Sixth Annual Meet-
ing, 1975, pp. 277.
23. Haggendal, E., and Johansson, B., Effects of arterial carbon
dioxide tension and 02 saturation on cerebral blood flow auto-
regulation in dogs, Acta Physiologica Scandinavica, 66 (1965)
27-53.
24. Heaton, G. M., and Bachelard, H. S., The kinetic properties of
hexose transport into synaptosomes from guniea pig cerebral
cortex, Journal of Neurochemistry, 21 (1973) 1099-1108.
25. Ito, U., Spatz, M., Walker, J. T., Jr., and Klatzo, I., Exper-
imental cerebral ischemia in Mongolian gerbils. I. Light micro-
scopic observations, Acta Neuropathologica (1975) 32: 209-223.
26. Ito, U., Mrsulja, B. B., Fujimoto, T., Spatz, M., and Klatzo,
I., Effects of increased systemic blood pressure on brain tis-
sue subjected to ischemia, Proceedings of the International
Symposium on Pathophysiological, Biochemical and Morphological
Aspects of Cerebral Ischemia and Arterial Hypertension, (1976)
in press.
27. Johansson, B., Li,·· C.-L, Olsson, Y., and K1atzo, 1., The
effect of acute arterial hypertension on the blood-brain
barrier to protein tracers, Acta Neuropathologica, 16 (1970)
117-124. .
28. Joo, F., and Karushina, I., A procedure for the isolation of
capillaries from rat brain, Cytobios, 8 (1973) 41-48.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 493
John W. Olney
from the brain proper in its tendency to accumulate GLU from blood.
By injecting infant mice soc. with GLU, 2 mg/g, we induced a 4-fold
increase in GLU concentrations in the ARH whereas the immediately ad-
jacent ventromedial nucleus and the more remote lateral nucleus of
the thalamus exhibited no appreciable increase in GLU 2 30 It was
also noted that the time course of GLU uptake and accumulation in
ARH paralleled the time course of the neurodegenerative reaction in
that nucleus. We are currently examining threshold conditions for
inducing necrosis of ARH neurons in infant mice and have obtained
preliminary evidence that (1) acute necrosis of ARH neurons occurs
when ARH levels of GLU become 20% elevated and (2) that conditions
for inducing such ARH levels may be either a transient peaking of
plasma GLU to 20-fold resting levels or a more sustained elevation
at lower, yet to be determined levels (Perez, Olney, Martin and
Cannon, unpublished)u
Compound Potency
L-Glutamic Acid I
D-Glutamic Acid* I
L-Aspartic Acid 1
D-Aspartic Acid 1
L-Cysteine Sulfinic Acid* I
L-Cysteic Acid* 1
DL-Homocysteic Acid* 20
N-Methyl-DL-Aspartic Acid 100
Kainic Acid 200
10 0 050 0 35
0
21 1.00 0 080
45 1.50 1.20
60 2000 1.50
The lowest effective dose (LED) at any given age was established by
administering L-GLU either orally or SoCo to mice over a range of
doses and determining histologically the lowest dose which, in > 50%
of the animals treated at that dose (n = 6), produced at least 5 necro-
tic neuronal profiles in a representative section cut through the ARH
at its point of maximal damage (Olney and de Gubareff, unpublished).
Figs 0 2a-c o A small lesion in the ARH of a 21 day-old mouse (a) and
moderate sized lesion in the ARH of a 45 day-old mouse (b) induced by
oral GLU, 1 and 2 mg/g respectivelyo On the right (c) is a small
lesion in the ARH of a 15 day-old mouse 3 hours following oral Aspar-
tame, 205 mg/g. Neurons rendered necrotic (dark centers surrounded
by clear halos) appear identical to those killed by GLU in Figso 2a
and b (X 200)0 (Olney, Rhee, de Gubareff, unpublished)o
CYSTEINE NEUROTOXICITY
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
20. Olney, J.W., Rhee, V. and Ho, O.L., Kainic acid: A powerful
neurotoxic analogue of glutamate. Brain Res. 77 (1974) 507-512.
21. Olney, J.W., Sharpe, L.G. and de Gubareff, T., Excitotoxic
amino acids (Abstr.) Soc. Neurosciences, 1975.
22. Olney, J.W., Sharpe, r:-and Feigin, R., Glutamate induced brain
damage in primates. I. Neuropath. Expo Neurol. 31 (1972) 464-488.
23. Perez, V.J. and Olney, J.W., Accumulation of glutamic acid in
the arcuate nucleus following subcutaneous administration of the
amino acid. J. Neurochem. 19 (1972) 1777-1781.
