Brain Research, 517 (1990) 151-156 Elsevier
151
BRES 15483
Electrophysiological evidence for the existence of GABAA receptors in cultured frog melanotrophs Estelle Louiset 1, Frans H.M.M. van de Put 1'2, Marie-Christine Tonon 1, Corinne Basille 1, Bruce G. Jenks 2, Hubert Vaudry 1 and Lionel Cazin 1 ~Laboratoire d'Endocrinologie Mol~culaire, Unit~ Affili~e ~ I'INSERM, Universitd de Rouen, Mont-Saint-Aignan (France) and 2Laboratory of Animal Physiology, Faculty of Science, Catholic University, Nijmegen (The Netherlands) (Accepted 24 October 1989) Key words: Whole-cell patch-clamp; Chloride channel; Membrane potential; GABAA-receptor subtype; Pars intermedia cell
The neurotransminer GABA exerts a biphasic effect on a-melanocyte-stimulating hormone (a-MSH) secretion from pars intermedia cells: GABA induces a rapid and transient stimulation followed by a sustained inhibition of a-MSH release. In the present study, we have investigated the effect of GABA on the electrophysiological properties of frog melanotrophs in primary culture using the patch-clamp technique in the whole cell configuration. In all cells tested, GABA stimulated an inward current and induced depolarization. A transient period of intense firing was consistently observed at the onset of GABA administration. During the depolarization phase, the membrane potential reached a plateau corresponding to the CI- equilibrium potential. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the response evoked by GABA. The effect of GABA was abolished by the chloride channel blocker picrotoxin, and by antagonists of GABAA receptors (bicuculline and SR 95531). The depolarizing action of GABA was mimicked by muscimol, an agonist of GABA A receptors. Taken together, our results indicate that the rapid and transient stimulation of a-MSH release induced by GABA can be accounted for by activation of a chloride conductance which causes membrane depolarization. These data support the notion that the transient stimulation of a-MSH secretion induced by GABA can be accounted for by membrane depolarization which provokes activation of voltage-operated calcium channels. Since no evidence was found for GABA-induced hyperpolarization, the intracellular mechanisms leading to the strong inhibitory effect of GABA on a-MSH secretion remain to be elucidated.
INTRODUCTION The pars intermedia of the pituitary is supplied by a dense network of nerve fibers originating from the hypothalamus ~3'27. In mammals, the axon terminals, which make intimate contacts with the endocrine cells of the intermediate lobe, contain various classical neurotransmitters including catecholamines (mainly dopamine) g, serotonin 22 and y-aminobutyric acid ( G A B A ) 2°. In non-mammalian vertebrates, the pars intermedia is also innervated by fibers containing neuropeptides such as thyrotropin-releasing hormone 17A9'24, corticotropin-releasing factor 21'3°, mesotocinn,17 and neuropeptide y7,35, as well as classical neurotransmitters including dopamine 31 and G A B A ~,33. Most of the neuroendocrine signals contained in these nerve terminals influence the secretory activity of amphibian pituitary melanotrophs 16,29'3°. Since the secretion of melanotropic peptides is globally under negative hypothalamic control, the inhibitory factors (i.e. dopamine, G A B A and neuropeptide Y) likely play a pivotal role in the regulation of melanotropin secretion.
