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Characteristics of tryptophan accumulation by glial cells
Brain Research, 66 (1974) 253-263 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHARACTERISTICS OF TRYPTOPHAN CELLS
ACCUMULATION
253
BY G L I A L
ANNIE BAUMAN, SYLVIE BOURGOIN, PHILIPPE BENDA, JACQUES GLOWINSKI AND MICHEL HAMON
Laboratoire de Biologie Moldculaire, and Laboratoire de Neuropharmacologie Biochimique ( INSERM Ul14), Colldge de France, Paris 75005 (France) (Accepted July 16th, 1973)
SUMMARY
L-Tryptophan uptake was studied in C6 glial cells and found to be a very rapid and temperature dependent process. Kinetic analysis shows the presence of two saturable transport systems acting at different tryptophan concentrations. Both these high- and low-affinity processes were found in 3T3 fibroblast cultured cells, as well as in synaptosomes and cell suspensions obtained from rat cortex. No Na + requirement could be demonstrated in a wide range of tryptophan concentrations. The data presented here suggest that tryptophan accumulation in glial cells could be dependent on allosteric changes of the carrier, which might be relevant to the understanding of 5-HT synthesis regulation.
INTRODUCTION
The synthesis of serotonin (5-HT) in the central nervous system is affected by alterations of the concentration of the essential amino acid L-tryptophan (Try) 5,8,11. Since the turnover of the precursor amino acid is very rapid (its half-life is about 10 min) zS, its availability for 5-HT synthesis is largely dependent on its transport from blood to serotonergic neurons. Henn and Hamberger 16 have suggested that gila could play an important role in this process. Consequently, we have studied the characteristics of Try transport into a rat glial line, clone C64. Several specialized functions characteristic of the cell line C6 have already been described: synthesis of S-100 protein 3, production of glycerol-3-phosphate dehydrogenase and inducibility of this enzyme by hydrocortisone 6, accumulation of large amounts of 3',5'-cAMP in response to catecholamines 1°, and stimulation of adenylate cyclase activity by isoproterenol and dopamine is. Other cell lines such as fibroblasts
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A. BAUMANet al.
3T3-4 were used for comparison, as well as synaptosomal fractions and cell suspensions, both obtained from rat cortex. We found that Try is accumulated in all cases by two saturable processes: a high-affinity and a low-affinity mechanism. Attempts were made to characterize ion dependency as well as sensitivity to other aromatic amino acids and related amines. Presence of a high-affinity transport system for Try in glial cells and fibroblasts indicates that this process is not only operating in serotonergic neurons. MATERIAL
L-[3H]Try (3.2 Ci/mmole, generally labelled) was obtained from the Radiochemical Centre, Amersham; L-tryptophan from Merck; L-phenylalanine, N60 2dibutyryl adenosine 3',5'-cyclic phosphate, serotonin creatinine sulfate and tryptamine from Calbiochem; L-norepinephrine from Fluka; OL-p-chlorophenylalanine (pCPA) and DL-pCPA methyl ester (pCPA M.E.) from Rhtne-Poulenc; trypsin from Gibco. Cultured cells. Clone C6 glial cells, isolated from a rat tumor induced by Nnitrosomethylurea, and mouse fibroblast clone 3T3-4 were grown on glass in Ham F10 medium supplemented with 10~ fetal calf serum at 37 °C. Cells were scraped at the stationary phase with a rubber policeman, rinsed three times with Hanks solution to eliminate amino acids contained in the culture medium, and resuspended in the appropriate volume of Hanks solution. Cell viability was checked using trypan blue coloration. Synaptosomalfractions. Male Charles River rats (250-300 g) were decapitated. The cerebral cortex was quickly dissected in the cold (4 °C) and homogenized in 10 vol. of 0.32 M sucrose in a Potter-Elvehjem glass homogenizer fitted with a teflon pestle. Crude synaptosomal fraction (P2) and purified synaptosomes (fraction B) were prepared as described by Gray and Whittaker 13. Both preparations were resuspended in Hanks medium (20 ml/g of wet tissue) for uptake experiments. Cortical cell suspension (CCS). Thirty- to 40-day-old male Charles River rats were killed by decapitation. The cerebral cortex was dissected and chopped at 4 °C in 0.4 mm sided cubes by using a Mcllwain tissue slicer. Tissue disaggregation was performed by a 20-min incubation at 37 °C with 0.25 ~ trypsin. After centrifugation in Hanks solution containing 1 ~ bovine serum albumin, tissue fragments were rinsed in Hanks medium and disrupted according to the technique described by Hamberged 4. This consisted of passage through a syringe equipped with a 500-/zm pore nylon mesh, and filtration through successive nylon networks, with decreasing pore sizes; the last filtration, using a 50-#m pore size, led to a suspension of isolated cells. Centrifugation of the final filtrate at a very low speed (150 × g, 3 min) allowed the cell bodies to sediment, whereas most of the membrane fragments and synaptosomes remained in the supernatant. Microscopic observations and colorations (eosin, trypan blue) demonstrated the viability of the cells in the final suspension.
TRYPTOPHAN ACCUMULATION BY GLIAL CELLS
255
METHODS
Measurement of tryptophan accumulation Cultured cells and cortical cell suspensions. Aliquots of 2 4 × 106 cells, representing0.3-0.4 mg protein, in a final volume of 0.9 ml of Hanks medium, were preincubated for 5 rain at 37 °C under a constant stream of O2-CO2 (95:5~). [3H]Try (100 #l, 2.5 #Ci) was added and the incubation continued for time periods ranging from 15 sec to l0 min. For the kinetic analysis, incubations were carried out for 30 sec (a time chosen in the linear phase of Try accumulation) with various concentrations of Try from 1/zM up to 5 raM. When substances which might influence uptake were used, they were added at the beginning of the preincubation period, but amino acids were added simultaneously with [ZH]Try. The pH of the incubating medium was carefully kept between 7.3 and 7.4 in all conditions, throughout the incubation period. When Na + was removed, the isosmolarity was maintained by substituting choline and the pH was adjusted with Tris buffer. To stop the uptake, the test tubes were dipped into a melting ice bath, and filled with 8 ml of cold (0 °C) Hanks solution. After centrifugation, the pellet was rinsed twice with 2 ml of cold Hanks medium, which eliminated 15 ~ of the radioactivity originally accumulated. This effect was observed in all experiments, except in the study made with Ca2+-free media (see Results). Finally, the pellet was resuspended in 1.5 rnl of 0.05 M NaH2PO4/Na2HPO4 (pH 7) and submitted for 20 sec to ultrasonic disintegration (20 kHz, 40 W) in a sonimasse T 50 apparatus, fitted with a microtip. Radioactivity was estimated on 0.2 ml of the suspension by liquid scintillation counting in toluene phosphor using a Packard 3003 spectrometer. Proteins were estimated by the method of Lowry et al. ~3 using bovine serum albumin as a standard. When incubations lasted for l0 rain, ion exchange chromatography on a Dowex AG50-WX4 column 15 of an ethanol-water (74:16, v/v) extract of the cell pellet revealed that 90~ at least of the radioactivity was associated with authentic L-[aH]Try. The blank reaction was estimated by incubating samples at 0 °C instead of 37 °C. The radioactivity accumulated at 37 °C was at least 3 times higher than that of the corresponding blanks. In all cases, the results were expressed on the basis of the radioactivity remaining in pellets after the washings, as nmoles Try/rag protein/30 sec. Synaptosomalfractions. The procedure was exactly the same as described for cells until the end of the incubation. The synaptosornal suspension (1 ml of incubating medium ~- 8 ml cold Hanks solution) was centrifuged for 5 rain at 14,500 × g in a Sorvall RC2B centrifuge at 4 °C. The resulting pellet was rinsed with 3 ml of cold Hanks medium and recentrifuged as before. Finally, synaptosomes were suspended in 1.5 ml of 0.05 M NaH2POa/Na2HPO4 (pH 7) and lysed by sonication. Measurements and expression of results were similar to those used for cells. RESULTS
Time course of L-[3H_] Try accumulation Glial cells. C6 glial cells were incubated for time intervals ranging between 30
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Fig. 1. Time course o f Try accumulation in C6 glial cells. Cells were incubated for various times with 6 / ~ M T r y (a) or 1 m M T r y (b), either at 37 °C or at 0 °C. Each point corresponds to the total a m o u n t o f Try found in the cell pellets (mean o f 4 determinations). The active accumulation o f Try [(Try accumulated at 37 °C) - - (Try accumulated at 0 °C)] levels off within 2 rain at 6 # M T r y as well as at 1 mMTry.
