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Synthesis and release of serotonin by brain slices: Effect of ionic manipulations and cationic ionophores
Brain Research, 172 (1979) 461-469 © Elsevier/North-HollandBiomedicalPress
461
SYNTHESIS AND RELEASE OF SEROTONIN BY BRAIN SLICES: EFFECT OF IONIC MANIPULATIONS AND CATIONIC IONOPHORES
MARTHA L. ELKS, WILLIAM. W. YOUNGBLOOD and JOHN S. KIZER* Departments of Pharmacology and Medicine, and The Neurobiology Program and the Biological Sciences Research Center, University of North Carolina, School of Medicine, Chapel Hill, N.C. 27514 (U.S.A.)
(Accepted December21st, 1978)
SUMMARY The effect of various ionic manipulations and cationic ionophores on the rates of release and synthesis of serotonin were investigated in brain slices prepared from adult male rats. Two depolarizing, univalent cationic ionophores, gramacidin (10 #g/ml) and valinomycin (10 #g/ml), and the non-depolarizing, calcium-specific ionophore, A23187 (190/aM), stimulated both the release and synthesis of serotonin in this tissue preparation. Electrical field depolarization of the brain slices also stimulated both the release and synthesis of serotonin, effects which were completely blocked by a calciumfree media and 1 mM EGTA or by 10 mM Mg 2+. Lithium partially blocked the release of serotonin by the stimulated brain slices, but markedly augmented the rates of serotonin biosynthesis. Although electrical stimulation significantly increased the rate of [3H]tryptophan uptake by the slices, the increased rates of release and synthesis of serotonin following treatment of the tissues with the ionophores and lithium were not associated with increased rates of tryptophan uptake. Furthermore, despite the inhibition of the release and synthesis of serotonin by stimulated slices incubated in the presence of magnesium or in the absence of calcium, rates of tryptophan uptake by the stimulated brain slice remained increased. These results taken together with the results of other studies suggest that release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the ionic events attending depolarization and further argue than transcellular calcium fluxes may be of special importance in this regulation. These results do not support the postulate that regulation of serotonin release and synthesis is dependent upon substrate availability as a major variable.
* To whom correspondenceshould be addressed at: Biological Sciences Research Center, 220H, Division of Health Affairs, University of North Carolina, Chapel Hill, N.C. 27514 U.S.A.
462 INTRODUCTION Current evidence supports the proposal that rates of synthesis of serotonin in serotonergic neurons are closely coupled to the electrical activity of the cell. For example, stimulation of the midbrain raphe nuclei results in a rapid increase in the rate of serotonin synthesis in the terminal projections of these nuclei6,13. Conversely, abrupt decreases in the rates of serotonin synthesis in the terminal projections of serotonergic neurons are observed following acute transection of serotonergic fiber tractsL Recent studies suggest that acute alterations in the rate of serotonin biosynthesis which accompany changes in the electrical activity of the serotonergic neuron are independent of changes in tryptophan uptake or availability, and argue that other cellular events attending depolarization of the serotonergic neuron activate the serotonergic biosynthetic pathway - - perhaps through an activation of tryptophan hydroxylase5. Because of the recent suggestion that diavalent cations may play a role in the regulation of tryptophan hydroxylase in vitro 2 and the obvious central importance of ion fluxes in the cellular events attending depolarization, we systematically studied the effects of ionic manipulations on the rates of synthesis and release of serotonin in brain slices. Our findings form the basis of this report. METHODS
Preparation of slices Adult male Sprague-Dawley rats, weighing approximately 150-300 g were housed and fed under standard lighting and feeding conditions. Between 09.00 and 11.30 h the rats were decapitated and the brains quickly removed and placed in a beaker of cold buffer. Brain slices were harvested from each side of a parasagittal cut as previously described5, clamped in a Mcllwain9 tissue electrode and incubated in a modified Krebs-Henseleit buffer, continuously bubbled with warm humidified 95 ~o 02-5 ~ C O z for oxygenation and agitation. The composition of the buffer was as follows: NaC1, 140 mM; KC1, 4.75 mM; K2HPOa, 1.2 mM; MgClz, 1.2 mM; HEPES, 25 mM; glucose, 10 mM; CaC12, 0.75 raM, and ascorbate, 5 mM (added as an antioxident). When used, the concentration of pargy!ine was 5 × 10-5 M.
Experimental protocol To allow recovery from the trauma of isolation, tissue slices were preincubated for 15 min prior to being subjected to the following protocol: tissues were subjected to 3 experimental cycles, each consisting of 15 min rest and 15 min stimulation by electrical field depolarization. At the end of each interval, the buffer was removed for assay and immediately replaced with fresh warm oxygenated buffer. Electrical field depolarization was provided by alternating DC pulses, generated as previously describedL Lithium, magnesium, and EGTA, when used, were present in the incubation media throughout both the resting and stimulation phases of the cycle. Gramacidin, valinomycin (Sigma, St. Louis, Mo.) and the calcium ionophore A23187 (a generous gift of the Eli Lilly and Co., N. Chicago, I11.) were prepared in 50-fold
463 concentrates and added to the incubation media during the 5-min period corresponding to electrical field stimulation. Final concentrations were gramacidin (10 #g/ml), valinomycin (10 #g/ml) and A23187 (190 #M). During a drug stimulation period, exposure to the ionophore was substituted for the electrical field stimulation. Tissues incubated in 1 ~ dimethylsulphoxide served as a control for A23187. Following exposure to gramacidin, valinomycin and A23187 the tissues were quickly rinsed with incubation buffer to insure the absence of these compounds during the resting part of the cycle.