The involvement of G A B A in the regulation of the pars intermedia activity was first demonstrated by Davis and Hadley 8. In mammals, G A B A induces a biphasic effect on a-melanocyte-stimulating h o r m o n e (a-MSH) secretion: a transient stimulation followed by sustained inhibition 9'28. Electrophysiological studies on rat or porcine melanotrophs in primary culture, revealed that G A B A increases m e m b r a n e CI- permeability which causes a depolarization 1°'25'26. The effect of G A B A on chloride conductance is mimicked by isoguvacine 1° and muscimo126 and antagonized by bicuculline 1°'26, indicating that the response of pituitary cells is mediated through activation of G A B A A receptors. We have recently investigated the effect of G A B A on a - M S H release from the amphibian pars intermedia. In the toad X e n o p u s laevis, G A B A produces only inhibition of the secretory process 33'34 and the effect of G A B A appears to be mediated primarily through a G A B A A receptor mechanism 37. In contrast, in the frog Rana ridibunda, G A B A and G A B A A agonists induce a biphasic action on a - M S H secretion l'z. The question arises: are
Correspondence: L. Cazin, Laboratoire d'Endocrinologie Mol6culaire, CNRS URA 650, Unit6 Affili6e h I'INSERM, Facult6 des Sciences, Universit6 de Rouen, BP 118, F-76134 Mont-Saint-Aignan Cedex, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
152 both phases of biphasic phenomena a reflection of the action of a classical GABAA receptor? Electrophysiological techniques on cultured pars intermedia cells represent an ideal approach to answer this question. Using the patch-clamp technique, we have recently shown that frog melanotrophs spontaneously generate action potentials and we have identified the ionic currents underlying spontaneous electrical activity TM. The aim of the present study was to demonstrate the existence of GABA receptors on these cells and to determine the characteristics of the ionic conductances involved in the mechanism of action of GABA. MATERIALS AND METHODS Neurointermediate lobes were removed from 8 freshly decapitated male frogs Rana ridibunda and the pars intermedia cells were enzymatically dispersed, as previously described TM. The cells were plated at a density of 250,000 cells/ml on microwell Terasaki dishes containing Leibovitz medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (0.1 mg/ml kanamycine and 1% antibiotic-antimycotic solution). The cells were allowed to attach to the culture microwell for 24 h at 26 °C in a humidified atmosphere. The incubation medium was replaced by fresh medium every day. The cells were used for electrophysiologicai studies 2-7 days after plating. During electrophysiological recording, the cells were continuously superfused with standard Ringer's solution (in
mM: 112 NaCI, 2 KCI, 2 CaCI2, 15 HEPES) containing 11 mM glucose (pH adjusted to 7.4 with NaOH). The intracellular solution contained (in mM) 100 KCI, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES (pH adjusted to 7.4 with KOH). All recordings were obtained in the whole-cell configuration of the patch-clamp technique TM. In the present experimental conditions, the CIequilibrium potential was found to be 0 mV according to the Nernst equation. The current and voltage outputs were recorded from the patch-clamp amplifier (EPC 7, List-electronic, Darmstadt, ER.G.) on a digital video recorder (Sony, Japan), and later replayed for analysis on a Gould recorder (type 2200 S). OABA, muscimol or baclofen were administered for 3-10 s via pressure ejection from a glass pipette. For longer applications of agonists or antagonists, the drugs were added to the superfusion medium. Leibovitz medium, GABA, muscimol, bicuculline and picrotoxin were purchased from Sigma Chemical (St. Louis, MO), and baclofen from Ciba-Geigy (Basel). SR 95531 was obtained through the courtesy of Dr. K. Bizi6re (Sanofi, MontpeUier, France). Kanamycine and the antibiotic-antimycotic solution were supplied by Boehringer (Meylan, France).
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Fig. 1. Effect of G A B A on membrane potential in the whole-cell current clamp mode. GABA (10 .4 M) was ejected for 3 s (arrows). The resting potential was -40 inV. A: GABA induced a potential jump towards 0 mV which corresponds to the CI- equilibrium potential in the present experimental conditions. Hyperpolarizing pulses (100 pA; 400 ms) applied at 5-s intervals showed that GABA induced a dramatic increase of membrane conductance. B: dependence of GABA-evoked responses on the imposed levels of membrane potential. C: relationship between membrane potential and the amplitude of the response showing that the reversal potential was 0 mV.
F~ ' ~ , ~ ~ t Fig. 2. Effects of GABA and picrotoxin on membrane current (A-C) and membrane potential (D-F) in the whole-cell configuration. The membrane currents were measured from one cell and the membrane potentials from another cell. A: inward current activated by a 3-s ejection of GABA (10 -4 M; arrow) on a cell held at -40 inV. B: superfusion with picrotoxin (10 4 M) for 50 s (horizontal bar) totally abolished this current. C: during the washout, the effect of GABA was totally recovered. D: depolarization induced by a 3-s ejection of GABA. E: during superfusion with picrotoxin, GABA was ineffective. F: GABA-evoked depolarization during the washout.