sec and 60 min in the presence of 6 # M Try. The accumulation of the amino acid was very rapid and reached a plateau within 2 min after the onset of the incubation (Fig. la). The maximal level of Try accumulation (0.5-0.7 nmoles of Try/mg protein) remained unchanged for at least 60 min. In the presence of 1 m M Try, the same pattern was observed (Fig. lb). In both cases, the initial phase of linear increase in the [3H]Try content of the cells lasted for less than 1 min. Consequently, further kinetic analyses were done with incubation periods of 30 sec. Fibroblasts. When performed under the same conditions, incubations of fibroblasts (clone 3T3) with 6 # M Try revealed a similar time course for uptake. Moreover, the maximal levels of Try accumulation reached in both cases were very similar. Cortical cell suspension (CCS). [3H]Try was rapidly taken up by cortical cell suspensions in the same way as by cultured cells. Accumulation was linear for less than 90 sec, and reached a plateau within 150 sec for amino acid concentrations ranging from 1.3 # M to 1 raM. However, the maximal level of Try accumulated, when Try extracellular concentration was 6 #M, corresponded to 0.10 nmoles/mg protein. This is 6 times lower than that observed in cultured cells. Synaptosomalfractions. [3H]Try was rapidly accumulated in synaptosomes (Pz and B fractions). The plateau reached within 3 min corresponded to 0.20 nmoles/mg protein when Try concentration in the incubating medium was 6 #M.
Temperature dependency When incubations were made at 0 °C, Try accumulation was strongly reduced in all cultured cells and cortical preparations studied (Table I). The small amount of Try accumulated under these conditions was probably the result of passive diffusion since no saturable component could be observed when the amino acid concentrations were increased from 1/zM to 5 mM. Consequently, quantities of Try accumulated at 0 °C were considered as blanks and subtracted from those obtained at 37 °C. When
257
TRYPTOPHAN ACCUMULATIONBY GLIAL CELLS TABLE I EFFECTOFTEMPERATUREONTRYPTOPHANACCUMULATIONIN CORTICALCELLSUSPENSION
These values are the mean of 4 determinations and correspond to the total amount of Try found in the pellets, after two washings (tryptophan concentration: 0.1 mM).
Incubation temperature (°C)
Try accumulated (nmoles/mg/30 sec)
0
0.46 2.26 0.46
living cells 37 ~ heated cells (10 min at 50 °C)
cultured cells or cortical preparations (Table I) were incubated for 10 min at 50 °C, Try accumulation studied in such preparations was irreversibly abolished. Kinetic parameters Cultured cells. W h e n estimated within a wide range o f amino acid concentrations, the initial velocity (during the first 30 sec) o f Try accumulation in C6 glial cells appeared to change in a rather complicated pattern. According to the LineweaverBurk representation, a double reciprocal plot produced curves which could be resolved into two straight lines (Fig. 2). W h e n Try concentrations varied f r o m 0.1 m M to 5 m M , the uptake of the amino acid followed Michaelis-Menten kinetics, and could be characterized by two constants Km and Vmax. F o r concentrations lower than 0.1 m M ,
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point is the mean of 4 determinations.
A. BAUMANet al.
258 TABLE II gm
AND Vmax VALUES FOR TRYPTOPHAN UPTAKE BY CELL LINES AND CORTICAL PREPARATIONS
Incubations were performed at 37 °C for 30 sec, in presence of Try concentrations ranging from 5/~M to 3 mM. Each value is the mean of 3 experiments.
Preparations
Cell lines C6 glial cells 3T3 fibroblasts Corticalpreparations CCS P2 Synaptosomes
High-affinity system
Low-affinity system
Kml (#M)
Vmaxl (nrnoles/mg/30 sec)
Kin2 (mM)
Vmax2 (nmoles/mg/30 sec)
7.0 6.9
1.48 1.20
0.8 1.2
3.6 7.5
59.0 58.5 52.0
1.85 1.50 2.76
1.1 0.8 0.9
5.0 5.1 7.1
the uptake process, still described by a straight line, was characterized by different constants which were found lower than the previous ones. F r o m these results a lowaffinity transport process for Try (Kmz, Vmax2) could be distinguished from a highaffinity system (Kin1, Vmaxl). As shown in Table II, Try uptake process in 3T3 fibroblasts displayed similar kinetic characteristics to those of cloned glial cells. Double reciprocal plots for the accumulation of Try in the two types of cells (C6, 3T3) demonstrated the existence of both a high-affinity and a low-affinity transport system acting at different Try concentrations. Synaptosomes and cortical cell suspensions. N o significant differences could be observed between these two cortical preparations. As in cultured cells, Try transport appeared to depend on two different processes when amino acid concentrations were distributed over the same range as previously. Although the maximal velocity (Vnaaxl) was similar to that observed in cultured cells, the high affinity process had a Km 1 value 9 times higher in cortical preparations. On the other hand, the low affinity process was the same in both cortical preparations and in cloned cells (Table II).