Assay methods' At the end of the incubation, tissue tryptophan was measured by modification5 of the method of Denckla and Dewey4 and tissue protein by the method of Lowrys. Tissue and buffer samples were assayed for serotonin by modifications5 of the methods of Saavedra et al.lZ. Estimates of tryptophan uptake and rates of serotonin synthesis were determined by methods as previously described 5. Statistics Data were tested for inhomogeneity of variance by the Bartlett's test or by the Ftest. In most instances a systematic inhomogeneity of variance existed due to a linear relationship between the magnitude of the mean and magnitude of the standard deviation. In these cases, natural log transformation was carried out before analysis to stabilize the variance. Where appropriate, data were analyzed using Student's t-test, or one-way analysis of variance with repeated measures, Significant differences following analysis of variance were identified with the Sheffe multiple comparison procedure 1°, 14
RESULTS
In confirmation of our previous studiesS, both electrical field depolarization and 50 mM potassium in the incubation media stimulated the release of serotonin and simultaneously increased rates of serotonin synthesis in brain slices (data not presented). Similarly, the addition of the depolarizing, univalent cation ionophores 7, gramacidin and valinomycin stimulated the release of serotonin from brain slices from basal values of approximately 40 4- 10 fmol/mg protein/min to as high as 306 4- 54 fmol/mg protein/mg-1 (Table I). These increases in the rate of serotonin release following exposure to the depolarizing ionophores were also accompanied by increases in the rates of serotonin synthesis from values of approximately 12 pmol/mg protein/h to as high as 19 pmol/mg protein/h (Table I). In order to evaluate the possible role of calcium in the regulation of serotonin synthesis and release following depolarization, a relatively calcium specific, nondepolarizing ionophorO1, A23187 was added to the incubation media. This calcium ionophore dramatically increased the rate of serotonin release from basal values of 64 4- 24 fmol/mg protein/min to as high as 496 4- 124 fmol/mg protein/min. The rates of serotonin synthesis were also dramatically increased from basal values of 12.2 4- 1.2 to
464 TABLE I Release and synthesis of serotonin following exposure to brain slices" to either gramacidin (10 #g/ml) or valinomycin (10 i~g/ml)
Release expressed in this and all subsequent tables as X 4- S.E.M. fmol/mg protein/rain. Synthesis expressed in this and all subsequent tables as .X 4- S.E.M. pmol/mg protein/hr. Tryptophan cone = 0. Pargyline cone = 0. n -- 6 for all determinations. Observation period
Gramacidin
BasalI Drug I Basal II Drug lI
40 295 37 296
Valinomycin
Serotonin release
4± 44-
10 55** 8 87**
Synthesis
Serotonin release
Synthesis
12.4 4- 2.0 18.4 4- 1.5"
75 306 41 196
11.9 ± 2.1 19.1 ± 1.2"
± 20 -+- 54** 4- 9 ~_ 63**
* Significantly different from control value, P _< 0.05. ** Significantly different from control value, P < 0.01.
22.3 ± 1.4 p m o l / m g p r o t e i n / h (P < 0.01) (Table II). Both the rates of release o f serotonin a n d the rate of serotonin biosynthesis in the presence of the calcium ionophore, A23187, were significantly higher t h a n similar values for release a n d synthesis of serotonin following electrical field depolarization or the addition of the u n i v a l e n t depolarizing ionophores (P < 0.01). To further investigate the i m p o r t a n c e of calcium in the i n i t i a t i o n of the events leading to increases in the rates of release a n d synthesis of serotonin, b r a i n slices were subjected to electrical field depolarization in the presence of a calcium-free media a n d 1 m M E G T A . I n the absence of calcium a n d in the presence of E G T A , electrical field depolarization failed to stimulate the release of serotonin a n d failed to increase rates of serotonin biosynthesis i n the b r a i n slice p r e p a r a t i o n (Table III). F u r t h e r m o r e , the a d d i t i o n of 10 m M m a g n e s i u m to the i n c u b a t i o n media d u r i n g electrical field
TABLE II Release and synthesis of serotonin in brain slices following exposure to A23187 (190 # M )
n = 6 for all determinations. Observation period
Serotonin release
Serotonin synthesis
Basal I A23187 I Basal II A23187II Basal III A23187 III
64 496 70 470 59 250
12.2 4- 1.2 22.3 4- 1.4"
* P < 0.01.
444444-
24 124" 13 114" 11 37*
465 TABLE III Inhibition of release and synthesis of serotonin in brain slices exposed to EGTA and calcium-free medium during electrical field stimulation
n -- 6 for all determinations. Observation period
Release
Synthesis
Control
Basal I Stimulation I BasalII Stimulation II Basal III Stimulation III
86 346 101 349 110 496
4. 4. ± ± 54.
20 31"* 18 52** 14 60**
Calcium-free
Control
Calcium-free
118 125 124 115 110 135
16.2 4- 1.3
9.2 4- 1.2"
:~ 4. 4. 4. 4. 4.