24. Pollin, W., Cardon, P. and Kety, S., Schizophrenic patients fed
amino acid loads with Iproniazid. Science 175 (1961) 1365-1366.
25. Spaide, J., Davis, J. and Himwich, J., Plasma amino acids in
schizophrenic patients fed methionine or cysteine loads with a
monoamine oxidase inhibitor. Am. I. C1in. Nutr. 24 (1971) 1053-
1059.
26. Stegink, L., Filer, L., Baker, G., Glutamate metabolism in the
neonatal pig. J. Nutr. 103 (1973) 1138-1145.
27. Stegink, L., FiIer~ and Baker, G. Glutamate effect on plasma
and breast milk amino acid levels in lactating women. Proc. Soc.
Exp. !!£!. Med. 149 (1972) 838-841. --
28. Stegink, L., Pitkin, R., Reynolds, W., Filer, L., Boaz, D.,
Brummel, M., Placental transfer of glutamate and its metabolites
in the primate. Am. I. Obstet. i Gyn. 122 (1975) 70-78.
29. Van Harreveld, A. and Fifkova, E., Light and electronmicroscopic
changes in central nervous tissue after electrophoretic injection
of glutamate. Exp. Mo1ec. Patho1. 15 (1971) 61-81.
PHYSIOPATHOLOGY OF THE BLOOD-BRAIN BARRIER
Michael W. Bradbury
Department of Physiology
King's College
London WC2R 2LS
507
508 M.W. BRADBURY
90
TOTAL WATER
I + t
"i
a;
~
ICF
..
OJ 60
~ ......
:§
Cl
0
-------.
~
ECF
"-
E 30 .........
a:
w
f-
«
:;:
0
180 210 240 270
Most workers who have studied the water content of brain during
osmotic disturbances have also estimated the content of the monovalent
ions, chloride, sodium and potassium. As might be expected there is
little net loss or gain of electrolyte during either acute hypona-
traemia or hypernatraemia ll ,15. During the chronic disturbances
there are, however, marked shifts of some or all of the major
electrolytes and no doubt these shifts playa part in volume control.
1
In chronic h pernatraemia sodium and chloride correspondingly
increase in brain 5,7. Again the gain seems just about adequate to
maintain the chloride space constant.
TABLE 1
Hyponatraemia in Rats
NORMAL CHANGE %
SERUM
(mM!l or mOsm)
SKELETAL MUSCLE
(mM!Kg dry wt)
Na 98 + 8 67 ± 9 -32
K 459 ±29 441 ± 18 -4
Swelling +12
BRAIN
TABLE 2
Hypernatraemia in Cats
PLASMA (mM/ 1) :
SKELETAL MUSCLE
(mM/Kg dry st)
Cl 50 + 2 46 ±
2 -8.0
Na 75 + 3 74 ± 6 -1.3
K 433 + 14 420 + 5 -3.0
Swelling -13.6
BRAIN
3·0
CONCLUSION
REFERENCES
15. Holliday, M.A., Kalayci, M.N., and Harrah, J., Factors that
limit brain volume changes in response to acute and sustained
hyper- and hyponatremia, J. Clin. Invest., 47 (1968) 1916-1928.
16. Jelsma, L.F., and Mcqueen, J.D., Effect of water restriction
on brain water, J. Neurosurg.,26 (1967) 35-40.
17. Klatzo, I., Neuropathological aspects of brain oedema,
J. Neuropath. Exp. Neurol., 26 (1967) 1-14.
18. Swinyard, E.A., Effect of extracellular electrolyte depletion
on brain electrolyte pattern and electro-shock seizure threshold.
Amer. J. Physiol., 156 (1949) 163-169.
19. Woodbury, D.M., Effect of acute hyponatremia on distribution
of water and electrolytes in various tissues of the rat,
Amer. J. Physiol., 185 (1956) 281-286.
2.0. Yannet, H., Changes in brain resulting from depletion of
extracellular electrolyte, Amer. J. Physiol., 128 (1940)
683-689.
21. Yudilevich, D.L., and Rose, N. De, Blood-brain transfer of
glucose and other molecules measured by rapid indicator
dilution, Amer. J. Physiol., 220 (1971) 841-846.
BRAIN BARRIER PATHOLOGY IN ACUTE ARTERIAL HYPERTENSION
Barbro Johansson
517
518 B. JOHANSSON
ion pressure 11 •
C Br
300 % I-
200 % I-
100 % I-
Exp. No . I
CONCLUSIONS
REFERENCES
12. Folkow, B., Hallback, M., Lundgren, Y., Sivertsson, R., and
Weiss, L., Importance of adaptive changes in vascular design
for establishment of primary hypertension, studied in man and
in soo~taneously hypertensive rats, Circulat. Res., 32~33:
Supple 1 (1973) 2-13.