Effect of ions Complete removal of N a + ions - - replaced by isosmolar choline - - changed neither the low-affinity, nor the high-affinity process (Table III) for Try transport in all types of cloned cells. Similarly, preincubation of cells with 0.1 m M ouabain for 15 min at 37 °C, in order to block the N a + pump, did not affect Try accumulation between 6 # M and 1 m M Try. As shown in Table III, when the K + concentration was increased from 5 m M to 50 m M - - simultaneously with an isosmolar decrease of N a + ions - - Try accumulation was not changed in cultured cells, but significantly reduced in cortical preparations.
TRYPTOPHAN
ACCUMULATION
259
BY G L I A L C E L L S
TABLE III EFFECTS OF MULATION
Na +, K + AND f a g+ CONCENTRATIONS
IN THE INCUBATING MEDIUM ON TRYPTOPHAN ACCU-
Results are expressed as ~ of the Try accumulation in preparations incubated 30 sec in normal Hanks medium. Each value is the mean of 4 determinations (100% = 0.43 nmoles/mg/30 sec for C6 glial cells, 0.55 nmoles/mg/30 sec for 3T3 fibroblasts, 0.17 nmoles/mg/30 sec for SCC and 0.13 nmoles/mg/30 sec for P2 at Try -- 6 #M). n.d. = non-determined. Preparations
Na +free
50 mM K +
Ca2+free
Cell lines C6 glial ceils 3T3 fibroblasts
103 102
93 97
37 51
Cortical preparations CCS Synaptosomes
n.d. n.d.
66 68
n.d. 110
TABLE IV EFFECT OF LACK OF C a 2+ IN INCUBATING AND WASHING MEDIA ON TRY ACCUMULATION IN 3 T 3 EIBROBLASTS
Cells were incubated with 5.8 ktM Try for 30 sec at 37 °C in normal or Ca2+ free Hanks medium and subsequently washed twice with either medium at 0 °C. Values are the mean of 4 determinations. Incubation medium (37 °C)
Washingmedium (0 °C)
Control Ca 2+ free Control
Control Ca 2+ free Ca 2+ free
Tryptophan In cells (nmoles/mg)
In washings (nmoles/mg)
Total (nmoles/mg)
0.567 0.289 0.320
0.088 0.353 0.423
0.655 0.642 0.743
U p t a k e of Try in cell lines was n o t modified by d o u b l i n g the Ca 2+ c o n c e n t r a t i o n in the i n c u b a t i n g m e d i u m for all types of preparations. T r y a c c u m u l a t i o n i n synaptosomes was n o t modified either in the absence of Ca 2+, whether 1 m M E G T A was added or not. O n the contrary, T r y a c c u m u l a t i o n in cultured cells was strongly decreased in Ca 2+ free m e d i u m (Table III): this effect can be attributed to a n e n h a n c e d passive diffusion of the a m i n o acid, since increased levels of T r y were f o u n d in washings of fibroblasts which were made with Ca 2+ free m e d i u m at 0 °C (Table IV). Effect o f other amino acids W h e n added in the i n c u b a t i n g m e d i u m simultaneously with [ZH]Try (5 × 10-6 M), p h e n y l a l a n i n e (10 -5 M), tyrosine (10 -5 M ) a n d p C P A (10 -5 M ) inhibited the a c c u m u l a t i o n of the labelled a m i n o acid in cultured cells ( - - 3 5 ~ ) as well as in cortical p r e p a r a t i o n s ( - - 3 0 ~ ) . P h e n y l a l a n i n e was a competitive i n h i b i t o r of the high-affinity a n d of the low-affinity (Fig. 3) Try t r a n s p o r t processes in C6 glial cells.
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Fig. 4. Effect o f p C P A M.E. on Try accumulation by cortical cell suspension. Incubations were done in two different ranges of Try concentrations, a: high-affinity process. Try varies from 6 # M to 40 /~M. Control ( A); 50 f f M p C P A M.E. (0). b: low-affinity process. Try varies from 0.5 m M t o 2 raM; control ( i ) , 1 m M p C P A M.E. (0). Blanks were subtracted; each point is the mean of 4 determinations. The double reciprocal plot shows that pCPA M.E. stimulates Try accumulation without changing the maximal velocity.
In contrast, pCPA-methyl ester (pCPA M.E.), the soluble derivative ofpCPA, instead of inhibiting Try uptake, slightly increased the accumulation of this amino acid both in cultured cells and in the cortical cell suspension: this effect was seen for the low-affinity process (Fig. 4b) as well as for the high-affinity one (Fig. 4a). There was no change in Try uptake in synaptosomal preparations.
Effect of other compounds Norepinephrine and the two indoleamines, serotonin and tryptamine, added even at high concentrations (1 mM), did not alter Try transport into glial cells. Dibutyryl cyclic AMP (0.1-1 raM)did not affect Try accumulation neither in cultured cells, nor in cortical preparations.
TRYPTOPHAN ACCUMULATION BY GLIAL CELLS
261
DISCUSSION
Tryptophan transport in C6 glial cell line was found to be a very rapid process, the maximal level of intracellular Try being reached within 2 min. In determining the kinetic parameters of Try accumulation, we observed two transport systems with different velocities and affinities for the amino acid. Time course and kinetic parameters of the low affinity process in C6 clone were identical to that of 3T3 fibroblasts and of synaptosomes. On the contrary, when highaffinity processes are considered, Km for the C6 and 3T3 lines (7/~M) differed from the figures in synaptosomes (52 # M ) by one order of magnitude. Kinetic parameters for both Try uptake processes were found to be identical in crude synaptosomal fraction (P2) and in purified synaptosomes (Table I); moreover, our values are in agreement with published data2, 21. The presence of the same high-affinity transport for Try in glial cells and fibroblasts indicates that this process is not only operating in serotonergic neurons, but also in other cells. Moreover, Try uptake by a high-affinity process (Kin: 15 # M ) has been observed in preliminary experiments in a cholinergic (S-20) as well as in an adrenergic (N1E-115) neuroblastoma clone (generously supplied by M. Nirenberg). These data are in agreement with recent reports22: Try accumulation in synaptosomal preparations obtained from rat forebrains is not changed after destruction of serotonergic pathways by raphe lesions. In view of the fact that the C6 glial cell line did not differ in any respect from the 3T3 fibroblasts, as far as Try accumulation is concerned, we investigated the role of possible alterations of membrane properties due to growth in vitro. We then studied freshly separated cortical cell suspensions, and found that their behavior was similar to that of the cloned cells or of synaptosomes: when we considered parameters such as value of Km1 and changes in Try accumulation after K + increase in the incubating medium, cortical cell suspension seemed to have a synaptosome-like reactivity; but their sensitivity to pCPA M.E. was the same as cloned ceils. Moreover, such a mixture of cells, either viable or non-viable, is likely to be contaminated with intact nuclei and particulate debris 1. Thence, one would suspect that some types of membranes in this preparation are different from that of synaptosomes. Whereas cloned lines are growing as isolated cells, with free diffusion of amino acids between cells and the medium, it is tempting to consider Try accumulation in slices, because they constitute a more organized material with various types of neural cells in close contact and an extracellular compartment, evoking the in vivo situation. While no differences are seen for the inhibitory effect of phenylalanine and pCPA in slices ~°, synaptosomes 12, or cells on the two Try transport systems, marked differences exist for other parameters: (1) in slices, Try accumulation increases gradually for at least 30 min 20, the KmI value being around 0.1 m M (Hery et al., personal communication). (2) It is reduced in Na + free medium19, 20 contrary to synaptosomes 12 or cells (Table III). (3) Whereas incubation in Ca e+ free medium slightly increases Try accumulation in brain slices (unpublished observations), it has no effect on synaptosomal uptake 12 (Table III); furthermore, it reduces Try accumulation in cloned cells, by enhancing passive diffusion of the amino acid (Table IV). (4) In high K + concentration,
