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Transport of L-proline by rat brain slices
Brain Research, 102 (1976) 143-151 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
143
T R A N S P O R T OF L - P R O L I N E BY R A T B R A I N SLICES
V. J. BALCAR*, G. A. R. JOHNSTON AND A. L. STEPHANSON
Department of Pharmacology, Australian National University, Canberra (Australia) (Accepted July 8th, 1975)
SUMMARY
L-Proline is taken up into slices of rat cerebral cortex by a structurally specific 'high affinity' system which is absolutely dependent on sodium ions. The system mediating L-proline uptake in homogenates of cerebral cortex is associated with osmotically sensitive particles of the same equilibrium density as synaptosomes. Based on tissue-medium ratios, the uptake of L-proline is most efficient in slices of cerebral cortex and of hypothalamus, and least efficient in slices of cerebellum. L-Proline taken up into slices of cerebral cortex can be released from these slices by an increased potassium ion concentration in a calcium-dependent manner. These observations on L-proline transport into and out of brain slices are consistent with L-proline furtctioning as a synaptJc transmitter in that they are analogous to observations on the transport of transmitter amino acids such as GABA.
INTRODUCTION
There is circumstantial evidence that L-proline may act as an inhibitory transmitter in the mammalian CNS. L-Proline inhibits the firing of neurones in the cerebellum 12 and spinal cord s when administered microelectrophoretically. In the spinal cord but not in the cerebellum, this inhibitory action of e-proline can be antagonised by strychnine. Furthermore L-proline can displace radioactive strychnine bound to membranes isolated from spinal cord z4. L-Proline antagonises the change in transparency of the isolated chick retina induced by L-glutamate 23. A structurally specific, sodium-dependent, 'high affinity' uptake system has been described for L-proline into brain synaptosomes and slices4,6, 20, and the potassium-stimulated release of radioactive L-proline from brain slices has been reported ts. The present study examines * Present address: Centre de Neurochimie, Centre National de la Recherche Scientifique, Strasbourg, France.
144 possible inhibitors, sodium dependence, regional and subcellular distribution of the L-proline uptake system in. and the release of radioactive L-proline ['t'om, rat brutal slices. MATERIALS AND METHODS
Uptake of radioactive L-proline into rat brain slices The effects of inhibitors of the uptake of L-proline were measured using slices (0.1 mm × 0.1 mm ~ 2 mm) of cerebral grey matter from adult Wistav rats incubated at 37 °C in freshly oxygenated Krebs-Ringer mediuma, 4. Inhibitors were preincubated for 15 rain with the slices at 37 °C and uptake of radioactivity measured 4 rain after the addition of tritiated L-proline (0.1 #Ci per incubation vessel, equivalent to 0.006 ,uM exogenous L-proline). Essentially no metabolism of L-proline is observed under these conditions 4. The concentration of inhibitor causing 50% inhibition of uptake of radioactivity (ICs0) was determined by varying the concentration of inhibitor over a range of values. The percentage inhibition found at each concentration was expressed in terms of the probability that it differed from 50 % assuming a normal distribution. A least squares regression analysis was made of these probability data versus the logarithm of the inhibitor concentration to provide an estimate :[: S.E.M. of the inhibitor concentration producing 50 % inhibition of uptake compared with uptake observed in the absence of inhibitor. This analysis agreed with the method of graphic analysis used previously in which the inhibitor concentration was plotted against the per cent inhibition of uptake using log probability graph paper 3. The regional distribution of the proline transport system was studied on slices prepared from the various regions of rat brain dissected according to Glowinski and Iversen 13. The subcellular distribution of radioactive L-proline taken up into subcellular particles of rat cerebral cortex was studied by homogenising the tissue in cold 0.32 M sucrose (l ml/100 mg tissue) under conditions known to yield intact synaptosomes ~6. Nuclei and unbroken cells were removed from this homogenate by centrifugation at 1000 x g for 10 min and samples (0.5 ml, equivalent to 50 mg of original tissue) of the supernatant incubated in medium (10 ml) for 15 rain at 37 °C, then tritiated i.proline (5/~Ci) added and incubation continued for another 4 min. The samples were then rapidly cooled to 4 °C and centrifuged at 100,000 × g for 20 rain. The resulting pellets were washed with cold medium (5 ml) and carefully suspended in cold 0.32 M sucrose (5 ml). Each sample was then layered on to a 20 ml continuous linear density gradient (0.6-1.2 M sucrose) and centrifuged at 50,000 )( g for 120 rain in a swinging bucket rotor. After centrifugation, the tubes were pierced at the bottom and 50 fractions (each of 20 drops) were collected in scintillation vials. Water (t ml) and scintillator (10 ml, Bray's solution) were added and the radioactivity measured. To obtain 'osmotically shocked' preparations the final pellet was suspended in cold water instead of 0.32 M sucrose to lyse osmotically sensitive structures. In several experiments Tris.HCl buffer (50 raM; pH 7.3) was used to replace the sodium phosphate buffer in the Krebs-Ringer solution. The dependence of the
145 uptake of radioactive proline on the sodium ion concentration was then assessed by substituting varying amounts of the sodium chloride in the medium with choline chloride.