153 RESULTS The effect of G A B A on pituitary melanotrophs has been studied in 54 cultured cells. At the resting potential, all of them responded to G A B A application with a depolarization which was rapid in onset and reversible. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the depolarization phase induced by G A B A (Fig. 1A). Typically, G A B A induced a membrane potential jump towards 0 mV, a value corresponding to the C1- equilibrium potential (ECI-) in the present experimental conditions. When the membrane potential was held at this value, the application of G A B A led to an increase of membrane conductance without any detectable membrane potential variation (data not shown). To further demonstrate the chloride dependency of G A B A evoked depolarization, the relationship between the membrane potential and the amplitude of the response was studied. As illustrated in Fig. 1B, for imposed negative potentials, G A B A gave rise to depolarization while for positive potentials, G A B A induced hyperpolarization. The response amplitude-membrane potential relationship was linear and the deduced reversal potential value corresponded to the EC1- (0 mV). The involvement of chloride ions was also demonstrated by current and voltage recordings performed in the presence of the chloride channel blocker picrotoxin (10-4 M). AS shown in Fig. 2, G A B A induced an inward current which was totally suppressed by picrotoxin. Thus, it appeared that the inward current evoked by G A B A was carried by chloride ions leaving the cell. Picrotoxin sensitivity was also found in voltage recordings, although a slight depolarizing effect of G A B A was still observed in the presence of the chloride channel blocker (Fig. 2E). G A B A A receptor antagonists (bicuculline or SR 95531) and agonist (muscimol) were used to study the
pharmacological profile of the G A B A receptors in our cell model. Fig. 3 illustrates the inhibitory effect of bicuculline (10 -4 M) on GABA-induced depolarization. The onset of bicuculline exposure was followed by transient fluctuations of the membrane potential and afterwards, the voltage trace became more stable. The specific G A B A A antagonist SR 95531 (10 -4 M) did not modify either the membrane potential or the membrane conductance but blocked the effect of G A B A ; this blocking effect was markedly reduced if the cell had been previously exposed to G A B A (Fig. 4). The depolarization produced by G A B A was mimicked by the selective G A B A A agonist muscimol (Fig. 5). The profile of the response to muscimol was identical to that obtained with
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Fig. 4. Effect of GABA in the presence or absence of the GABAA antagonist SR 95531 on membrane potential (A-C) and membrane current (D,E). A: during superfusion with SR 95531 (10-4 M; horizontal bar), GABA (3.3 x 10-4 M; arrow) did not induce any effect. Hyperpolarizing pulses (100 pA; 400 ms) applied at 5-s intervals showed no change in membrane conductance. B: effect of GABA during washout showing both depolarization and increased membrane conductance. C: when the cell was perifused again with SR 95531, the depolarizing effect of GABA was only partially reduced. D: voltage-clamp recording from another cell held at -40 mV. GABA (10-4 M) had no effect during superfusion with SR 95531. E: during the washout, GABA evoked an inward current.