18 34 14 7 9 17
* P -< 0.05. ** P < 0.01.
depolarization also blocked the release of serotonin from the tissue a n d prevented the increases in serotonin synthesis occurring with depolarization (Table IV). I n further studies the effect of 1 m M lithium on the release of serotonin following electrical field depolarization was examined. Electrical s t i m u l a t i o n released significantly less serotonin in the presence of l i t h i u m t h a n in the presence of a n o r m a l i n c u b a t i o n media (Table V). Nevertheless, despite the moderate i n h i b i t i o n of the release of serotonin in the presence of lithium, the rate of serotonin synthesis was dramatically increased over the rate of serotonin synthesis observed in stimulated tissue i n c u b a t e d in the absence of lithium (25.3 ± 2.5 vs 17.5 4- 1.9 p m o l / m g p r o t e i n / h ; P < 0.01). I n the presence of b o t h lithium a n d E G T A , electrical field depolarization failed to stimulate a n y release of serotonin into the i n c u b a t i o n media. F u r t h e r m o r e , E G T A completely blocked the increase in serotonin synthesis observed following electrical
TABLE IV Inhibition of release and synthesis of serotonin in brain slices exposed to 10 m M Mg 2+ during electrical field depolarization
n : 6 for all determinations. Observation period
Release
Synthesis
Control
Basal I Stimulation I BasallI Stimulation II * P < 0.02. ** P < 0.01.
78 250 64 278
444. 4-
14 40 12 32
10 m M Mg ~+
Control
l O m M M g 2+
59 43 39 50
16.2 4. 1.2
12.6 4- 0.8*
4, ± 44-
17 10'* 9 17'*
466 TABLE V Effect of lithium ( 1 raM) on the release and synthesis of serotonin by brain slices following electrical field stimulation Observation period
Basal I Stimulation I Basal II Stimulation II Serotoninsynthesis*
Release Normal media
(n)
Lithium (1 raM)
(n)
Lithium (1 raM) (n) + EGTA (l mM) (0 Ca 2+)
140 276 68 244 17.5
(6) (6) (6) (6) (6)
68 178 23 170 25.3
(6) (6) (6) (6) (6)
114 63 70 88 8.4
4- 20 -4- 63** 4- 28 4- 17"* 4- 1.9
4± 4± 4-
9 41 9 12 2.5
444± -4-
25 16 20 22 1.1
(5) (5) (5) (5) (5)
* Each value in this line significantly differs from the other two (P _< 0.01). ** Significantly greater than corresponding value for lithium treatment and for lithium 4- EGTA ( e <_ 0.01). d e p o l a r i z a t i o n o f tissue slices i n c u b a t e d in the presence o f lithium. Thus, a calciumfree m e d i u m b l o c k e d b o t h the release a n d increase in synthesis o f s e r o t o n i n o b s e r v e d following electrical d e p o l a r i z a t i o n o f tissue slices t r e a t e d with lithium. I n further studies, the effects o f electrical field d e p o l a r i z a t i o n , lithium a n d E G T A , a n d the calcium i o n o p h o r e A23187 on t r y p t o p h a n u p t a k e were d e t e r m i n e d (Table VI). In the presence o f n o r m a l i n c u b a t i o n m e d i a electrical field d e p o l a r i z a t i o n increased the rate o f t r y p t o p h a n u p t a k e f r o m 310 4- 13 to 445 ~ 22 p m o l / m g protein. A l t h o u g h the presence o f E G T A in the i n c u b a t i o n m e d i a c o m p l e t e l y b l o c k e d the release a n d biosynthesis o f s e r o t o n i n following electrical d e p o l a r i z a t i o n , the increased rate o f t r y p t o p h a n u p t a k e was u n d i m i n i s h e d . L i t h i u m (1 m M ) also a p p e a r e d to have n o significant effect u p o n the rate o f t r y p t o p h a n u p t a k e b y the s t i m u l a t e d b r a i n slice TABLE VI Effect of electrical field depolarization, lithium, A23187 and calcium + EGT.4 on tryptophan uptake by brain slices
Tryptophan uptake expressed as pmol/mg protein. Treatment
Normal media No calcium ÷ EGTA Lithium (1 mM) A23187(absent)* A23187(present)
Uptake Nonstimulated
( n)
Electrically stimulated
( n)
310 ± 13
(6)
445 ± 22**
(6)
335 324 424 384
(6) (5) (6) (6)
506 4- 32** 471 4- 20** ---
(6) (5)
4- 16 4- 18 4-4- 14 4- 24
* 1 ~ DMSO served as control (see Methods). ** Significantly different from corresponding control value (P < 0.05).