Release of radioactive L-proline from rat brain slices The release of radioactive L-proline was studied essentially by the same method used to study the release of radioactive GABA and glycine 1°. Slices of cerebral grey matter were prepared as above and incubated at 37 °C in medium (500 mg of slices in 5 ml of medium) for 15 rain. Radioactive L-proline (0.5 #Ci) was then added and incubation continued for a further 4 rain. The slices were collected by vacuum filtration on glass fibre filter discs (Whatman GF/A, 2.5 cm diameter) and washed with 10 ml of warm medium. The filter discs were transferred rapidly to filter holders (Swinnex 25, Millipore Corp.) and perfused with freshly oxygenated medium at 37 °C at a rate of 0.5 ml/min. After perfusing for 30-35 rain with normal medium, the perfusion medium was changed to 'high potassium' medium containing 44.75 m M potassium chloride instead of 4.75 mM for 10 rain before changing back to normal medium. The extent of metabolism of radioactive L-proline during the course of the release experiments was examined by column and thin layer chromatography. Samples of the perfusate prior to and during the potassium stimulation, and 0.5 N aqueous trichloroacetic acid extracts of the tissue slices at the end of the perfusion were applied to small columns of Dowex 50W × 4, (200-400 mesh), washed with 0.01 N hydrochloric acid and water, then eluted with 2 N ammonia solution. Aliquots of the acid and water washes were counted to determine radioactivity in acidic metabolites. The alkaline eluents were evaporated to dryness in vacuo at 37 °C, resuspended in small volumes of water and analysed by thin layer chromatography on microcrystalline
H
COOH
PROtlNE
NORLEUCINE
OOH
H
CH 3CH2CHzCH21CHCOOH NHz
PIPECOLIC ACID
c.~c.c.,c.coo, 2-AMINO-5-CHLORO4.HEXENOIC ACID
CH3CH'CH ~ICHCOOH NHz
AZETIDINE-2-CARBOXYLIC ACID
CROTYLGLYCINE
~COOH H
NORVALINE
BAIKIAIN
c.~c.c.,c.coo.
(c.~),c.c.,,c,coo.
ALLYLGLYCINE
COOH
H
CH 3CI"IccCHCH 2CHCOOH NH2
H
CH2CHzCH)
CONIINE
LEUCINE
CH z~ .CCH2.CHCOOH C;H3 ~IH~
y-METHALLYLGLYCiNE
Fig. 1. Structures of proline and some of the substances that inhibit the uptake of radioactive proline into rat brain slices.
146 TABLE l I N H I B 1 T O R S O F T H E U P T A K E O F R A D I O A ( ' T I V E I , - P R O L I N E IN R A T B R A I N S L I C E S
Slices of cerebral cortical grey matter were preincubated with inhibitor (0.1 mM) for 15 rain, a|ld uptake measured after incubation for a further 4 min with radioactive L-proline (0.006/~M). Results for ~ inhibition are mean values ! S.E. of quadruplicate experiments. Results for the ICs~ vatuc.s were calculated as described in the text.
Inhibitor
Inhibition (°,oJ
IC.~o(ltM)
L-Norleucine 94 :i 5 20 ± 2 DL-Crotylglycine 86 :!: 7 42 ± 7 DL-Pipecolicacid 91 :[:: 2 44l: 3 L-Norvaline 64 :i: 2 53 ± 7 L-Baikiain 00 :: 4 55 :[: 6 DL-2-Amino-5-chloro-4-hexenoicacid 55 ~ 9 53 ~ 8 DL-Vinylglycine 54 ! 10 DL-Allylglycine 52 i: 3 61 :- 6 L-Azaserine 45 :: 2 DL-2-Amino-4-hexynoic acid 44 :: 3 4-n-Pentylpiperidine 44 3 t-Leucine 41 ]. 4 94± 9 t.-Azetidine-2-carboxylic acid 38 5 155 3_- 41 oL-Coniine 386 oL-Thioproline 35 : 5 L-2-Amino-4,5-hexadienoic acid 35 ~- 5 OL-7-Methallylglycine 31 _ 10 203 :}_ 26 DL-Cycloleucine 31 ! 2 DL-3,4-Dehydroproline 29 :L 5 L-5-Hydroxypipecolic acid 28 ~- 3 4-n-Butylpiperidine 24 :!: 3 The following were inactive at 0.1 mM: o-cycloserine, L-glutamine, helodine, l,hydroxy-3-amino-2pyrrolidone, DL-fl-hydroxyvaline, kainic acid, kojic acid, pemoline, o-proline.
cellulose (Avicel) plates developed with n-butanol-acetone-diethytamine-water (20:20:3:10) or n-butanol-acetic acid-water (8:5:1). Sources o f compounds
[5-3HJL-Proline, specific activity 18 Ci/mmole, was purchased from The Radiochemical Centre, Amersham. De-2-Amino-5-chloro-4-hexenoic acid, e-2-amino-4,5-hexadienoic acid, DL-2amino-4-hexynoic acid and helodine were gifts from Dr. W. S. Chilton, Seattle. DLVinylglycine was a gift from Dr. P. Friis, Copenhagen. 4-n-Butyl- and 4-n-pentylpiperidine were prepared by Mr. B. Twitchin. Pemoline and 1-hydroxy-3-amino-2pyrrolidone were prepared according to the procedures of Traube and Ascher 22 and Smrt et al. 2t respectively. All other substances were purchased from commercial suppliers. RESULTS
Inhibitors of the uptake of radioactive L-proline A variety of amino acids structurally related to L-proline (Fig. t) inhibited the
147 TABLE II T I S S U E - - M E D I U M RATIOS F O R T H E U P T A K E OF R A D I O A C T I V E L - P R O L I N E I N SLICES OF V A R I O U S R E G I O N S OF RAT BRAIN
The uptake of radioactive L-proline (0.006/zM) was measured over 4 rain at 37 °C for slices of rat brain dissected according to Glowinski and Iversen 13.
Region
Tissue-medium ratio ( ~: S.E. for quadruplicate experiments)
Cerebral cortex Hypothalamus Midbrain Striatum Medulla Cerebellum
6.50 ± 0.57 5.04 -5=0.60 3.50 ± 0.65 2.78 5z 0.53 1.76 ± 0.17 1.41 ± 0.09
uptake o f radioactive L-proline (Table I). The most active inhibitors were the open chain c o m p o u n d s L-norleucine and its dehydro derivative DL-crotylglycine. A n u m b e r o f cyclic c o m p o u n d s also inhibited L-proline uptake with DL-pipecolic acid being the most potent. Three piperidine derivatives (coniine, 4-n-pentylpiperidine and 4-nbutylpiperidine), which are not amino acids, inhibited L-proline uptake.