262
A. BAUMANet al.
Try accumulation is reduced in slices (unpublished observations) and in both cortical preparations (Table III) as a consequence of Try leakage induced by membrane depolarization; on the other hand, we saw no effect on cloned cells. (5) Eventually, while addition of dibutyryl cAMP increases Try accumulation in slicesg, 15, we saw no effect on our preparations. These differences stress the importance of membrane interactions between different cell types. Finally, our findings show that the Kin1 value for cortical preparations is in the range of physiological tryptophan concentrations (21 /~M in brain tissue11), while that of cultured cells is close to the concentration of Try in F10 supplemented with 10~ fetal calf serum (4 #M). Moreover, the Lineweaver-Burk representation gives a two-linear section curve for Try accumulation, even for a homogenous cell population (cloned cells): this has been shown to occur in allosteric negative cooperativityT, 24. Of particular interest are then the findings ~7 showing that Try accumulation in brain slices fluctuates following a nycthemeral rhythm, and induces parallel modifications in 5-HT synthesis. We would suggest that in physiological conditions, where glial cells are located between capillaries and neurons, variations of plasmatic free Try could modify the Try carrier activity in glial cells, thence leading to changes in 5-HT synthesis. ACKNOWLEDGEMENTS
The authors would like to thank Mrs. E. Rosenbaum for her excellent technical assistance. This research was supported by grants from DGRST and INSERM. REFERENCES 1 BARKLEY,D. S., RAKIC, L. L., CHAFFEE,J. K., AND WONG, D. L., Cell separation by velocity sedimentation of post-natal mouse cerebellum, J. Cell Physiol., 81 (1973) 271-279. 2 BELIN, M. F., ET PUJOL, J. F., Transport synaptosomal du tryptophane et de la tyrosine c6r6brale. Existence de systSmes de capture d'affinit6 diff6rente, Experientia (Basel), 29 (1973) 411-413. 3 BENDA, P., LIGHTBODY,J., SATO, G., LEVINE, L., AND SWEET, W. H., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 4 BENDA, P., SOMEDA,K., MESSER, J., AND SWEET, W. H., Morphological and immunochemical studies of rat glial tumors and clonal strains propagated in culture, J. Neurosurg., 34 (1971) 310320. 5 CARLSSON,A., AND LINDQVIST, M., The effect of L-tryptophan and some psychotropic drugs on the formation of 5-hydroxytryptophan in the mouse brain in vivo, J. Neurol. Transmission, 33 (1972) 23-43. 6 DEVELLIS,J., AND INGLISH,D., Effect of cortisol and epinephrine on the biochemical differentiation of cloned glial cells in culture and of the developing rat brain, SecondInt. Meeting Soc. Neurochem., (1969) 151. 7 ENGEL, P. C., AND FERDINAND, W., The significance of abrupt transitions in Lineweaver-Burk plots with particular reference to glutamate dehydrogenase. Negative and positive cooperativity in catalytic rate constants, Biochem. J., 131 (1973) 97-105. 8 FERNSTROM,J. D., AND WURTMAN,R. J., Brain serotonin content: Physiological dependence on plasma Try levels, Science, 173 (1971) 149-152. 9 FORN, J., TAGLIAMONTE,P., TAGLIAMONTE,A., AND GESSA,G. L., Stimulation by dibutyryl cyclic AMP of serotonin synthesis and tryptophan transport in brain slices, Nature New Biol., 237 (1972) 245-247.
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10 GILMAN, A. G., AND N1RENBERG, M., Effect of catecholamines on the adenosine 3',5'-.cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2165-2168. 11 GRAHAME-SMITH,D. G., Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan, J. Neurochem., 18 (1971) 1053-1066. 12 GRAHAME-SMITH,D. G., AND PARFITT, A. G., Tryptophan transport across the synaptosomal membrane, J. Neurochem., 17 (1970) 1339-1353. 13 GRAY, E. G., AND WHITTAKER,V. P., The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.) , 96 (1962) 79-88. 14 HAMBERGER,A., Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex, Brain Research, 31 (1971) 169-178. 15 HAMON,M., BOURGOIN,S., AND GLOWINSKI,J., Feed-back of 5-HT synthesis in rat striatal slices, J. Neurochem., 20 (1973) 1727-1745. 16 Ht/NN, F. A., AND HAMBERGER,A., Glial cell function: uptake of transmitter substances, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2686--2690. 17 HERY, F., ROUER, E., AND GLOWINSKI, J., Daily variations of serotonin metabolism in the rat brain, Brain Research, 43 (1972) 445-465. 18 .lARD,S., ~.EMONT, J., AND BENDA, P., Adenylate cyclase, phosphodiesterases and protein kinase of rat glial cells in culture, FEBS Letters, 26 (1972) 344-348. 19 JOANNY,P., BARBOSA,E., ET CORRIOL,.l., Accumulation active de quelques acides amin6s dans les coupes de cerveau de Rat, J. Physiol. (Paris), 60 (1968) 265. 20 KtELY, M., AND SOURKES, T. L., Transport of L-tryptophan into slices of rat cerebral cortex, J. Neurochem., 19 (1972) 2863-2872. 21 KNAPP, S., AND MANDELL,A. J., Narcotic drugs: effects on the serotonin biosynthetic systems of the brain, Science, 177 (1972) 1209-1211. 22 KUHAR, M. H., ROTH, R. H., AND AGHAJANIAN,G. K., Synaptosomes from forebrains of rats with midbrain raphe lesions: selective reduction of serotonin uptake, J. Pharmacol. exp. Ther., 181 (1972) 3~45. 23 LOWRY,O. H., ROSEBROUGH,N..l., FARR, A. L., AND RANDALL,R. J., Protein measurement with the folin Phenol reagent, J. biol. Chem., 193 (1951) 265-275. 24 TEIPEL, J., AND KOSHLAND,O. E., JR., The significance of intermediary plateau regions in enzyme saturation curves, Biochemistry, 8 (1969) 4656-4663. 25 TYCE, G. M., FLOCK, E. V., OWEN, C. A., JR., STOBIE,G. H. C., AND DAVID, C., 5-Hydroxyindole metabolism in the brain after hepatectomy, Biochem. Pharmacol., 16 (1967) 979-992.