467 preparations, even though serotonin release was moderately inhibited and serotonin synthesis stimulated by depolarization in the presence of lithium. Finally, although the relatively specific calcium ionophore, A23187 markedly stimulated both the release and synthesis of serotonin, there was no significant increase in the rate of tryptophan uptake in the drug-treated slice compared to the control slice. It is important to note that the basal unstimulated value for the rate of tryptophan uptake in the presence of 1 ~ dimethylsulphoxide (a control for the calcium ionophore) was significantly increased. DISCUSSION Previous studies have suggested that the release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the events surrounding depolarization1,1a. In a previous study we have demonstrated that tryptophan uptake and availability are not significant variables in the regulation of serotonin release and synthesis following electrical field depolarizationL The purpose of the present study was to explore the cellular ionic events attending depolarization of the serotonergic neuron and to determine the possible importance of specific ion fluxes in the regulation of serotonin release a n d synthesis by the serotonergic neuron. Gramacidin and valinomycin stimulated the release and biosynthesis of serotonin in brain slices. Both of these compounds are univalent cation ionophores which are thought to induce depolarization of excitable membranes by increasing transmembrane conductances for either sodium or potassium 7. Although the exact mechanism of action of these two compounds is not clear, their ability to stimulate both release and synthesis in brain slices suggests that a means of depolarization other than potassium or application of an electrical field also activate the mechanisms controlling the regulation of tryptophan hydroxylase. The relatively specific calcium ionophore, A23187, has been shown to increase the intracellular conductances for calcium without significantly altering the resting membrane potential of the excitable celP t. In our preparation, A23187 markedly increased both the rates of serotonin release and serotonin biosynthesis, arguing that transcellular fluxes of calcium may be of importance in regulating the release of serotonin and increasing the rate of serotonin synthesis. Further support for the hypothesis that the transcellular fluxes of calcium attending depolarization are responsible for increasing both serotonin biosynthesis and its rate of release is provided by the inhibitory effects of EGTA and magnesium on the release and synthesis of serotonin following electrical stimulation of the slice. Both the absence of calcium and the presence of magnesium blocked the stimulated increases in synthesis and release of serotonin arguing that the event responsible for the initiation of both these processes is a transcellular flux of calcium. Lithium is thought to have two major ionic effects upon the excitable cell; the first is a reduction of resting membrane potential and the second is an increase in intracellular calcium, perhaps as a result of the first action3. In electrically stimulated brain slices, we have demonstrated an inhibition of the rate of serotonin release in the
468 presence of 1 mM lithium, yet a dramatic enhancement of the rate of serotonin synthesis, demonstrating that although synthesis and release usually proceed 'hand-inhand' it is possible to clearly dissociate the two phenomena. Furthermore, the increase in serotonin biosynthesis induced by lithium'was completely blocked by E G T A further supporting the argument that intracellular calcium is important in the activation of the rate limiting step in the serotonin biosynthetic pathway. To provide further evidence for the dissociation between tryptophan uptake and/or availability and the rate of serotonin biosynthesis and release, tryptophan uptake was examined in the absence of calcium, or in the presence of lithium or A23187. Although the absence of calcium prevented both the increase in serotonin synthesis and release during depolarization of the brain slice, the rate of tryptophan uptake was unaltered. Similarly, although lithium partially blocked the release of serotonin and markedly augmented the synthesis of serotonin accompanying depolarization of the slices, tryptophan uptake was unaltered. Finally, despite the marked stimulation of release and biosynthesis of serotonin by A23187, there was no significant difference between the rate of uptake of tryptophan between the treated and control brain slices. These data provide further evidence that the synthesis and release of serotonin are tightly coupled to the ionic events surrounding electrical depolarization and further suggest that the transcellular flux of calcium during depolarization serves to trigger the release of serotonin and activate the serotonergic biosynthetic pathway. Our results provide no support for the postulate 15 that these increases in serotonin biosynthesis and release are dependent upon substrate availability or uptake. In a previous study, we have found that the apparent Km for tryptophan in the stimulated brain slice is considerably reduced when compared to the unstimulated control. It is possible that following depolarization, transcellular fluxes of calcium increase the synthesis of serotonin through a direct or indirect allosteric activation of tryptophan hydroxylase. Proof of such a hypothesis will require further study, ACKNOWLEDGEMENTS We wish to acknowledge the support and encouragement of Dr. Morris A. Lipton, Director of the Biological Sciences Research Center, and the essential help of Ms. Marty Byrd and Ms. Pamela Bullock in the preparation of this manuscript. This work was supported by Grants MH2889 from N I M H , HD10570 from N I C H D and the BSRC Core Grant HD03110 from NICHD. J.S.K. is the recipient of a Research Scientist Career Development Award, MH-00114; and M.L.E. was supported by an N I H Neurobiology Fellowship, MH14277. REFERENCES 1 Anden, N.-E., Fuxe, K. and Hokfelt, T., The importance of nervous impulse flow for the depletion of monoamines from central neurons by some drugs, J. Pharm. PharmacoL, 18 (1966) 630-632. 2 Boadle-Biber, M. C., Effect of calcium on tryptophan hydroxylase from rat hind brain, Biochem. PharmacoL, 24 (1975) 1455-1460.