Regional distribution of L-proline uptake Tissue-medium ratios were measured after 4 min at 37 °C in the presence o f 0.006 # M radioactive e-proline, for slices prepared f r o m various regions o f rat brain. The highest tissue-medium ratios measured under these conditions were for slices of cerebral cortex and o f hypothalamus, and the lowest for slices o f cerebellum (Table II).
>- 20' > U
L-PROLINE UPTAKE INTO S U B C E L L U L A RPARTICLES OF "HOMOGENISED RAT CEREBRAL CORTEX
< lS. O O
<
10-
u0
~=
5-
"~...o o ~'~'~.-.~.~--~--=~ o
0.32 0.6
0".8 1,0 SUCROSE MOLARITY
.~.~a.~a~t~t~ .......
172
Fig. 2. Fractionation on a linear sucrose gradient (0.6-1.2 M sucrose) of radioactive L-proline accumulated by subcellular particles in a sucrose homogenate of rat cerebral cortex.Closed symbols: particles suspended in 0.32 M sucrose prior to density gradient centrifugation. Open symbols: particles suspended in water to lyse osmotically sensitive structures.
148
4. O
7 :E
|
3.
|
KE
OF RADIOACTIVE L-PROLINE
I--
6
20
;o
SODIUM I O N
go
do
;60
,~o
C O N C E N T R A T I O N (raM)
Fig. 3. Plot of tissue-medium ratio of radioactive L-proline versusthe sodium ion concentration in the incubation medium. Points are means ~ S.E. of quadruplicate experiments.
Subcellular distribution of accumulated radioactivity A major portion of the radioactivity taken up into subcellular particles of sucrose homogenates of rat cerebral cortex incubated with radioactive L-proline was associated with particles of equilibrium density equivalent to that of 0.9 M sucrose (Fig. 2). These particles were osmotically sensitive in that suspension in water instead of in 0.32 M sucrose prior to density gradient centrifugation resulted in a loss of radioactivity from these particles.
Sodium dependence of L-proline uptake The dependence of the uptake of radioactive L-proline into slices of cerebral cortex on the external sodium ion concentration was studied over the range 0-118 mM. L-Proline uptake was completely inhibited by lowering the sodium ion concentration below 10 m M (Fig. 3).
Release of radioactive L-proline The rate of release of radioactivity from slices of rat cerebral cortex preloaded with radioactive L-proline could be increased 102 ~ 1 2 ~ (peak percentage increase over immediate prestimulation release; mean -+- S.E. of 5 experiments) by raising the potassium concentration in the perfusing medium from 4.75 to 44.75 m M (Fig. 4). The effect of increased potassium concentration could be reduced to 29 ± 6 % by omission of calcium from the perfusing medium. These observations are similar to those of Mulder and Snyder 18 who found 104 ~ 18 % and 52 ± 2 % potassium stimulated release in the presence and absence of calcium respectively using thicker slices of rat cerebral cortex (0.5 m m × 1 mm × 1 mm). In the present experiments, the potassium-stimulated release of radioactive L-proline was less than that observed for radioactive GABA (505 :k 54 ~ ) under the same conditions but significantly higher than
149
2°3'22J2°;'2EX
1500,
100
g
~I"
"'c°~, I I_LI\
5@
high K+ FRACTION N U M B E R 2
4
6
8
10
N;IX,'Illl;I;tH$1#,
12
14
16
18
20
Fig. 4. Release o f radioactive L-proline from slices of rat cerebral cortex into the perfusion medium. Closed symbols: perfusion with control medium containing 1.77 m M CaC12. Open symbols: perfusion with medium containing no added calcium ions. Fractions of perfusate were collected every 2.8 rain; 'high K +' refers to the potassium concentration in the perfusion medium being increased to 44.75 m M for the period indicated by the broken lines. Points are means ± S.E. of 5 experiments.
that observed for radioactive glycine (34 :k 4 ~) or 2-amino-isobutyric acid (8 ~o :k 1) (ref. 10). The tissue retained 25 ~ of its initial radioactivity at the end of the perfusion experiments and more than 80 ~ of the retained radioactivity could be extracted with trichloroacetic acid and ran with authentic L-proline on thin layer chromatography. Similarly more than 80 ~ of the radioactivity in the perfusate prior to and during the potassium stimulation could be attributed to L-proline. DISCUSSION
The uptake of transmitter amino acids, such as GABA and L-glutamate, into brain slices and synaptosomal preparations appears to be characterised by being of a 'high affinity' type, i.e. having an apparent Km of the order of 10 #M, being absolutely dependent on sodium ions and which transports the amino acid into a compartment from which it can be released by potassium stimulation in a calcium-dependent manner as. Studies on the uptake and release of e-proline indicate that this amino acid is in these respects analogous to the transmitter amino acids. For this and other reasons 12 L-proline is being investigated as a possible transmitter in the mammalian CNS. The apparent efficiency of L-proline uptake shows marked regional variation. There are no available data regarding regional variation in the endogenous levels of L-proline in adult rat brain, so that it is not possible to judge if the apparent efficiency of uptake correlates with endogenous levels. In 10-day-old rats, the highest brain level of L-proline is found in the cerbral cortex~'(0.4/~mole/g) with lower levels in the cere-
150 bellum (10.3 #mole/g), midbrain and pons-medulla (0.2 #mole/g) 9, but the proline level~ are known to be lower in adult rat brain (0. l #mole/g) 1. Studies on the regional variation of glycine levels and uptake efficiency'',r~,t9 indicate a direct correlation between levels and uptake, and are in agreement with physiological evidence that glycine is more important as art inhibitory transmitter in the spinal cord and ports-medulla than in other CNS regionsL The present studies on the uptake of e-proline indicate that the cerebral cortex and hypothalamus are likely to be the brain regions most deserving of investigation with respect to a possible transmitter role for this amino acid. The cerebellum appears to be the least efficient brain region with respect to L-proline uptake, nonetheless microelectrophoretic and autoradiographic studies have suggested that L-proline could be an inhibitory transmitter in the cerebellumlL While the uptake of E-proline appears to be relatively structurally specific, there are interactions between L-proline uptake and the uptake of other amino acids. L-Proline (0. I raM) weakly inhibits (22 ~/,,) the 'high affinity' uptake of L-glutamine '~ whereas L-glutamine (0.1 m M ) does not inhibit the uptake of L-proline. L-Leucine (0.1 m M ) inhibits the uptake of L-proline (Table I) but L-proline (0.1 raM) does not inhibit the uptake of L-leucine 4. G A B A and nipecotic acid, a potent inhibitor of G A B A uptake, are weak inhibitors (18°/,i at 1 raM) and L-proline is a similarly weak inhibitor (18°~ at I raM) of G A B A uptake 17. The uptake of L-proline is more sensitive to inhibition by allylglycine and amino-oxyacetic acid than is the uptake of a variety of amino acids and amines ~,j~. Like allylglycine, 3 of the inhibitors of L-proline uptake described in the present study are convulsants; these are the piperidine derivatives coniine, 4-n-butylpiperidine and 4-n-pentylpiperidineV,lL These convulsants and some of the other inhibitors of L-proline uptake may be useful in further experiments directed towards assessing the possible role of L-proline as a transmitter. ACKNOWLEDGEMENTS
The authors are grateful to Drs. Chilton and Friis for gifts of chemicals, to Dr. C. J. A. G a m e for advice, and to Mr. B. Twitchin for assistance.