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G A B A , i.e. a voltage jump from the resting potential to 0 mV (EC1-), followed by arrest of action potentials. After washing with the standard saline solution, spontaneous action potentials reappeared progressively. The depolarizing effect of muscimol was also blocked by picrotoxin (Fig. 5B). However, when the cells were pre-exposed to muscimol, picrotoxin could not block muscimol-induced depolarization (Fig. 5C,D). DISCUSSION We have previously shown that G A B A modulates the secretory activity of the amphibian pars intermedia in vitro 1"2. In the present study, we provide evidence for a direct effect of G A B A on the electrophysiological activity of frog melanotrophs in primary culture. In concert with previous studies conducted with mammalian pars intermedia cells 1°'25'26, our data show that G A B A induces membrane depolarization associated with a decrease of membrane resistance. The effect of G A B A on membrane potential was mimicked by the G A B A A receptor agonist muscimol, and suppressed by the clas-
sical G A B A A antagonist bicuculline as well as by SR 95531; this latter substance being a member of a new class of G A B A g receptor antagonists 38. These observations are consistent with previous studies on a-MSH secretion which demonstrated the existence of typical G A B A A receptors in frog melanotrophs 1. Pharmacological and electrophysiological studies have clearly demonstrated that, in rat 26 and porcine pars intermedia cells 1°, as well as in rat lactotrophs 15, the action of G A B A is predominantly mediated through activation of G A B A A receptors. However, in rat melanotrophs, Taraskevich and Douglas 26 also reported an inhibitory effect of baclofen, a specific G A B A B agonist, on spontaneous action potentials. Recent studies have shown that, in the toad Xenopus laevis, G A B A B receptors play a major role in the mechanism of action of G A B A on melanotropin secretion 37. In this latter species, baclofen caused an inhibition of a-MSH release which was ascribed to a depression of the adenylate cyclase activity36. Although baclofen did not induce any change of membrane voltage or current in our cell preparation (data not shown), we cannot exclude a possible involvement of G A B A B receptors in these cells. In fact, perifusion experiments conducted with frog pars intermedia reveal that baclofen exhibits a weak inhibitory effect on a-MSH secretion 1. The apparent refractoriness of frog melanotrophs to baclofen, observed in the whole-cell configuration, could be accounted for by the dialysis phenomenon which is known to occur during whole-cell recording ~2. Altogether we conclude that the electrophysiological effects of G A B A observed in the present study can be ascribed to its action on G A B A A receptors. Electrophysiological studies on cultured neurons 6 and adrenal chromaffin cells 5 have established that the effect of G A B A on G A B A A receptors is predominantly mediated by a gated chloride channel which is integrally associated with the receptor 3'23. The chloride dependency of GABA-induced depolarization in our model was supported by the observation that G A B A caused a potential jump toward the calculated ECI-, regardless of the value of the holding potential. In addition, at holding potentials corresponding to ECI-, G A B A increased the membrane conductance without changing the membrane potential. The fact that the action of G A B A was suppressed by the Cl- channel blocker picrotoxin was also consistent with the view that the depolarizing action of G A B A resulted from an efflux of chloride ions. The voltage jump towards positive values induced by G A B A , generated a brief discharge of action potentials. Typically, the transient increase of firing was followed by a plateau response characterized by complete disappearance of electrical activity. Such a depolarization, which totally inactivates Na + channels and significantly reduces
155 Ca 2+ conductance TMcaused the arrest of firing during the p l a t e a u response. A n interesting p h e n o m e n o n with respect to the action of picrotoxin and, to a lesser extent that o f S R 95531, is that p r e - e x p o s u r e of the r e c e p t o r to agonists (muscimol and G A B A , respectively) r e d u c e d the ability of these antagonists to block agonist e v o k e d responses. A p p a r e n t l y , this p r e - e x p o s u r e induced an antagonist-insensitive state, possibly by changing the c o n f o r m a t i o n a l state of the receptor. Pharmacological studies using the in vitro perifusion m o d e l have shown that, in rat 28 and frog pars i n t e r m e d i a L32, G A B A induces a biphasic effect on a - M S H secretion: a brief stimulation of m e l a n o t r o p i n release followed by a sustained inhibition. In the frog, the transient stimulatory phase is suppressed by both G A B A A antagonists and chloride channel blockers ~. These d a t a strongly suggest that the first phase of the action of G A B A (transitory stimulation) can be attributed to activation of Cl- channels, leading to chloride efflux. Since b o t h nifedipine (a d i h y d r o p y r i d i n e Ca 2÷ channel b l o c k e r ) and t e t r o d o t o x i n (a blocker of voltage-sensitive N a ÷ channels) suppressed G A B A - i n d u c e d stimulation of a - M S H release 32, it a p p e a r s that the chloride conductance must cause activation of fast N a + channels, which
in turn induces action potentials and activates voltageo p e r a t e d calcium channels TM. A l t h o u g h t e t r o d o t o x i n abolished the electrical activity of these cells TM, it had no effect on G A B A - e v o k e d inhibition of a - M S H release (second phase of the response). Thus, the inhibitory phase of the secretory response cannot be r e g a r d e d as a direct consequence of the cessation of firing. In the present study, no evidence was found for G A B A - i n d u c e d hyperpolarization, even when G A B A was perfused for 1.5 min (data not shown). T h e r e f o r e , the inhibitory response could very well be m e d i a t e d through intracellular mechanisms, which in our studies is not reflected in any electrophysiological m e m b r a n e changes, possibly due to a dialysis p h e n o m e n o n 12.