469 3 Branisteanu, D. D. and Voile, R. L., Modification by lithium of transmitter release at the neuromuscular junction of the frog, J. Pharmacol. exp. Ther., 194 (1975) 262-269. 4 Denkla, W. D. and Dewey, H. K., The determination of tryptophan in plasma liver and urine, J. Lab. din. Med., 69 (1967) 160-169. 5 Elks, M. L. and Kizer, J. S., Release and biosynthesis of serotonin in brain slices: independence of tryptophan, Brain Research, 172 (1979) 471-486. 6 Herr, B. E. and Roth, R. H., The effect of acute raphe lesions on serotonin synthesis and metabolism in the rat forebrain and hippocampus, Brain Research, 110 (1976) 189-193. 7 Li, P. P. and White, T. D., Rapid effect of veratridine, tetrodotoxin, gramacidin D, valinomycin, and NaCN on the Na, K, and ATP content of synaptosomes J. Neurochem., 28 (1977) 967-975. 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265. 9 Mcllwain, H., Practical Neurochemistry, Churchill Livingstone, New York, 1975. 10 Neter, J. and Wasserman, W., In D. Irvin (Ed.), Applied Linear StatisticalModels, Richard Homewood, I11., 1974. 11 Pressman, B. C. Biological application of ionophores, Ann. Rev. Biochem., 45 (1976) 531-527. 12 Saavedra, J. M., Brownstein M. and Axelrod, J., A specific and sensitive enzymatic-isotopic microassay for 5-hydroxytryptamine in tissue, J. Pharmacol. exp. Ther. 186 (1973) 508-515. 13 Sheard, M. H. and Aghajanian, G. K., Stimulation of the midbrain raphe: effect of 5-HT metabolism, J. Pharmacol. exp. Ther., 163 (1968) 425-430. 14 Snedecor, G. W. and Cochran, W. G., Statistical Methods, Iowa State University Press, Ames, Iowa, 1967. 15 Wurtman, R. J. and Fernstrom, J. D., Control of brain neurotransmitter synthesis by precurscr availability and nutritional status, Biochem. Pharmacol., 25 (1976) 1691-1696.
461
SYNTHESIS AND RELEASE OF SEROTONIN BY BRAIN SLICES: EFFECT OF IONIC MANIPULATIONS AND CATIONIC IONOPHORES
MARTHA L. ELKS, WILLIAM. W. YOUNGBLOOD and JOHN S. KIZER* Departments of Pharmacology and Medicine, and The Neurobiology Program and the Biological Sciences Research Center, University of North Carolina, School of Medicine, Chapel Hill, N.C. 27514 (U.S.A.)
(Accepted December21st, 1978)
SUMMARY The effect of various ionic manipulations and cationic ionophores on the rates of release and synthesis of serotonin were investigated in brain slices prepared from adult male rats. Two depolarizing, univalent cationic ionophores, gramacidin (10 #g/ml) and valinomycin (10 #g/ml), and the non-depolarizing, calcium-specific ionophore, A23187 (190/aM), stimulated both the release and synthesis of serotonin in this tissue preparation. Electrical field depolarization of the brain slices also stimulated both the release and synthesis of serotonin, effects which were completely blocked by a calciumfree media and 1 mM EGTA or by 10 mM Mg 2+. Lithium partially blocked the release of serotonin by the stimulated brain slices, but markedly augmented the rates of serotonin biosynthesis. Although electrical stimulation significantly increased the rate of [3H]tryptophan uptake by the slices, the increased rates of release and synthesis of serotonin following treatment of the tissues with the ionophores and lithium were not associated with increased rates of tryptophan uptake. Furthermore, despite the inhibition of the release and synthesis of serotonin by stimulated slices incubated in the presence of magnesium or in the absence of calcium, rates of tryptophan uptake by the stimulated brain slice remained increased. These results taken together with the results of other studies suggest that release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the ionic events attending depolarization and further argue than transcellular calcium fluxes may be of special importance in this regulation. These results do not support the postulate that regulation of serotonin release and synthesis is dependent upon substrate availability as a major variable.
* To whom correspondenceshould be addressed at: Biological Sciences Research Center, 220H, Division of Health Affairs, University of North Carolina, Chapel Hill, N.C. 27514 U.S.A.
462 INTRODUCTION Current evidence supports the proposal that rates of synthesis of serotonin in serotonergic neurons are closely coupled to the electrical activity of the cell. For example, stimulation of the midbrain raphe nuclei results in a rapid increase in the rate of serotonin synthesis in the terminal projections of these nuclei6,13. Conversely, abrupt decreases in the rates of serotonin synthesis in the terminal projections of serotonergic neurons are observed following acute transection of serotonergic fiber tractsL Recent studies suggest that acute alterations in the rate of serotonin biosynthesis which accompany changes in the electrical activity of the serotonergic neuron are independent of changes in tryptophan uptake or availability, and argue that other cellular events attending depolarization of the serotonergic neuron activate the serotonergic biosynthetic pathway - - perhaps through an activation of tryptophan hydroxylase5. Because of the recent suggestion that diavalent cations may play a role in the regulation of tryptophan hydroxylase in vitro 2 and the obvious central importance of ion fluxes in the cellular events attending depolarization, we systematically studied the effects of ionic manipulations on the rates of synthesis and release of serotonin in brain slices. Our findings form the basis of this report. METHODS
Preparation of slices Adult male Sprague-Dawley rats, weighing approximately 150-300 g were housed and fed under standard lighting and feeding conditions. Between 09.00 and 11.30 h the rats were decapitated and the brains quickly removed and placed in a beaker of cold buffer. Brain slices were harvested from each side of a parasagittal cut as previously described5, clamped in a Mcllwain9 tissue electrode and incubated in a modified Krebs-Henseleit buffer, continuously bubbled with warm humidified 95 ~o 02-5 ~ C O z for oxygenation and agitation. The composition of the buffer was as follows: NaC1, 140 mM; KC1, 4.75 mM; K2HPOa, 1.2 mM; MgClz, 1.2 mM; HEPES, 25 mM; glucose, 10 mM; CaC12, 0.75 raM, and ascorbate, 5 mM (added as an antioxident). When used, the concentration of pargy!ine was 5 × 10-5 M.