REFERENCES 1 AGRAWAL, H. C., DAVIS, J. M., AND HIMWICH, W. A., Postnatal changes in free amino acid pool
of rat brain, J. Neurochem., 13 (1966) 607-615. 2 APRISON,M. H., SHANK,R. P., ANO DAVIDOFF,R. A., A comparison of the concentration of glycine, a transmitter suspect, in different areas of the brain and spinal cord in seven different vertebrates, Comp. Biochem. PhysioL, 28 (1969) 1345-1355. 3 BALCAR, V. J., AND JOHNSTON, G. A. R., The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 4 BALCAR, V. J., AND JOHNSTON, G. A. R., Allylglycine, an inhibitor of the uptake of L-leucine and L-proline in rat brain slices, Biochem, PharmacoL, 23 (1974) 821-827. 5 BALCAR,V. J., ANDJOHNSTON,G. A. R., High affinity uptake of L-glutamine in rat brain slices, J. Neurochem., 24 (1975) 875-879. 6 BENNETT,J. P., LOGAN,W. J., AND SNYDER,S. H., Amino acid neurotransmitter candidates: sodium-dependent high-affinity uptake by unique synaptosomal fractions, Science, 178 (1972) 997-999.
151 7 BOWMAN,W. C., AND SANGHVI, I. S., Pharmacological actions of hemlock (Conium macu&tum) alkaloids, J. Pharm. Pharmacol., 15 (1963) 1-24. 8 CURTIS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 9 DAVIES, L. P., AND JOHNSTON, G. A. R., Postnatal changes in the activities of serine hydroxymethyltransferase and glycine 2-oxoglutarate amino transferase in the rat central nervous system, J. Neurochem., 22 (1974) 107-112. 10 DAVIES, L. P., JOHNSTON, G. A. R., AND STEPHANSON, A. L., Postnatal changes in the potassiumstimulated calcium-dependent release of radioactive GABA and glycine from slices of rat central nervous tissue, J. Neurochem., 25 (1975) 387-392. 11 ESPLIN, D. W., AND ZABLOCKA-ESPLIN, B., Mechanisms of action ofconvulsants. In H. H. JASPER, A. A. WARD AND A. POPE (Eds.), Basic Mechanisms of the Epilepsies, Little Brown and Co., Boston, Mass., 1969, pp. 167-193. 12 FELIX, D., AND KONZLE, H., Iontophoretic and autoradiographic studies on the role of proline in nervous transmission, Pfliigers Arch. ges. Physiol., 350 (1974) 135-144. 13 GLOWlNSKL J., AND IVERSEN, L. L., Regional studies of catecholamines in the rat brain - - 1. The disposition of [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 14 JOHNSTON, G. A. R., AND BALCAR, V. J., Amino-oxyacetic acid: a relatively non-specific inhibitor of uptake of amino acids and amines by brain and spinal cord, J. Neurochem., 22 (1974) 609 610. 15 JOHNSTON, G. A. R., AND IVERSEN, L. L., Glycine uptake in rat central nervous system slices and homogenates: evidence for different uptake systems in spinal cord and cerebral cortex, J. Neurochem., 18 (1971) 1951-1961. 16 JOHNSTON, G. A. R., LLOYD, H. J., AND STONE, N., Liquid cation exchange extraction of cholinomimetic activity from brain, J. Neurochem., 15 (1968) 361-365. 17 KROGSGAARD-LARSEN,P., AND JOHNSTON, G. A. R., Inhibition of G A B A uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., in press. 18 MULDER, A. H., AND SNYDER, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308. 19 NEAL, M. J., The uptake of [14C]glycine by slices of mammalian spinal cord, J. Physiol. (Lond.), 215 (1971) 103-117. 20 PETERSON,N. A., AND RAGHUPATHY, E., Characteristics of amino acid accumulation by synaptosomal particles isolated from rat brain, J. Neurochem., 19 (1972) 1423-1438. 21 SMRT, J., BER~NEK,J., UND HOR.~,K, M., N-Hydroxypyrrolidon-(2), ein neuer Pyrrolidonderivattyp, Coll. Czech. chem. Commun., 24 (1959) 1672-1676. 22 TRAUaE, W., UNO ASCHER, R., [3ber das Isohydantoin 2-Imino-4-keto-tetrahydro-oxazol und seine Homologen, Chem. Ber., 46 (1913) 2077-2084. 23 VAN HARREVELD, A., AND FIFKOVA,E., Effects of amino acids on the isolated chicken retina, and on its response to glutamate stimulation, J. Neurochem., 20 (1973) 947-962. 24 YOUNG, A. B., AND SNYDER, S. H., Strychnine binding associated with glycine receptors of the central nervous system, Proe. nat. Aead. Sci. (Wash.), 70 (1973) 2832-2836.