13. Folkow, B., and Neil, E., Circulation Oxford University Press,
New York, London, Toronto (1971) p. 46 ff.
14. Frank, 0., Die Elastizitat der Flutgefasse, Z. Biol., 71 (1920)
255-272.
15. Gannushkina, I.V., Shafranova, V.P., Dad iany , L.N., and Ga1ayda,
T.V., Mechanisms related to decrease of CBF during acute in-
crease of arterial pressure in hypertensive animals. In, J.S.
Meyer, H. Lechner, M. Reivich and O. Eichhorn (Eds.) Cerebral
Vascular Disease, 6th International Conference, Saltzburg, 1972,
George Thieme Publishers, Stuttgart (1973) pp. 84-86.
16. Gannushkina, I.V., and Shafranova, V.P., Some aspects of
pathogenesis and pathomorphology of the hypertensive encephalo-
pathy. Abstract. Vllth Internation&l Congress of Neuropathology,
Budapest (1974) p. 100.
17. Giese, J., The pathogenesis of hypertensive vascular disease,
Munksgaard, Copenhagen (1966).
18. Giacomelli, F., Wiener, J., and Spiro, D., The cellular pathology
of experimental hypertension. V. Increased permeability of
cerebral arterial vessels, Amer. J. Path., 59 (1970 133-159.
19. Hansson, H.A., Johansson, B., and Blomstrand, C., Ultrastructural
studies on cerebrovascular permeability in acute hhpertension,
Acta neuropath. (BerlJ, 32 (1975) 187-198.
27. Johansson, B., and Henning, M., The clinical effect of acute
blood pressure increase in awake rats. A comparison between
normotensive and spontaneously hypertensive rats, (to be
published) •
28. Johansson, B., Li, C.-L., Olsson, Y., and Klatzo, I., The
effect of acute arterial hypertension on the blood-brain barrier
to protein tracers, Acta neuropath (Berl.) 16 (1970) 117-124.
29. Johansson, B., and Linder, L.-E., Blood brain barrier dysfunction
in acute arterial hypertension induced by clamping of the
thoracic aorta, Acta neurol. scand. 50 (1974) 360-365.
30. Mathew, N.T., Meyer, J.S.) and Hrastnik, F., Vasospasm
versue ''breakthrough'' in the pathogenesis of hypertensive
encephalopathy, In, M. Harper, B. Jennett, D. Miller and
J. Rowan, Blood Flow and Metabolism in the Brain.
Churchill Livingstone, Edinburgh (1975) p. 5.17-5.21.
31. Okamoto, K., and Aoki, K., Development of a strain of spontaneous-
ly hypertensive rats, Jap. Circulat. J., 27 (1963) 282-293.
32. Okamoto, K., Yamori, Y., and Nagaoka, A., Establishment of the
stroke-prone spontaneously hypertensive rat (SHR), Circulat. Res.
Supple 1 34 (1974) 143-153.
33. Reese, T.S., and Karnovsky, M.J., Fine structural localization
of a blood-brain barrier to exogenous peroxidase, J. Cell Biol.,
34 (1967) 207-217.
34. Robertson, A.L., and Khairallah, P.A., Effects of angiotensin
II and some analogues on vascular permeability in the rabbit,
Circulat. Res. 31 (1972) 923-931.
35. Rodda, R., and Denny-Brown, D., The cerebral arerioles in
experimental hypertension. II. The development of arteriolo-
necrosis, Amer. J. Path. 49 (1966) 365-381.
36. Skinhoj, E., and Strandgaard, S., Pathogenesis of hypertensive
encephalopathy, Lancet, I (1973) 461-462.
37. Smirk, F.H., and Hall, W.H., Inherited hypertension in rats,
Nature (London), 182 (1958) 727-728.
38. Steinwall, 0., and Klatzo, I., Selective vulnerability of the
blood brain barrier in chemically induced lesion, J. Neuropath.
expo Neurol., 25 (1966) 542-559.
39. Strandgaard, S., Olesen, J., Skinhoj, E., and Lassen, N.A.,
Autoregulation of brain circulation in severe arterial hyper-
tension, Brit. med. J.l(1973) 507-510.