REFERENCES
Neuroendocrinology, 26 (1978) 277-282. 9 Demeneix, B.A., DesauUes, E., Feltz, P. and Loeffler, J.P., Dual population of GABA A and GABA n receptors in rat pars intermediate demonstrated by release of a-MSH caused by barium ions, Br. J. Pharmacol., 82 (1984) 183-190. 10 Demeneix, B.A., Taleb, O., Loeffler, J.P. and Feltz, P., GABA g receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release, Neuroscience, 17 (1986) 1275-1285. 11 Dierickx, K. and Vandesande, E, Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibers in the pars intermedia of the amphibian hypophysis, Cell Tissue Res., 174 (1976) 25-53. 12 Dufy, B., Jaken, S. and Barker, J.L., Intracellular Ca 2÷dependent protein kinase C activation mimics delayed effects of thyrotropin-releasing hormone on clonal pituitary cell excitability, Endocrinology, 121 (1987) 793-802. 13 Fuxe, K., Cellular localization of monoamines in the median eminence and infundibular stem of some mammals, Z. Zellforsch., 61 (1964) 710-724. 14 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, EG., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pflagers Arch., 39 (1981) 85-100. 15 Inenaga, K. and Mason, W.T., y-Aminobutyric acid modulates chloride channel activity in cultured primary bovine lactotrophs, Neuroscience, 23 (1987) 649-660. 16 Jenks, B.G., Verburg-van Kemenade, B.M.L. and Martens, G.M.J., Proopiomelanocortin the amphibian pars intermedia: a neuroendocrine model system. In M.E. Hadley, The Melanotropic Peptides, C.R.C. Press New York, 1988, pp. 103-126. 17 Lamacz, M., Hindelang, C., Tonon, M.C., Vaudry, H. and Stoeckel, M.E., Three distinct TRH-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for colocalization of TRH and mesotocin in fibers innervating pars intermedia cells, Neuroscience, 32 (1989) 451-462.
1 Adjeroud, S., Tonon, M.C., Lamacz, M., Leneveu, E., Stoeckel, M.E., Tappaz, M.L., Cazin, L., Danger, J.M., Bernard, C. and Vaudry, H., GABAergic control of a-melanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro, Brain Res. Bull., 17 (1986) 717-723. 2 Adjeroud, S., Tonon, M.C., Leneveu, E., Lamacz, M., Danger, J.M., Gouteux, L., Cazin, L. and Vaudry, H., The benzodiazepine agonist clonazepam potentiates the effects of y-aminobutyric acid on a-MSH release from neurointermediate lobes in vitro, Life Sci., 40 (1987) 1881-1887. 3 Barker, J.L. and Owen, D.G., Electrophysiological pharmacology of GABA and diazepam in cultured CNS neurons. In R.W. Olsen and J.C. Venter (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Alan R. Liss, New York, 1986, pp. 135-156. 4 Bjfrklund, A., Falck, B., Hromek, E, Owman, C. and West, K.A., Identification and terminal distribution of the tuberohypophyseal monoamine fiber system in the rat by means of stereotaxic and microspectrofluorimetric techniques, Brain Research, 17 (1970) 1-23. 5 Bormann, J. and Clapham, D.E., y-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2168-2172. 6 Bormann, J., Hamill, O.P. and Sakmann, B., Mechanism of anion permeation through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones, J. Physiol. (Lond.), 385 (1987) 243-286. 7 Danger, J.M., Guy, J., Benyamina, M., J6gou, S., Leboulenger, E, C6t6, J., Tonon, M.C., Pelletier, G. and Vaudry, H., Localization and identification of neuropeptide Y (NPY)-like immunoreactivity in the frog brain, Peptides, 6 (1985) 12251236. 8 Davis, M.D. and Hadley, M.E., Pars intermedia electrical potentials: changes in spike frequency induced by regulatory factors of melanocyte-stimulating hormone (MSH) secretion,
Acknowledgements. This research was supported by grants from INSERM (86-4016), the Commission of the European Communities (87 300 445), the Minist~re des Affaires Etrang~res and the Minist6re de la Recherche et de la Technologie (R6seau Europ6an de Laboratoires) and the Conseil R6gional de Haute-Normandie. We are indebted to Dr. K. Bizi6re, SANOFI-Research, Montpellier, France for the kind gift of SR 95531. EH.M.M.V.D.E was a recipient of a fellowship from the Commission of the European Communities (ERASMUS program no. ICP-88-NL-0122/actie 2). We thank Mrs. Catherine Buquet and Hel6ne Dudouit for expert technical assistance and Mrs. Sabrina Moreau for typing the manuscript.