143
T R A N S P O R T OF L - P R O L I N E BY R A T B R A I N SLICES
V. J. BALCAR*, G. A. R. JOHNSTON AND A. L. STEPHANSON
Department of Pharmacology, Australian National University, Canberra (Australia) (Accepted July 8th, 1975)
SUMMARY
L-Proline is taken up into slices of rat cerebral cortex by a structurally specific 'high affinity' system which is absolutely dependent on sodium ions. The system mediating L-proline uptake in homogenates of cerebral cortex is associated with osmotically sensitive particles of the same equilibrium density as synaptosomes. Based on tissue-medium ratios, the uptake of L-proline is most efficient in slices of cerebral cortex and of hypothalamus, and least efficient in slices of cerebellum. L-Proline taken up into slices of cerebral cortex can be released from these slices by an increased potassium ion concentration in a calcium-dependent manner. These observations on L-proline transport into and out of brain slices are consistent with L-proline furtctioning as a synaptJc transmitter in that they are analogous to observations on the transport of transmitter amino acids such as GABA.
INTRODUCTION
There is circumstantial evidence that L-proline may act as an inhibitory transmitter in the mammalian CNS. L-Proline inhibits the firing of neurones in the cerebellum 12 and spinal cord s when administered microelectrophoretically. In the spinal cord but not in the cerebellum, this inhibitory action of e-proline can be antagonised by strychnine. Furthermore L-proline can displace radioactive strychnine bound to membranes isolated from spinal cord z4. L-Proline antagonises the change in transparency of the isolated chick retina induced by L-glutamate 23. A structurally specific, sodium-dependent, 'high affinity' uptake system has been described for L-proline into brain synaptosomes and slices4,6, 20, and the potassium-stimulated release of radioactive L-proline from brain slices has been reported ts. The present study examines * Present address: Centre de Neurochimie, Centre National de la Recherche Scientifique, Strasbourg, France.
144 possible inhibitors, sodium dependence, regional and subcellular distribution of the L-proline uptake system in. and the release of radioactive L-proline ['t'om, rat brutal slices. MATERIALS AND METHODS
Uptake of radioactive L-proline into rat brain slices The effects of inhibitors of the uptake of L-proline were measured using slices (0.1 mm × 0.1 mm ~ 2 mm) of cerebral grey matter from adult Wistav rats incubated at 37 °C in freshly oxygenated Krebs-Ringer mediuma, 4. Inhibitors were preincubated for 15 rain with the slices at 37 °C and uptake of radioactivity measured 4 rain after the addition of tritiated L-proline (0.1 #Ci per incubation vessel, equivalent to 0.006 ,uM exogenous L-proline). Essentially no metabolism of L-proline is observed under these conditions 4. The concentration of inhibitor causing 50% inhibition of uptake of radioactivity (ICs0) was determined by varying the concentration of inhibitor over a range of values. The percentage inhibition found at each concentration was expressed in terms of the probability that it differed from 50 % assuming a normal distribution. A least squares regression analysis was made of these probability data versus the logarithm of the inhibitor concentration to provide an estimate :[: S.E.M. of the inhibitor concentration producing 50 % inhibition of uptake compared with uptake observed in the absence of inhibitor. This analysis agreed with the method of graphic analysis used previously in which the inhibitor concentration was plotted against the per cent inhibition of uptake using log probability graph paper 3. The regional distribution of the proline transport system was studied on slices prepared from the various regions of rat brain dissected according to Glowinski and Iversen 13. The subcellular distribution of radioactive L-proline taken up into subcellular particles of rat cerebral cortex was studied by homogenising the tissue in cold 0.32 M sucrose (l ml/100 mg tissue) under conditions known to yield intact synaptosomes ~6. Nuclei and unbroken cells were removed from this homogenate by centrifugation at 1000 x g for 10 min and samples (0.5 ml, equivalent to 50 mg of original tissue) of the supernatant incubated in medium (10 ml) for 15 rain at 37 °C, then tritiated i.proline (5/~Ci) added and incubation continued for another 4 min. The samples were then rapidly cooled to 4 °C and centrifuged at 100,000 × g for 20 rain. The resulting pellets were washed with cold medium (5 ml) and carefully suspended in cold 0.32 M sucrose (5 ml). Each sample was then layered on to a 20 ml continuous linear density gradient (0.6-1.2 M sucrose) and centrifuged at 50,000 )( g for 120 rain in a swinging bucket rotor. After centrifugation, the tubes were pierced at the bottom and 50 fractions (each of 20 drops) were collected in scintillation vials. Water (t ml) and scintillator (10 ml, Bray's solution) were added and the radioactivity measured. To obtain 'osmotically shocked' preparations the final pellet was suspended in cold water instead of 0.32 M sucrose to lyse osmotically sensitive structures. In several experiments Tris.HCl buffer (50 raM; pH 7.3) was used to replace the sodium phosphate buffer in the Krebs-Ringer solution. The dependence of the
145 uptake of radioactive proline on the sodium ion concentration was then assessed by substituting varying amounts of the sodium chloride in the medium with choline chloride.