CHARACTERISTICS OF TRYPTOPHAN CELLS
ACCUMULATION
253
BY G L I A L
ANNIE BAUMAN, SYLVIE BOURGOIN, PHILIPPE BENDA, JACQUES GLOWINSKI AND MICHEL HAMON
Laboratoire de Biologie Moldculaire, and Laboratoire de Neuropharmacologie Biochimique ( INSERM Ul14), Colldge de France, Paris 75005 (France) (Accepted July 16th, 1973)
SUMMARY
L-Tryptophan uptake was studied in C6 glial cells and found to be a very rapid and temperature dependent process. Kinetic analysis shows the presence of two saturable transport systems acting at different tryptophan concentrations. Both these high- and low-affinity processes were found in 3T3 fibroblast cultured cells, as well as in synaptosomes and cell suspensions obtained from rat cortex. No Na + requirement could be demonstrated in a wide range of tryptophan concentrations. The data presented here suggest that tryptophan accumulation in glial cells could be dependent on allosteric changes of the carrier, which might be relevant to the understanding of 5-HT synthesis regulation.
INTRODUCTION
The synthesis of serotonin (5-HT) in the central nervous system is affected by alterations of the concentration of the essential amino acid L-tryptophan (Try) 5,8,11. Since the turnover of the precursor amino acid is very rapid (its half-life is about 10 min) zS, its availability for 5-HT synthesis is largely dependent on its transport from blood to serotonergic neurons. Henn and Hamberger 16 have suggested that gila could play an important role in this process. Consequently, we have studied the characteristics of Try transport into a rat glial line, clone C64. Several specialized functions characteristic of the cell line C6 have already been described: synthesis of S-100 protein 3, production of glycerol-3-phosphate dehydrogenase and inducibility of this enzyme by hydrocortisone 6, accumulation of large amounts of 3',5'-cAMP in response to catecholamines 1°, and stimulation of adenylate cyclase activity by isoproterenol and dopamine is. Other cell lines such as fibroblasts
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A. BAUMANet al.
3T3-4 were used for comparison, as well as synaptosomal fractions and cell suspensions, both obtained from rat cortex. We found that Try is accumulated in all cases by two saturable processes: a high-affinity and a low-affinity mechanism. Attempts were made to characterize ion dependency as well as sensitivity to other aromatic amino acids and related amines. Presence of a high-affinity transport system for Try in glial cells and fibroblasts indicates that this process is not only operating in serotonergic neurons. MATERIAL
L-[3H]Try (3.2 Ci/mmole, generally labelled) was obtained from the Radiochemical Centre, Amersham; L-tryptophan from Merck; L-phenylalanine, N60 2dibutyryl adenosine 3',5'-cyclic phosphate, serotonin creatinine sulfate and tryptamine from Calbiochem; L-norepinephrine from Fluka; OL-p-chlorophenylalanine (pCPA) and DL-pCPA methyl ester (pCPA M.E.) from Rhtne-Poulenc; trypsin from Gibco. Cultured cells. Clone C6 glial cells, isolated from a rat tumor induced by Nnitrosomethylurea, and mouse fibroblast clone 3T3-4 were grown on glass in Ham F10 medium supplemented with 10~ fetal calf serum at 37 °C. Cells were scraped at the stationary phase with a rubber policeman, rinsed three times with Hanks solution to eliminate amino acids contained in the culture medium, and resuspended in the appropriate volume of Hanks solution. Cell viability was checked using trypan blue coloration. Synaptosomalfractions. Male Charles River rats (250-300 g) were decapitated. The cerebral cortex was quickly dissected in the cold (4 °C) and homogenized in 10 vol. of 0.32 M sucrose in a Potter-Elvehjem glass homogenizer fitted with a teflon pestle. Crude synaptosomal fraction (P2) and purified synaptosomes (fraction B) were prepared as described by Gray and Whittaker 13. Both preparations were resuspended in Hanks medium (20 ml/g of wet tissue) for uptake experiments. Cortical cell suspension (CCS). Thirty- to 40-day-old male Charles River rats were killed by decapitation. The cerebral cortex was dissected and chopped at 4 °C in 0.4 mm sided cubes by using a Mcllwain tissue slicer. Tissue disaggregation was performed by a 20-min incubation at 37 °C with 0.25 ~ trypsin. After centrifugation in Hanks solution containing 1 ~ bovine serum albumin, tissue fragments were rinsed in Hanks medium and disrupted according to the technique described by Hamberged 4. This consisted of passage through a syringe equipped with a 500-/zm pore nylon mesh, and filtration through successive nylon networks, with decreasing pore sizes; the last filtration, using a 50-#m pore size, led to a suspension of isolated cells. Centrifugation of the final filtrate at a very low speed (150 × g, 3 min) allowed the cell bodies to sediment, whereas most of the membrane fragments and synaptosomes remained in the supernatant. Microscopic observations and colorations (eosin, trypan blue) demonstrated the viability of the cells in the final suspension.
TRYPTOPHAN ACCUMULATION BY GLIAL CELLS
255
METHODS
Measurement of tryptophan accumulation Cultured cells and cortical cell suspensions. Aliquots of 2 4 × 106 cells, representing0.3-0.4 mg protein, in a final volume of 0.9 ml of Hanks medium, were preincubated for 5 rain at 37 °C under a constant stream of O2-CO2 (95:5~). [3H]Try (100 #l, 2.5 #Ci) was added and the incubation continued for time periods ranging from 15 sec to l0 min. For the kinetic analysis, incubations were carried out for 30 sec (a time chosen in the linear phase of Try accumulation) with various concentrations of Try from 1/zM up to 5 raM. When substances which might influence uptake were used, they were added at the beginning of the preincubation period, but amino acids were added simultaneously with [ZH]Try. The pH of the incubating medium was carefully kept between 7.3 and 7.4 in all conditions, throughout the incubation period. When Na + was removed, the isosmolarity was maintained by substituting choline and the pH was adjusted with Tris buffer. To stop the uptake, the test tubes were dipped into a melting ice bath, and filled with 8 ml of cold (0 °C) Hanks solution. After centrifugation, the pellet was rinsed twice with 2 ml of cold Hanks medium, which eliminated 15 ~ of the radioactivity originally accumulated. This effect was observed in all experiments, except in the study made with Ca2+-free media (see Results). Finally, the pellet was resuspended in 1.5 rnl of 0.05 M NaH2PO4/Na2HPO4 (pH 7) and submitted for 20 sec to ultrasonic disintegration (20 kHz, 40 W) in a sonimasse T 50 apparatus, fitted with a microtip. Radioactivity was estimated on 0.2 ml of the suspension by liquid scintillation counting in toluene phosphor using a Packard 3003 spectrometer. Proteins were estimated by the method of Lowry et al. ~3 using bovine serum albumin as a standard. When incubations lasted for l0 rain, ion exchange chromatography on a Dowex AG50-WX4 column 15 of an ethanol-water (74:16, v/v) extract of the cell pellet revealed that 90~ at least of the radioactivity was associated with authentic L-[aH]Try. The blank reaction was estimated by incubating samples at 0 °C instead of 37 °C. The radioactivity accumulated at 37 °C was at least 3 times higher than that of the corresponding blanks. In all cases, the results were expressed on the basis of the radioactivity remaining in pellets after the washings, as nmoles Try/rag protein/30 sec. Synaptosomalfractions. The procedure was exactly the same as described for cells until the end of the incubation. The synaptosornal suspension (1 ml of incubating medium ~- 8 ml cold Hanks solution) was centrifuged for 5 rain at 14,500 × g in a Sorvall RC2B centrifuge at 4 °C. The resulting pellet was rinsed with 3 ml of cold Hanks medium and recentrifuged as before. Finally, synaptosomes were suspended in 1.5 ml of 0.05 M NaH2POa/Na2HPO4 (pH 7) and lysed by sonication. Measurements and expression of results were similar to those used for cells. RESULTS
Time course of L-[3H_] Try accumulation Glial cells. C6 glial cells were incubated for time intervals ranging between 30
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Fig. 1. Time course o f Try accumulation in C6 glial cells. Cells were incubated for various times with 6 / ~ M T r y (a) or 1 m M T r y (b), either at 37 °C or at 0 °C. Each point corresponds to the total a m o u n t o f Try found in the cell pellets (mean o f 4 determinations). The active accumulation o f Try [(Try accumulated at 37 °C) - - (Try accumulated at 0 °C)] levels off within 2 rain at 6 # M T r y as well as at 1 mMTry.