40. Strandgaard, S., MacKenzie, E.T., Sengupta, D., Rowan, J.O.,
Lassen, N.A., and Harper, A.M., The upper limit for autoregulat-
ion of cerebral blood flow in the baboon, Circulat. Res., 34
(1974) 435-440.
41. Westergaard, E., Brightman, M.S., Transport of proteins across
normal cerebral arterioles, J. compo Neurol. 152 (1973) 17-44.
42. Westergaard, E., and Br~ndsted, H.E., The effect of acute hyper-
gens ion on the vesicular transport of proteins in cerebral
vessels. Abstract. Vllth International Congress of Neuro-
pathology, Budapt'lst (1974) p. 322.
AUTHOR INDEX
529
SUBJECT INDEX
CHLORIDE DESIPRAMINE
control of brain fluid volume, effect on norepinephrine
510-514 efflux, 300-302
CHOLINE DESMETHYLIMIPRAMINE
high affinity uptake, 199, 205 effects on induced release
uptake kinetics in nerve cell of biogenic amines, 327-329
cultures, 185, 201-204
DEVELOPMENT
COMPARTMENTS changes in amino acid transport,
exchange in vivo and in 408-409
vitro, 402 - - - in enzyme patterns,
405-406
CONVULSIONS in ions, spaces, 407
amino acid uptake 446-449
DIAMINOBUTYRIC ACID
CORPUS STRIATUM effect on GABA exchange, 280
distribution of dopamine uptake effect on glial transport of
in, 339, 340 GABA, 229
synaptosomes, dopamine fluxes
in, 397-402 DOPAMINE
release of amines from, 323- distribution of uptake, in brain,
332 339-341
fluxes in synaptosome "beds",
CORTEX effects of amphetamine, 398-401
cerebral, demonstration of newly synthesized, release from
dopaminergic terminals, 337 synaptosome "beds", 398
distribution of dopamine release from synaptosomes
uptake, 341 effects of amphetamine, 398,
synaptosomes, release of 400, 323, 326
amines, 323-327 effect of K+, 398, 401
effect of 8-phenylethylamine
CRAYFISH derivatives, 330, 331
CNS connective, movements of uptake in cortex, 338, 339
Na+ and K+, 155-161 uptake and release in synapto-
somes, 398, 401
CYSTEINE uptake as an index of dopaminer-
brain damage on oral intake, gic innervation, 338, 341, 342
499, 502
DRUGS
2-DEOXY-GLUCOSE distribution between tissues and
abnormal transport through the CNS and partition coefficients,
barrier, 480, 481, 483-485 106-107, 447-461
altered capillary uptake in uptake mechanisms 447-461
vitro, 489, 490 -
uptake in synaptosomes from
ischemic brains, 485, 486
536 INDEX
ETHANOL GLUCOSE
partition coefficient and analogs, extracellular diffus-
penetration, 108 ion, membrane transport, 269
passage, effect of hypoxia
EVANS BLUE and pC0 2 tension, 484,
abnormal penetration into 486-488
brain, 42-47 uptake in brain slices, 268,
penetration into brain vessels 269
during hypertension, 522 uptake in slices from
ischemic brains, 270
EXTRACELLULAR SPACE depletion, and amino acid
in brain slices, 266, 267,419 transport, 377, 378, 381
after convulsions, 468 influx and efflux in the living
brain, 55-59, 69
GANGLIA kinetics of transport, 134, 135
release and exchange of amino during hypoglycemia, 136
acids in, 171, 172 during anoxia, 136-138
structure and function, 166 relation between transport and
uptake of GABA and glutamate utilization in vivo, 56-59
into, 167-172 transport across the blood-
brain barrier, chemiostatic
GLIA model, 141
buffer for K+, crayfish CNS, 159- comparison with erythrocyte,
161 141-144
vertebrate studies, 152-155 mechanisms, 138-141
function in ganglia, 175 model for translocation, 144,
isolated, accumulation of radio- 145
active amino acids, 223, 224 structure-activity relations,
homoexchange and hetero- 141-144
exchange of GABA, 229, 230 transport, in perfused brain, 133-
metabolic and ionic requir~ 145
ments for amino acid uptake, effect of insulin, 69
226 through brain capillaries,
release of GABA from, 228 81-83
transport of amino acids, 222-
230 GLUTAMATE DECARBOXYLASE
responses to high K+ concentrat- inhibition, relationship to the
ions, 378 onset of convulsions, 386
INDEX 537
TRYPTOPHAN
transport, effect of insulin,
89-93
p-TYRAMINE
effects on the release of amines
from synaptosomes, 329-333
TYROSINE
transport from blood to brain,
effect of insulin, 89-93