Release of radioactive L-proline from rat brain slices The release of radioactive L-proline was studied essentially by the same method used to study the release of radioactive GABA and glycine 1°. Slices of cerebral grey matter were prepared as above and incubated at 37 °C in medium (500 mg of slices in 5 ml of medium) for 15 rain. Radioactive L-proline (0.5 #Ci) was then added and incubation continued for a further 4 rain. The slices were collected by vacuum filtration on glass fibre filter discs (Whatman GF/A, 2.5 cm diameter) and washed with 10 ml of warm medium. The filter discs were transferred rapidly to filter holders (Swinnex 25, Millipore Corp.) and perfused with freshly oxygenated medium at 37 °C at a rate of 0.5 ml/min. After perfusing for 30-35 rain with normal medium, the perfusion medium was changed to 'high potassium' medium containing 44.75 m M potassium chloride instead of 4.75 mM for 10 rain before changing back to normal medium. The extent of metabolism of radioactive L-proline during the course of the release experiments was examined by column and thin layer chromatography. Samples of the perfusate prior to and during the potassium stimulation, and 0.5 N aqueous trichloroacetic acid extracts of the tissue slices at the end of the perfusion were applied to small columns of Dowex 50W × 4, (200-400 mesh), washed with 0.01 N hydrochloric acid and water, then eluted with 2 N ammonia solution. Aliquots of the acid and water washes were counted to determine radioactivity in acidic metabolites. The alkaline eluents were evaporated to dryness in vacuo at 37 °C, resuspended in small volumes of water and analysed by thin layer chromatography on microcrystalline
H
COOH
PROtlNE
NORLEUCINE
OOH
H
CH 3CH2CHzCH21CHCOOH NHz
PIPECOLIC ACID
c.~c.c.,c.coo, 2-AMINO-5-CHLORO4.HEXENOIC ACID
CH3CH'CH ~ICHCOOH NHz
AZETIDINE-2-CARBOXYLIC ACID
CROTYLGLYCINE
~COOH H
NORVALINE
BAIKIAIN
c.~c.c.,c.coo.
(c.~),c.c.,,c,coo.
ALLYLGLYCINE
COOH
H
CH 3CI"IccCHCH 2CHCOOH NH2
H
CH2CHzCH)
CONIINE
LEUCINE
CH z~ .CCH2.CHCOOH C;H3 ~IH~
y-METHALLYLGLYCiNE
Fig. 1. Structures of proline and some of the substances that inhibit the uptake of radioactive proline into rat brain slices.
146 TABLE l I N H I B 1 T O R S O F T H E U P T A K E O F R A D I O A ( ' T I V E I , - P R O L I N E IN R A T B R A I N S L I C E S
Slices of cerebral cortical grey matter were preincubated with inhibitor (0.1 mM) for 15 rain, a|ld uptake measured after incubation for a further 4 min with radioactive L-proline (0.006/~M). Results for ~ inhibition are mean values ! S.E. of quadruplicate experiments. Results for the ICs~ vatuc.s were calculated as described in the text.
Inhibitor
Inhibition (°,oJ
IC.~o(ltM)
L-Norleucine 94 :i 5 20 ± 2 DL-Crotylglycine 86 :!: 7 42 ± 7 DL-Pipecolicacid 91 :[:: 2 44l: 3 L-Norvaline 64 :i: 2 53 ± 7 L-Baikiain 00 :: 4 55 :[: 6 DL-2-Amino-5-chloro-4-hexenoicacid 55 ~ 9 53 ~ 8 DL-Vinylglycine 54 ! 10 DL-Allylglycine 52 i: 3 61 :- 6 L-Azaserine 45 :: 2 DL-2-Amino-4-hexynoic acid 44 :: 3 4-n-Pentylpiperidine 44 3 t-Leucine 41 ]. 4 94± 9 t.-Azetidine-2-carboxylic acid 38 5 155 3_- 41 oL-Coniine 386 oL-Thioproline 35 : 5 L-2-Amino-4,5-hexadienoic acid 35 ~- 5 OL-7-Methallylglycine 31 _ 10 203 :}_ 26 DL-Cycloleucine 31 ! 2 DL-3,4-Dehydroproline 29 :L 5 L-5-Hydroxypipecolic acid 28 ~- 3 4-n-Butylpiperidine 24 :!: 3 The following were inactive at 0.1 mM: o-cycloserine, L-glutamine, helodine, l,hydroxy-3-amino-2pyrrolidone, DL-fl-hydroxyvaline, kainic acid, kojic acid, pemoline, o-proline.
cellulose (Avicel) plates developed with n-butanol-acetone-diethytamine-water (20:20:3:10) or n-butanol-acetic acid-water (8:5:1). Sources o f compounds
[5-3HJL-Proline, specific activity 18 Ci/mmole, was purchased from The Radiochemical Centre, Amersham. De-2-Amino-5-chloro-4-hexenoic acid, e-2-amino-4,5-hexadienoic acid, DL-2amino-4-hexynoic acid and helodine were gifts from Dr. W. S. Chilton, Seattle. DLVinylglycine was a gift from Dr. P. Friis, Copenhagen. 4-n-Butyl- and 4-n-pentylpiperidine were prepared by Mr. B. Twitchin. Pemoline and 1-hydroxy-3-amino-2pyrrolidone were prepared according to the procedures of Traube and Ascher 22 and Smrt et al. 2t respectively. All other substances were purchased from commercial suppliers. RESULTS
Inhibitors of the uptake of radioactive L-proline A variety of amino acids structurally related to L-proline (Fig. t) inhibited the
147 TABLE II T I S S U E - - M E D I U M RATIOS F O R T H E U P T A K E OF R A D I O A C T I V E L - P R O L I N E I N SLICES OF V A R I O U S R E G I O N S OF RAT BRAIN
The uptake of radioactive L-proline (0.006/zM) was measured over 4 rain at 37 °C for slices of rat brain dissected according to Glowinski and Iversen 13.
Region
Tissue-medium ratio ( ~: S.E. for quadruplicate experiments)
Cerebral cortex Hypothalamus Midbrain Striatum Medulla Cerebellum
6.50 ± 0.57 5.04 -5=0.60 3.50 ± 0.65 2.78 5z 0.53 1.76 ± 0.17 1.41 ± 0.09
uptake o f radioactive L-proline (Table I). The most active inhibitors were the open chain c o m p o u n d s L-norleucine and its dehydro derivative DL-crotylglycine. A n u m b e r o f cyclic c o m p o u n d s also inhibited L-proline uptake with DL-pipecolic acid being the most potent. Three piperidine derivatives (coniine, 4-n-pentylpiperidine and 4-nbutylpiperidine), which are not amino acids, inhibited L-proline uptake.