sec and 60 min in the presence of 6 # M Try. The accumulation of the amino acid was very rapid and reached a plateau within 2 min after the onset of the incubation (Fig. la). The maximal level of Try accumulation (0.5-0.7 nmoles of Try/mg protein) remained unchanged for at least 60 min. In the presence of 1 m M Try, the same pattern was observed (Fig. lb). In both cases, the initial phase of linear increase in the [3H]Try content of the cells lasted for less than 1 min. Consequently, further kinetic analyses were done with incubation periods of 30 sec. Fibroblasts. When performed under the same conditions, incubations of fibroblasts (clone 3T3) with 6 # M Try revealed a similar time course for uptake. Moreover, the maximal levels of Try accumulation reached in both cases were very similar. Cortical cell suspension (CCS). [3H]Try was rapidly taken up by cortical cell suspensions in the same way as by cultured cells. Accumulation was linear for less than 90 sec, and reached a plateau within 150 sec for amino acid concentrations ranging from 1.3 # M to 1 raM. However, the maximal level of Try accumulated, when Try extracellular concentration was 6 #M, corresponded to 0.10 nmoles/mg protein. This is 6 times lower than that observed in cultured cells. Synaptosomalfractions. [3H]Try was rapidly accumulated in synaptosomes (Pz and B fractions). The plateau reached within 3 min corresponded to 0.20 nmoles/mg protein when Try concentration in the incubating medium was 6 #M.
Temperature dependency When incubations were made at 0 °C, Try accumulation was strongly reduced in all cultured cells and cortical preparations studied (Table I). The small amount of Try accumulated under these conditions was probably the result of passive diffusion since no saturable component could be observed when the amino acid concentrations were increased from 1/zM to 5 mM. Consequently, quantities of Try accumulated at 0 °C were considered as blanks and subtracted from those obtained at 37 °C. When
257
TRYPTOPHAN ACCUMULATIONBY GLIAL CELLS TABLE I EFFECTOFTEMPERATUREONTRYPTOPHANACCUMULATIONIN CORTICALCELLSUSPENSION
These values are the mean of 4 determinations and correspond to the total amount of Try found in the pellets, after two washings (tryptophan concentration: 0.1 mM).
Incubation temperature (°C)
Try accumulated (nmoles/mg/30 sec)
0
0.46 2.26 0.46
living cells 37 ~ heated cells (10 min at 50 °C)
cultured cells or cortical preparations (Table I) were incubated for 10 min at 50 °C, Try accumulation studied in such preparations was irreversibly abolished. Kinetic parameters Cultured cells. W h e n estimated within a wide range o f amino acid concentrations, the initial velocity (during the first 30 sec) o f Try accumulation in C6 glial cells appeared to change in a rather complicated pattern. According to the LineweaverBurk representation, a double reciprocal plot produced curves which could be resolved into two straight lines (Fig. 2). W h e n Try concentrations varied f r o m 0.1 m M to 5 m M , the uptake of the amino acid followed Michaelis-Menten kinetics, and could be characterized by two constants Km and Vmax. F o r concentrations lower than 0.1 m M ,
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A. BAUMANet al.
258 TABLE II gm
AND Vmax VALUES FOR TRYPTOPHAN UPTAKE BY CELL LINES AND CORTICAL PREPARATIONS
Incubations were performed at 37 °C for 30 sec, in presence of Try concentrations ranging from 5/~M to 3 mM. Each value is the mean of 3 experiments.
Preparations
Cell lines C6 glial cells 3T3 fibroblasts Corticalpreparations CCS P2 Synaptosomes
High-affinity system
Low-affinity system
Kml (#M)
Vmaxl (nrnoles/mg/30 sec)
Kin2 (mM)
Vmax2 (nmoles/mg/30 sec)
7.0 6.9
1.48 1.20
0.8 1.2
3.6 7.5
59.0 58.5 52.0
1.85 1.50 2.76
1.1 0.8 0.9
5.0 5.1 7.1
the uptake process, still described by a straight line, was characterized by different constants which were found lower than the previous ones. F r o m these results a lowaffinity transport process for Try (Kmz, Vmax2) could be distinguished from a highaffinity system (Kin1, Vmaxl). As shown in Table II, Try uptake process in 3T3 fibroblasts displayed similar kinetic characteristics to those of cloned glial cells. Double reciprocal plots for the accumulation of Try in the two types of cells (C6, 3T3) demonstrated the existence of both a high-affinity and a low-affinity transport system acting at different Try concentrations. Synaptosomes and cortical cell suspensions. N o significant differences could be observed between these two cortical preparations. As in cultured cells, Try transport appeared to depend on two different processes when amino acid concentrations were distributed over the same range as previously. Although the maximal velocity (Vnaaxl) was similar to that observed in cultured cells, the high affinity process had a Km 1 value 9 times higher in cortical preparations. On the other hand, the low affinity process was the same in both cortical preparations and in cloned cells (Table II).
Effect of ions Complete removal of N a + ions - - replaced by isosmolar choline - - changed neither the low-affinity, nor the high-affinity process (Table III) for Try transport in all types of cloned cells. Similarly, preincubation of cells with 0.1 m M ouabain for 15 min at 37 °C, in order to block the N a + pump, did not affect Try accumulation between 6 # M and 1 m M Try. As shown in Table III, when the K + concentration was increased from 5 m M to 50 m M - - simultaneously with an isosmolar decrease of N a + ions - - Try accumulation was not changed in cultured cells, but significantly reduced in cortical preparations.