Experimental protocol To allow recovery from the trauma of isolation, tissue slices were preincubated for 15 min prior to being subjected to the following protocol: tissues were subjected to 3 experimental cycles, each consisting of 15 min rest and 15 min stimulation by electrical field depolarization. At the end of each interval, the buffer was removed for assay and immediately replaced with fresh warm oxygenated buffer. Electrical field depolarization was provided by alternating DC pulses, generated as previously describedL Lithium, magnesium, and EGTA, when used, were present in the incubation media throughout both the resting and stimulation phases of the cycle. Gramacidin, valinomycin (Sigma, St. Louis, Mo.) and the calcium ionophore A23187 (a generous gift of the Eli Lilly and Co., N. Chicago, I11.) were prepared in 50-fold
463 concentrates and added to the incubation media during the 5-min period corresponding to electrical field stimulation. Final concentrations were gramacidin (10 #g/ml), valinomycin (10 #g/ml) and A23187 (190 #M). During a drug stimulation period, exposure to the ionophore was substituted for the electrical field stimulation. Tissues incubated in 1 ~ dimethylsulphoxide served as a control for A23187. Following exposure to gramacidin, valinomycin and A23187 the tissues were quickly rinsed with incubation buffer to insure the absence of these compounds during the resting part of the cycle.
Assay methods' At the end of the incubation, tissue tryptophan was measured by modification5 of the method of Denckla and Dewey4 and tissue protein by the method of Lowrys. Tissue and buffer samples were assayed for serotonin by modifications5 of the methods of Saavedra et al.lZ. Estimates of tryptophan uptake and rates of serotonin synthesis were determined by methods as previously described 5. Statistics Data were tested for inhomogeneity of variance by the Bartlett's test or by the Ftest. In most instances a systematic inhomogeneity of variance existed due to a linear relationship between the magnitude of the mean and magnitude of the standard deviation. In these cases, natural log transformation was carried out before analysis to stabilize the variance. Where appropriate, data were analyzed using Student's t-test, or one-way analysis of variance with repeated measures, Significant differences following analysis of variance were identified with the Sheffe multiple comparison procedure 1°, 14
RESULTS
In confirmation of our previous studiesS, both electrical field depolarization and 50 mM potassium in the incubation media stimulated the release of serotonin and simultaneously increased rates of serotonin synthesis in brain slices (data not presented). Similarly, the addition of the depolarizing, univalent cation ionophores 7, gramacidin and valinomycin stimulated the release of serotonin from brain slices from basal values of approximately 40 4- 10 fmol/mg protein/min to as high as 306 4- 54 fmol/mg protein/mg-1 (Table I). These increases in the rate of serotonin release following exposure to the depolarizing ionophores were also accompanied by increases in the rates of serotonin synthesis from values of approximately 12 pmol/mg protein/h to as high as 19 pmol/mg protein/h (Table I). In order to evaluate the possible role of calcium in the regulation of serotonin synthesis and release following depolarization, a relatively calcium specific, nondepolarizing ionophorO1, A23187 was added to the incubation media. This calcium ionophore dramatically increased the rate of serotonin release from basal values of 64 4- 24 fmol/mg protein/min to as high as 496 4- 124 fmol/mg protein/min. The rates of serotonin synthesis were also dramatically increased from basal values of 12.2 4- 1.2 to
464 TABLE I Release and synthesis of serotonin following exposure to brain slices" to either gramacidin (10 #g/ml) or valinomycin (10 i~g/ml)
Release expressed in this and all subsequent tables as X 4- S.E.M. fmol/mg protein/rain. Synthesis expressed in this and all subsequent tables as .X 4- S.E.M. pmol/mg protein/hr. Tryptophan cone = 0. Pargyline cone = 0. n -- 6 for all determinations. Observation period
Gramacidin
BasalI Drug I Basal II Drug lI
40 295 37 296
Valinomycin
Serotonin release
4± 44-
10 55** 8 87**
Synthesis
Serotonin release
Synthesis
12.4 4- 2.0 18.4 4- 1.5"
75 306 41 196
11.9 ± 2.1 19.1 ± 1.2"
± 20 -+- 54** 4- 9 ~_ 63**
* Significantly different from control value, P _< 0.05. ** Significantly different from control value, P < 0.01.
22.3 ± 1.4 p m o l / m g p r o t e i n / h (P < 0.01) (Table II). Both the rates of release o f serotonin a n d the rate of serotonin biosynthesis in the presence of the calcium ionophore, A23187, were significantly higher t h a n similar values for release a n d synthesis of serotonin following electrical field depolarization or the addition of the u n i v a l e n t depolarizing ionophores (P < 0.01). To further investigate the i m p o r t a n c e of calcium in the i n i t i a t i o n of the events leading to increases in the rates of release a n d synthesis of serotonin, b r a i n slices were subjected to electrical field depolarization in the presence of a calcium-free media a n d 1 m M E G T A . I n the absence of calcium a n d in the presence of E G T A , electrical field depolarization failed to stimulate the release of serotonin a n d failed to increase rates of serotonin biosynthesis i n the b r a i n slice p r e p a r a t i o n (Table III). F u r t h e r m o r e , the a d d i t i o n of 10 m M m a g n e s i u m to the i n c u b a t i o n media d u r i n g electrical field
TABLE II Release and synthesis of serotonin in brain slices following exposure to A23187 (190 # M )
n = 6 for all determinations. Observation period
Serotonin release
Serotonin synthesis
Basal I A23187 I Basal II A23187II Basal III A23187 III
64 496 70 470 59 250
12.2 4- 1.2 22.3 4- 1.4"
* P < 0.01.