Regional distribution of L-proline uptake Tissue-medium ratios were measured after 4 min at 37 °C in the presence o f 0.006 # M radioactive e-proline, for slices prepared f r o m various regions o f rat brain. The highest tissue-medium ratios measured under these conditions were for slices of cerebral cortex and o f hypothalamus, and the lowest for slices o f cerebellum (Table II).
>- 20' > U
L-PROLINE UPTAKE INTO S U B C E L L U L A RPARTICLES OF "HOMOGENISED RAT CEREBRAL CORTEX
< lS. O O
<
10-
u0
~=
5-
"~...o o ~'~'~.-.~.~--~--=~ o
0.32 0.6
0".8 1,0 SUCROSE MOLARITY
.~.~a.~a~t~t~ .......
172
Fig. 2. Fractionation on a linear sucrose gradient (0.6-1.2 M sucrose) of radioactive L-proline accumulated by subcellular particles in a sucrose homogenate of rat cerebral cortex.Closed symbols: particles suspended in 0.32 M sucrose prior to density gradient centrifugation. Open symbols: particles suspended in water to lyse osmotically sensitive structures.
148
4. O
7 :E
|
3.
|
KE
OF RADIOACTIVE L-PROLINE
I--
6
20
;o
SODIUM I O N
go
do
;60
,~o
C O N C E N T R A T I O N (raM)
Fig. 3. Plot of tissue-medium ratio of radioactive L-proline versusthe sodium ion concentration in the incubation medium. Points are means ~ S.E. of quadruplicate experiments.
Subcellular distribution of accumulated radioactivity A major portion of the radioactivity taken up into subcellular particles of sucrose homogenates of rat cerebral cortex incubated with radioactive L-proline was associated with particles of equilibrium density equivalent to that of 0.9 M sucrose (Fig. 2). These particles were osmotically sensitive in that suspension in water instead of in 0.32 M sucrose prior to density gradient centrifugation resulted in a loss of radioactivity from these particles.
Sodium dependence of L-proline uptake The dependence of the uptake of radioactive L-proline into slices of cerebral cortex on the external sodium ion concentration was studied over the range 0-118 mM. L-Proline uptake was completely inhibited by lowering the sodium ion concentration below 10 m M (Fig. 3).
Release of radioactive L-proline The rate of release of radioactivity from slices of rat cerebral cortex preloaded with radioactive L-proline could be increased 102 ~ 1 2 ~ (peak percentage increase over immediate prestimulation release; mean -+- S.E. of 5 experiments) by raising the potassium concentration in the perfusing medium from 4.75 to 44.75 m M (Fig. 4). The effect of increased potassium concentration could be reduced to 29 ± 6 % by omission of calcium from the perfusing medium. These observations are similar to those of Mulder and Snyder 18 who found 104 ~ 18 % and 52 ± 2 % potassium stimulated release in the presence and absence of calcium respectively using thicker slices of rat cerebral cortex (0.5 m m × 1 mm × 1 mm). In the present experiments, the potassium-stimulated release of radioactive L-proline was less than that observed for radioactive GABA (505 :k 54 ~ ) under the same conditions but significantly higher than
149
2°3'22J2°;'2EX
1500,
100
g
~I"
"'c°~, I I_LI\
5@
high K+ FRACTION N U M B E R 2
4
6
8
10
N;IX,'Illl;I;tH$1#,
12
14
16
18
20
Fig. 4. Release o f radioactive L-proline from slices of rat cerebral cortex into the perfusion medium. Closed symbols: perfusion with control medium containing 1.77 m M CaC12. Open symbols: perfusion with medium containing no added calcium ions. Fractions of perfusate were collected every 2.8 rain; 'high K +' refers to the potassium concentration in the perfusion medium being increased to 44.75 m M for the period indicated by the broken lines. Points are means ± S.E. of 5 experiments.
that observed for radioactive glycine (34 :k 4 ~) or 2-amino-isobutyric acid (8 ~o :k 1) (ref. 10). The tissue retained 25 ~ of its initial radioactivity at the end of the perfusion experiments and more than 80 ~ of the retained radioactivity could be extracted with trichloroacetic acid and ran with authentic L-proline on thin layer chromatography. Similarly more than 80 ~ of the radioactivity in the perfusate prior to and during the potassium stimulation could be attributed to L-proline. DISCUSSION
The uptake of transmitter amino acids, such as GABA and L-glutamate, into brain slices and synaptosomal preparations appears to be characterised by being of a 'high affinity' type, i.e. having an apparent Km of the order of 10 #M, being absolutely dependent on sodium ions and which transports the amino acid into a compartment from which it can be released by potassium stimulation in a calcium-dependent manner as. Studies on the uptake and release of e-proline indicate that this amino acid is in these respects analogous to the transmitter amino acids. For this and other reasons 12 L-proline is being investigated as a possible transmitter in the mammalian CNS. The apparent efficiency of L-proline uptake shows marked regional variation. There are no available data regarding regional variation in the endogenous levels of L-proline in adult rat brain, so that it is not possible to judge if the apparent efficiency of uptake correlates with endogenous levels. In 10-day-old rats, the highest brain level of L-proline is found in the cerbral cortex~'(0.4/~mole/g) with lower levels in the cere-
150 bellum (10.3 #mole/g), midbrain and pons-medulla (0.2 #mole/g) 9, but the proline level~ are known to be lower in adult rat brain (0. l #mole/g) 1. Studies on the regional variation of glycine levels and uptake efficiency'',r~,t9 indicate a direct correlation between levels and uptake, and are in agreement with physiological evidence that glycine is more important as art inhibitory transmitter in the spinal cord and ports-medulla than in other CNS regionsL The present studies on the uptake of e-proline indicate that the cerebral cortex and hypothalamus are likely to be the brain regions most deserving of investigation with respect to a possible transmitter role for this amino acid. The cerebellum appears to be the least efficient brain region with respect to L-proline uptake, nonetheless microelectrophoretic and autoradiographic studies have suggested that L-proline could be an inhibitory transmitter in the cerebellumlL While the uptake of E-proline appears to be relatively structurally specific, there are interactions between L-proline uptake and the uptake of other amino acids. L-Proline (0. I raM) weakly inhibits (22 ~/,,) the 'high affinity' uptake of L-glutamine '~ whereas L-glutamine (0.1 m M ) does not inhibit the uptake of L-proline. L-Leucine (0.1 m M ) inhibits the uptake of L-proline (Table I) but L-proline (0.1 raM) does not inhibit the uptake of L-leucine 4. G A B A and nipecotic acid, a potent inhibitor of G A B A uptake, are weak inhibitors (18°/,i at 1 raM) and L-proline is a similarly weak inhibitor (18°~ at I raM) of G A B A uptake 17. The uptake of L-proline is more sensitive to inhibition by allylglycine and amino-oxyacetic acid than is the uptake of a variety of amino acids and amines ~,j~. Like allylglycine, 3 of the inhibitors of L-proline uptake described in the present study are convulsants; these are the piperidine derivatives coniine, 4-n-butylpiperidine and 4-n-pentylpiperidineV,lL These convulsants and some of the other inhibitors of L-proline uptake may be useful in further experiments directed towards assessing the possible role of L-proline as a transmitter. ACKNOWLEDGEMENTS
The authors are grateful to Drs. Chilton and Friis for gifts of chemicals, to Dr. C. J. A. G a m e for advice, and to Mr. B. Twitchin for assistance.