TRYPTOPHAN
ACCUMULATION
259
BY G L I A L C E L L S
TABLE III EFFECTS OF MULATION
Na +, K + AND f a g+ CONCENTRATIONS
IN THE INCUBATING MEDIUM ON TRYPTOPHAN ACCU-
Results are expressed as ~ of the Try accumulation in preparations incubated 30 sec in normal Hanks medium. Each value is the mean of 4 determinations (100% = 0.43 nmoles/mg/30 sec for C6 glial cells, 0.55 nmoles/mg/30 sec for 3T3 fibroblasts, 0.17 nmoles/mg/30 sec for SCC and 0.13 nmoles/mg/30 sec for P2 at Try -- 6 #M). n.d. = non-determined. Preparations
Na +free
50 mM K +
Ca2+free
Cell lines C6 glial ceils 3T3 fibroblasts
103 102
93 97
37 51
Cortical preparations CCS Synaptosomes
n.d. n.d.
66 68
n.d. 110
TABLE IV EFFECT OF LACK OF C a 2+ IN INCUBATING AND WASHING MEDIA ON TRY ACCUMULATION IN 3 T 3 EIBROBLASTS
Cells were incubated with 5.8 ktM Try for 30 sec at 37 °C in normal or Ca2+ free Hanks medium and subsequently washed twice with either medium at 0 °C. Values are the mean of 4 determinations. Incubation medium (37 °C)
Washingmedium (0 °C)
Control Ca 2+ free Control
Control Ca 2+ free Ca 2+ free
Tryptophan In cells (nmoles/mg)
In washings (nmoles/mg)
Total (nmoles/mg)
0.567 0.289 0.320
0.088 0.353 0.423
0.655 0.642 0.743
U p t a k e of Try in cell lines was n o t modified by d o u b l i n g the Ca 2+ c o n c e n t r a t i o n in the i n c u b a t i n g m e d i u m for all types of preparations. T r y a c c u m u l a t i o n i n synaptosomes was n o t modified either in the absence of Ca 2+, whether 1 m M E G T A was added or not. O n the contrary, T r y a c c u m u l a t i o n in cultured cells was strongly decreased in Ca 2+ free m e d i u m (Table III): this effect can be attributed to a n e n h a n c e d passive diffusion of the a m i n o acid, since increased levels of T r y were f o u n d in washings of fibroblasts which were made with Ca 2+ free m e d i u m at 0 °C (Table IV). Effect o f other amino acids W h e n added in the i n c u b a t i n g m e d i u m simultaneously with [ZH]Try (5 × 10-6 M), p h e n y l a l a n i n e (10 -5 M), tyrosine (10 -5 M ) a n d p C P A (10 -5 M ) inhibited the a c c u m u l a t i o n of the labelled a m i n o acid in cultured cells ( - - 3 5 ~ ) as well as in cortical p r e p a r a t i o n s ( - - 3 0 ~ ) . P h e n y l a l a n i n e was a competitive i n h i b i t o r of the high-affinity a n d of the low-affinity (Fig. 3) Try t r a n s p o r t processes in C6 glial cells.
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Fig. 4. Effect o f p C P A M.E. on Try accumulation by cortical cell suspension. Incubations were done in two different ranges of Try concentrations, a: high-affinity process. Try varies from 6 # M to 40 /~M. Control ( A); 50 f f M p C P A M.E. (0). b: low-affinity process. Try varies from 0.5 m M t o 2 raM; control ( i ) , 1 m M p C P A M.E. (0). Blanks were subtracted; each point is the mean of 4 determinations. The double reciprocal plot shows that pCPA M.E. stimulates Try accumulation without changing the maximal velocity.
In contrast, pCPA-methyl ester (pCPA M.E.), the soluble derivative ofpCPA, instead of inhibiting Try uptake, slightly increased the accumulation of this amino acid both in cultured cells and in the cortical cell suspension: this effect was seen for the low-affinity process (Fig. 4b) as well as for the high-affinity one (Fig. 4a). There was no change in Try uptake in synaptosomal preparations.
Effect of other compounds Norepinephrine and the two indoleamines, serotonin and tryptamine, added even at high concentrations (1 mM), did not alter Try transport into glial cells. Dibutyryl cyclic AMP (0.1-1 raM)did not affect Try accumulation neither in cultured cells, nor in cortical preparations.
TRYPTOPHAN ACCUMULATION BY GLIAL CELLS
261
DISCUSSION
Tryptophan transport in C6 glial cell line was found to be a very rapid process, the maximal level of intracellular Try being reached within 2 min. In determining the kinetic parameters of Try accumulation, we observed two transport systems with different velocities and affinities for the amino acid. Time course and kinetic parameters of the low affinity process in C6 clone were identical to that of 3T3 fibroblasts and of synaptosomes. On the contrary, when highaffinity processes are considered, Km for the C6 and 3T3 lines (7/~M) differed from the figures in synaptosomes (52 # M ) by one order of magnitude. Kinetic parameters for both Try uptake processes were found to be identical in crude synaptosomal fraction (P2) and in purified synaptosomes (Table I); moreover, our values are in agreement with published data2, 21. The presence of the same high-affinity transport for Try in glial cells and fibroblasts indicates that this process is not only operating in serotonergic neurons, but also in other cells. Moreover, Try uptake by a high-affinity process (Kin: 15 # M ) has been observed in preliminary experiments in a cholinergic (S-20) as well as in an adrenergic (N1E-115) neuroblastoma clone (generously supplied by M. Nirenberg). These data are in agreement with recent reports22: Try accumulation in synaptosomal preparations obtained from rat forebrains is not changed after destruction of serotonergic pathways by raphe lesions. In view of the fact that the C6 glial cell line did not differ in any respect from the 3T3 fibroblasts, as far as Try accumulation is concerned, we investigated the role of possible alterations of membrane properties due to growth in vitro. We then studied freshly separated cortical cell suspensions, and found that their behavior was similar to that of the cloned cells or of synaptosomes: when we considered parameters such as value of Km1 and changes in Try accumulation after K + increase in the incubating medium, cortical cell suspension seemed to have a synaptosome-like reactivity; but their sensitivity to pCPA M.E. was the same as cloned ceils. Moreover, such a mixture of cells, either viable or non-viable, is likely to be contaminated with intact nuclei and particulate debris 1. Thence, one would suspect that some types of membranes in this preparation are different from that of synaptosomes. Whereas cloned lines are growing as isolated cells, with free diffusion of amino acids between cells and the medium, it is tempting to consider Try accumulation in slices, because they constitute a more organized material with various types of neural cells in close contact and an extracellular compartment, evoking the in vivo situation. While no differences are seen for the inhibitory effect of phenylalanine and pCPA in slices ~°, synaptosomes 12, or cells on the two Try transport systems, marked differences exist for other parameters: (1) in slices, Try accumulation increases gradually for at least 30 min 20, the KmI value being around 0.1 m M (Hery et al., personal communication). (2) It is reduced in Na + free medium19, 20 contrary to synaptosomes 12 or cells (Table III). (3) Whereas incubation in Ca e+ free medium slightly increases Try accumulation in brain slices (unpublished observations), it has no effect on synaptosomal uptake 12 (Table III); furthermore, it reduces Try accumulation in cloned cells, by enhancing passive diffusion of the amino acid (Table IV). (4) In high K + concentration,