444444-
24 124" 13 114" 11 37*
465 TABLE III Inhibition of release and synthesis of serotonin in brain slices exposed to EGTA and calcium-free medium during electrical field stimulation
n -- 6 for all determinations. Observation period
Release
Synthesis
Control
Basal I Stimulation I BasalII Stimulation II Basal III Stimulation III
86 346 101 349 110 496
4. 4. ± ± 54.
20 31"* 18 52** 14 60**
Calcium-free
Control
Calcium-free
118 125 124 115 110 135
16.2 4- 1.3
9.2 4- 1.2"
:~ 4. 4. 4. 4. 4.
18 34 14 7 9 17
* P -< 0.05. ** P < 0.01.
depolarization also blocked the release of serotonin from the tissue a n d prevented the increases in serotonin synthesis occurring with depolarization (Table IV). I n further studies the effect of 1 m M lithium on the release of serotonin following electrical field depolarization was examined. Electrical s t i m u l a t i o n released significantly less serotonin in the presence of l i t h i u m t h a n in the presence of a n o r m a l i n c u b a t i o n media (Table V). Nevertheless, despite the moderate i n h i b i t i o n of the release of serotonin in the presence of lithium, the rate of serotonin synthesis was dramatically increased over the rate of serotonin synthesis observed in stimulated tissue i n c u b a t e d in the absence of lithium (25.3 ± 2.5 vs 17.5 4- 1.9 p m o l / m g p r o t e i n / h ; P < 0.01). I n the presence of b o t h lithium a n d E G T A , electrical field depolarization failed to stimulate a n y release of serotonin into the i n c u b a t i o n media. F u r t h e r m o r e , E G T A completely blocked the increase in serotonin synthesis observed following electrical
TABLE IV Inhibition of release and synthesis of serotonin in brain slices exposed to 10 m M Mg 2+ during electrical field depolarization
n : 6 for all determinations. Observation period
Release
Synthesis
Control
Basal I Stimulation I BasallI Stimulation II * P < 0.02. ** P < 0.01.
78 250 64 278
444. 4-
14 40 12 32
10 m M Mg ~+
Control
l O m M M g 2+
59 43 39 50
16.2 4. 1.2
12.6 4- 0.8*
4, ± 44-
17 10'* 9 17'*
466 TABLE V Effect of lithium ( 1 raM) on the release and synthesis of serotonin by brain slices following electrical field stimulation Observation period
Basal I Stimulation I Basal II Stimulation II Serotoninsynthesis*
Release Normal media
(n)
Lithium (1 raM)
(n)
Lithium (1 raM) (n) + EGTA (l mM) (0 Ca 2+)
140 276 68 244 17.5
(6) (6) (6) (6) (6)
68 178 23 170 25.3
(6) (6) (6) (6) (6)
114 63 70 88 8.4
4- 20 -4- 63** 4- 28 4- 17"* 4- 1.9
4± 4± 4-
9 41 9 12 2.5
444± -4-
25 16 20 22 1.1
(5) (5) (5) (5) (5)
* Each value in this line significantly differs from the other two (P _< 0.01). ** Significantly greater than corresponding value for lithium treatment and for lithium 4- EGTA ( e <_ 0.01). d e p o l a r i z a t i o n o f tissue slices i n c u b a t e d in the presence o f lithium. Thus, a calciumfree m e d i u m b l o c k e d b o t h the release a n d increase in synthesis o f s e r o t o n i n o b s e r v e d following electrical d e p o l a r i z a t i o n o f tissue slices t r e a t e d with lithium. I n further studies, the effects o f electrical field d e p o l a r i z a t i o n , lithium a n d E G T A , a n d the calcium i o n o p h o r e A23187 on t r y p t o p h a n u p t a k e were d e t e r m i n e d (Table VI). In the presence o f n o r m a l i n c u b a t i o n m e d i a electrical field d e p o l a r i z a t i o n increased the rate o f t r y p t o p h a n u p t a k e f r o m 310 4- 13 to 445 ~ 22 p m o l / m g protein. A l t h o u g h the presence o f E G T A in the i n c u b a t i o n m e d i a c o m p l e t e l y b l o c k e d the release a n d biosynthesis o f s e r o t o n i n following electrical d e p o l a r i z a t i o n , the increased rate o f t r y p t o p h a n u p t a k e was u n d i m i n i s h e d . L i t h i u m (1 m M ) also a p p e a r e d to have n o significant effect u p o n the rate o f t r y p t o p h a n u p t a k e b y the s t i m u l a t e d b r a i n slice TABLE VI Effect of electrical field depolarization, lithium, A23187 and calcium + EGT.4 on tryptophan uptake by brain slices
Tryptophan uptake expressed as pmol/mg protein. Treatment
Normal media No calcium ÷ EGTA Lithium (1 mM) A23187(absent)* A23187(present)
Uptake Nonstimulated
( n)
Electrically stimulated
( n)
310 ± 13
(6)
445 ± 22**
(6)
335 324 424 384
(6) (5) (6) (6)
506 4- 32** 471 4- 20** ---
(6) (5)
4- 16 4- 18 4-4- 14 4- 24
* 1 ~ DMSO served as control (see Methods). ** Significantly different from corresponding control value (P < 0.05).