156 18 Louiset, E., Cazin, L., Lamacz, M., Tonon, M.C. and Vaudry, H., Patch-clamp study of the ionic currents underlying action potentials in cultured frog pituitary melanotrophs, Neuroendocrinology, 48 (1988) 507-515. 19 Mimnagh, K.M., Bolaffi, J.L., Montgomery, N.M. and Kaltenbach, J.C., Thyrotopin-releasing hormone (TRH): immunohistochemical distribution in tadpole and frog brain, Gen. Comp. EndocrinoL, 66 (1987) 394-404. 20 Oertel, W.H., Mugnaini, E., Tappaz, M.L., Weise, V.K., Dahl, A.L., Schmechel, D.E. and Kopin, I.J., Central GABAergic innervation of neurointermediate pituitary lobe: biochemical and immunocytochemical study in the rat, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 675-679. 21 Oliverau, M. and Olivereau, J., Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish, Peptides, 9 (1988) 13-21. 22 Saland, L.C., Wallace, J.A. and Comunas, F., Serotoninimmunoreactive nerve fibers of the rat pituitary: effects of anticatecholamine and antiserotonin drugs on staining patterns, Brain Research, 368 (1986) 310-318. 23 Schofield, P.R., Davelizon, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, M., Rhee, L.M., Ramachandran, J., Reale, V., Glencorese, T.A., Seeburg, EM. and Barnard, E.A., Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family, Nature (Lond.), 328 (1987) 221-227. 24 Seki, T., Nakai, Y., Shioda, S., Mitsuma, T. and Kikuyama, S., Distribution of immunoreactive thyrotropin-releasing hormone in the forebrain and hypophysis of the bullfrog, Rana catesbeiana, Cell Tissue Res., 233 (1983) 507-516. 25 Taraskevich, P.S. and Douglas, W.W., GABA directly affects electrophysiological properties of pituitary pars intermedia cells, Nature (Lond.), 299 (1982) 733-734. 26 Taraskevich, P.J. and Douglas, W.W., Pharmacological and ionic features of y-aminobutyric acid receptors influencing electrical properties of melanotrophs isolated from the rat pars intermedia, Neuroscience, 14 (1985) 301-308. 27 Terlou, M. and Ploemacher, R.E., The distribution of monoamines in the tel-, di- and mesencephalon of Xenopus laevis tadpoles, with special reference to the hypothalamo-hypophysial system, Z. Zellforsch., 137 (1973) 521-540. 28 Tomiko, S.A., Taraskevich, P.S. and Douglas, W.W., GABA acts directly on cells of pituitary pars intermedia to alter hormone output, Nature (Lond.), 301 (1983) 706-707. 29 Tonon, M.C., Leroux, P., Stoeckel, M.E., J6gou, S., Pelletier, G. and Vaudry, H., Catecholaminergic control of a-melanocyte-
stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro: evidence for direct stimulation of a-MSH release by thyrotropin-releasing hormone, Endocrinology, 112 (1983) 133-141. 30 Tonon, M.C., Burlet, A., Lauber, M., Cuet, P., J6gou, S., Gouteux, L., Ling, N. and Vaudry, H., Immunohistochemicai localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda, Neuroendocrinology , 40 (1985) 109-119. 