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of rat brain, J. Neurochem., 13 (1966) 607-615. 2 APRISON,M. H., SHANK,R. P., ANO DAVIDOFF,R. A., A comparison of the concentration of glycine, a transmitter suspect, in different areas of the brain and spinal cord in seven different vertebrates, Comp. Biochem. PhysioL, 28 (1969) 1345-1355. 3 BALCAR, V. J., AND JOHNSTON, G. A. R., The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 4 BALCAR, V. J., AND JOHNSTON, G. A. R., Allylglycine, an inhibitor of the uptake of L-leucine and L-proline in rat brain slices, Biochem, PharmacoL, 23 (1974) 821-827. 5 BALCAR,V. J., ANDJOHNSTON,G. A. R., High affinity uptake of L-glutamine in rat brain slices, J. Neurochem., 24 (1975) 875-879. 6 BENNETT,J. P., LOGAN,W. J., AND SNYDER,S. H., Amino acid neurotransmitter candidates: sodium-dependent high-affinity uptake by unique synaptosomal fractions, Science, 178 (1972) 997-999.
151 7 BOWMAN,W. C., AND SANGHVI, I. S., Pharmacological actions of hemlock (Conium macu&tum) alkaloids, J. Pharm. Pharmacol., 15 (1963) 1-24. 8 CURTIS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 9 DAVIES, L. P., AND JOHNSTON, G. A. R., Postnatal changes in the activities of serine hydroxymethyltransferase and glycine 2-oxoglutarate amino transferase in the rat central nervous system, J. Neurochem., 22 (1974) 107-112. 10 DAVIES, L. P., JOHNSTON, G. A. R., AND STEPHANSON, A. L., Postnatal changes in the potassiumstimulated calcium-dependent release of radioactive GABA and glycine from slices of rat central nervous tissue, J. Neurochem., 25 (1975) 387-392. 11 ESPLIN, D. W., AND ZABLOCKA-ESPLIN, B., Mechanisms of action ofconvulsants. In H. H. JASPER, A. A. WARD AND A. POPE (Eds.), Basic Mechanisms of the Epilepsies, Little Brown and Co., Boston, Mass., 1969, pp. 167-193. 12 FELIX, D., AND KONZLE, H., Iontophoretic and autoradiographic studies on the role of proline in nervous transmission, Pfliigers Arch. ges. Physiol., 350 (1974) 135-144. 13 GLOWlNSKL J., AND IVERSEN, L. L., Regional studies of catecholamines in the rat brain - - 1. The disposition of [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 14 JOHNSTON, G. A. R., AND BALCAR, V. J., Amino-oxyacetic acid: a relatively non-specific inhibitor of uptake of amino acids and amines by brain and spinal cord, J. Neurochem., 22 (1974) 609 610. 15 JOHNSTON, G. A. R., AND IVERSEN, L. L., Glycine uptake in rat central nervous system slices and homogenates: evidence for different uptake systems in spinal cord and cerebral cortex, J. Neurochem., 18 (1971) 1951-1961. 16 JOHNSTON, G. A. R., LLOYD, H. J., AND STONE, N., Liquid cation exchange extraction of cholinomimetic activity from brain, J. Neurochem., 15 (1968) 361-365. 17 KROGSGAARD-LARSEN,P., AND JOHNSTON, G. A. R., Inhibition of G A B A uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., in press. 18 MULDER, A. H., AND SNYDER, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308. 19 NEAL, M. J., The uptake of [14C]glycine by slices of mammalian spinal cord, J. Physiol. (Lond.), 215 (1971) 103-117. 20 PETERSON,N. A., AND RAGHUPATHY, E., Characteristics of amino acid accumulation by synaptosomal particles isolated from rat brain, J. Neurochem., 19 (1972) 1423-1438. 21 SMRT, J., BER~NEK,J., UND HOR.~,K, M., N-Hydroxypyrrolidon-(2), ein neuer Pyrrolidonderivattyp, Coll. Czech. chem. Commun., 24 (1959) 1672-1676. 22 TRAUaE, W., UNO ASCHER, R., [3ber das Isohydantoin 2-Imino-4-keto-tetrahydro-oxazol und seine Homologen, Chem. Ber., 46 (1913) 2077-2084. 23 VAN HARREVELD, A., AND FIFKOVA,E., Effects of amino acids on the isolated chicken retina, and on its response to glutamate stimulation, J. Neurochem., 20 (1973) 947-962. 24 YOUNG, A. B., AND SNYDER, S. H., Strychnine binding associated with glycine receptors of the central nervous system, Proe. nat. Aead. Sci. (Wash.), 70 (1973) 2832-2836.