467 preparations, even though serotonin release was moderately inhibited and serotonin synthesis stimulated by depolarization in the presence of lithium. Finally, although the relatively specific calcium ionophore, A23187 markedly stimulated both the release and synthesis of serotonin, there was no significant increase in the rate of tryptophan uptake in the drug-treated slice compared to the control slice. It is important to note that the basal unstimulated value for the rate of tryptophan uptake in the presence of 1 ~ dimethylsulphoxide (a control for the calcium ionophore) was significantly increased. DISCUSSION Previous studies have suggested that the release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the events surrounding depolarization1,1a. In a previous study we have demonstrated that tryptophan uptake and availability are not significant variables in the regulation of serotonin release and synthesis following electrical field depolarizationL The purpose of the present study was to explore the cellular ionic events attending depolarization of the serotonergic neuron and to determine the possible importance of specific ion fluxes in the regulation of serotonin release a n d synthesis by the serotonergic neuron. Gramacidin and valinomycin stimulated the release and biosynthesis of serotonin in brain slices. Both of these compounds are univalent cation ionophores which are thought to induce depolarization of excitable membranes by increasing transmembrane conductances for either sodium or potassium 7. Although the exact mechanism of action of these two compounds is not clear, their ability to stimulate both release and synthesis in brain slices suggests that a means of depolarization other than potassium or application of an electrical field also activate the mechanisms controlling the regulation of tryptophan hydroxylase. The relatively specific calcium ionophore, A23187, has been shown to increase the intracellular conductances for calcium without significantly altering the resting membrane potential of the excitable celP t. In our preparation, A23187 markedly increased both the rates of serotonin release and serotonin biosynthesis, arguing that transcellular fluxes of calcium may be of importance in regulating the release of serotonin and increasing the rate of serotonin synthesis. Further support for the hypothesis that the transcellular fluxes of calcium attending depolarization are responsible for increasing both serotonin biosynthesis and its rate of release is provided by the inhibitory effects of EGTA and magnesium on the release and synthesis of serotonin following electrical stimulation of the slice. Both the absence of calcium and the presence of magnesium blocked the stimulated increases in synthesis and release of serotonin arguing that the event responsible for the initiation of both these processes is a transcellular flux of calcium. Lithium is thought to have two major ionic effects upon the excitable cell; the first is a reduction of resting membrane potential and the second is an increase in intracellular calcium, perhaps as a result of the first action3. In electrically stimulated brain slices, we have demonstrated an inhibition of the rate of serotonin release in the
468 presence of 1 mM lithium, yet a dramatic enhancement of the rate of serotonin synthesis, demonstrating that although synthesis and release usually proceed 'hand-inhand' it is possible to clearly dissociate the two phenomena. Furthermore, the increase in serotonin biosynthesis induced by lithium'was completely blocked by E G T A further supporting the argument that intracellular calcium is important in the activation of the rate limiting step in the serotonin biosynthetic pathway. To provide further evidence for the dissociation between tryptophan uptake and/or availability and the rate of serotonin biosynthesis and release, tryptophan uptake was examined in the absence of calcium, or in the presence of lithium or A23187. Although the absence of calcium prevented both the increase in serotonin synthesis and release during depolarization of the brain slice, the rate of tryptophan uptake was unaltered. Similarly, although lithium partially blocked the release of serotonin and markedly augmented the synthesis of serotonin accompanying depolarization of the slices, tryptophan uptake was unaltered. Finally, despite the marked stimulation of release and biosynthesis of serotonin by A23187, there was no significant difference between the rate of uptake of tryptophan between the treated and control brain slices. These data provide further evidence that the synthesis and release of serotonin are tightly coupled to the ionic events surrounding electrical depolarization and further suggest that the transcellular flux of calcium during depolarization serves to trigger the release of serotonin and activate the serotonergic biosynthetic pathway. Our results provide no support for the postulate 15 that these increases in serotonin biosynthesis and release are dependent upon substrate availability or uptake. In a previous study, we have found that the apparent Km for tryptophan in the stimulated brain slice is considerably reduced when compared to the unstimulated control. It is possible that following depolarization, transcellular fluxes of calcium increase the synthesis of serotonin through a direct or indirect allosteric activation of tryptophan hydroxylase. Proof of such a hypothesis will require further study, ACKNOWLEDGEMENTS We wish to acknowledge the support and encouragement of Dr. Morris A. Lipton, Director of the Biological Sciences Research Center, and the essential help of Ms. Marty Byrd and Ms. Pamela Bullock in the preparation of this manuscript. This work was supported by Grants MH2889 from N I M H , HD10570 from N I C H D and the BSRC Core Grant HD03110 from NICHD. J.S.K. is the recipient of a Research Scientist Career Development Award, MH-00114; and M.L.E. was supported by an N I H Neurobiology Fellowship, MH14277. REFERENCES 1 Anden, N.-E., Fuxe, K. and Hokfelt, T., The importance of nervous impulse flow for the depletion of monoamines from central neurons by some drugs, J. Pharm. PharmacoL, 18 (1966) 630-632. 2 Boadle-Biber, M. C., Effect of calcium on tryptophan hydroxylase from rat hind brain, Biochem. PharmacoL, 24 (1975) 1455-1460.
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