262
A. BAUMANet al.
Try accumulation is reduced in slices (unpublished observations) and in both cortical preparations (Table III) as a consequence of Try leakage induced by membrane depolarization; on the other hand, we saw no effect on cloned cells. (5) Eventually, while addition of dibutyryl cAMP increases Try accumulation in slicesg, 15, we saw no effect on our preparations. These differences stress the importance of membrane interactions between different cell types. Finally, our findings show that the Kin1 value for cortical preparations is in the range of physiological tryptophan concentrations (21 /~M in brain tissue11), while that of cultured cells is close to the concentration of Try in F10 supplemented with 10~ fetal calf serum (4 #M). Moreover, the Lineweaver-Burk representation gives a two-linear section curve for Try accumulation, even for a homogenous cell population (cloned cells): this has been shown to occur in allosteric negative cooperativityT, 24. Of particular interest are then the findings ~7 showing that Try accumulation in brain slices fluctuates following a nycthemeral rhythm, and induces parallel modifications in 5-HT synthesis. We would suggest that in physiological conditions, where glial cells are located between capillaries and neurons, variations of plasmatic free Try could modify the Try carrier activity in glial cells, thence leading to changes in 5-HT synthesis. ACKNOWLEDGEMENTS
The authors would like to thank Mrs. E. Rosenbaum for her excellent technical assistance. This research was supported by grants from DGRST and INSERM. REFERENCES 1 BARKLEY,D. S., RAKIC, L. L., CHAFFEE,J. K., AND WONG, D. L., Cell separation by velocity sedimentation of post-natal mouse cerebellum, J. Cell Physiol., 81 (1973) 271-279. 2 BELIN, M. F., ET PUJOL, J. F., Transport synaptosomal du tryptophane et de la tyrosine c6r6brale. Existence de systSmes de capture d'affinit6 diff6rente, Experientia (Basel), 29 (1973) 411-413. 3 BENDA, P., LIGHTBODY,J., SATO, G., LEVINE, L., AND SWEET, W. H., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 4 BENDA, P., SOMEDA,K., MESSER, J., AND SWEET, W. H., Morphological and immunochemical studies of rat glial tumors and clonal strains propagated in culture, J. Neurosurg., 34 (1971) 310320. 5 CARLSSON,A., AND LINDQVIST, M., The effect of L-tryptophan and some psychotropic drugs on the formation of 5-hydroxytryptophan in the mouse brain in vivo, J. Neurol. Transmission, 33 (1972) 23-43. 6 DEVELLIS,J., AND INGLISH,D., Effect of cortisol and epinephrine on the biochemical differentiation of cloned glial cells in culture and of the developing rat brain, SecondInt. Meeting Soc. Neurochem., (1969) 151. 7 ENGEL, P. C., AND FERDINAND, W., The significance of abrupt transitions in Lineweaver-Burk plots with particular reference to glutamate dehydrogenase. Negative and positive cooperativity in catalytic rate constants, Biochem. J., 131 (1973) 97-105. 8 FERNSTROM,J. D., AND WURTMAN,R. J., Brain serotonin content: Physiological dependence on plasma Try levels, Science, 173 (1971) 149-152. 9 FORN, J., TAGLIAMONTE,P., TAGLIAMONTE,A., AND GESSA,G. L., Stimulation by dibutyryl cyclic AMP of serotonin synthesis and tryptophan transport in brain slices, Nature New Biol., 237 (1972) 245-247.
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10 GILMAN, A. G., AND N1RENBERG, M., Effect of catecholamines on the adenosine 3',5'-.cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2165-2168. 11 GRAHAME-SMITH,D. G., Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan, J. Neurochem., 18 (1971) 1053-1066. 12 GRAHAME-SMITH,D. G., AND PARFITT, A. G., Tryptophan transport across the synaptosomal membrane, J. Neurochem., 17 (1970) 1339-1353. 13 GRAY, E. G., AND WHITTAKER,V. P., The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.) , 96 (1962) 79-88. 14 HAMBERGER,A., Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex, Brain Research, 31 (1971) 169-178. 15 HAMON,M., BOURGOIN,S., AND GLOWINSKI,J., Feed-back of 5-HT synthesis in rat striatal slices, J. Neurochem., 20 (1973) 1727-1745. 16 Ht/NN, F. A., AND HAMBERGER,A., Glial cell function: uptake of transmitter substances, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2686--2690. 17 HERY, F., ROUER, E., AND GLOWINSKI, J., Daily variations of serotonin metabolism in the rat brain, Brain Research, 43 (1972) 445-465. 18 .lARD,S., ~.EMONT, J., AND BENDA, P., Adenylate cyclase, phosphodiesterases and protein kinase of rat glial cells in culture, FEBS Letters, 26 (1972) 344-348. 19 JOANNY,P., BARBOSA,E., ET CORRIOL,.l., Accumulation active de quelques acides amin6s dans les coupes de cerveau de Rat, J. Physiol. (Paris), 60 (1968) 265. 20 KtELY, M., AND SOURKES, T. L., Transport of L-tryptophan into slices of rat cerebral cortex, J. Neurochem., 19 (1972) 2863-2872. 21 KNAPP, S., AND MANDELL,A. J., Narcotic drugs: effects on the serotonin biosynthetic systems of the brain, Science, 177 (1972) 1209-1211. 22 KUHAR, M. H., ROTH, R. H., AND AGHAJANIAN,G. K., Synaptosomes from forebrains of rats with midbrain raphe lesions: selective reduction of serotonin uptake, J. Pharmacol. exp. Ther., 181 (1972) 3~45. 23 LOWRY,O. H., ROSEBROUGH,N..l., FARR, A. L., AND RANDALL,R. J., Protein measurement with the folin Phenol reagent, J. biol. Chem., 193 (1951) 265-275. 24 TEIPEL, J., AND KOSHLAND,O. E., JR., The significance of intermediary plateau regions in enzyme saturation curves, Biochemistry, 8 (1969) 4656-4663. 25 TYCE, G. M., FLOCK, E. V., OWEN, C. A., JR., STOBIE,G. H. C., AND DAVID, C., 5-Hydroxyindole metabolism in the brain after hepatectomy, Biochem. Pharmacol., 16 (1967) 979-992.