31 Tonon M.C., Danger, J.M., Lamacz, M., Leroux, P., Adjeroud, S., Andersen, A.C., Verburg-van Kemenade, B.M.L., Jenks, B.G., Pelletier, G., Stoeckel, M.E., Burlet, A., Kupryszewski, G. and Vaudry, H., Multihormonal control of melanotropin secretion in cold-blooded vertebrates. In M.E. Hadley (Eds.), The Melanotropic Peptides, C.R.C. Press, New York, 1988, pp. 127-171. 32 Tonon, M.C., Adjeroud, S., Lamacz, M., Louiset, E., Danger, J.M., Desrues, L., Cazin, L., Nicolas, P. and Vandry, H., Central-type benzodiazepines and the octadecaneuropeptide (ODN) modulate the effects of y-aminobutyric acid on the release of a-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro, Neuroscience, 31 (1989) 485-493. 33 Verburg-van Kemenade, B.M.L., Jenks, B.G. and Driessen, A.G.Y., GABA and dopamine act directly on melanotrophs of Xenopus to inhibit MSH secretion, Brain Res. Bull., 17 (1986) 697-704. 34 Verburg-van Kemenade, B.M.L., Tappaz, M., Paut, L. and Jenks, B.G., GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence, Endocrinology, 118 (1986) 260-267. 35 Verburg-van Kemenade, B.M.L., Jenks, B.G., Danger, J.M., Vaudry, H., Pelletier, G. and Saint-Pierre, S., A NPY-like peptide may function as MSH-release inhibiting factor in Xenopus laevis, Peptides, 8 (1987) 61-67. 36 Verburg-van Kemenade, B.M.L., Jenks, B.G. and Houben, A.J.H.M., Regulation of cyclic AMP synthesis in amphibian melanotrope cells through catecholamine and GABA receptors, Life Sci., 40 (1987) 1859-1968. 37 Verburg-van Kemenade, B.M.L., Jenks, B.G., Lenssen, F.G.A. and Vaudry, H., Characterization of GABA receptors in the neurointermediate lobe of the amphibian Xenopus laevis, Endocrinology, 120 (1987) 622-628. 38 Wermuth, C.G. and Bizi~re, K., Pyridazinyl-GABA derivates: a new class of synthetic GABAA antagonists, Trends Pharmacol. Sci., 7 (1986) 421-424.
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Electrophysiological evidence for the existence of GABAA receptors in cultured frog melanotrophs Estelle Louiset 1, Frans H.M.M. van de Put 1'2, Marie-Christine Tonon 1, Corinne Basille 1, Bruce G. Jenks 2, Hubert Vaudry 1 and Lionel Cazin 1 ~Laboratoire d'Endocrinologie Mol~culaire, Unit~ Affili~e ~ I'INSERM, Universitd de Rouen, Mont-Saint-Aignan (France) and 2Laboratory of Animal Physiology, Faculty of Science, Catholic University, Nijmegen (The Netherlands) (Accepted 24 October 1989) Key words: Whole-cell patch-clamp; Chloride channel; Membrane potential; GABAA-receptor subtype; Pars intermedia cell
The neurotransminer GABA exerts a biphasic effect on a-melanocyte-stimulating hormone (a-MSH) secretion from pars intermedia cells: GABA induces a rapid and transient stimulation followed by a sustained inhibition of a-MSH release. In the present study, we have investigated the effect of GABA on the electrophysiological properties of frog melanotrophs in primary culture using the patch-clamp technique in the whole cell configuration. In all cells tested, GABA stimulated an inward current and induced depolarization. A transient period of intense firing was consistently observed at the onset of GABA administration. During the depolarization phase, the membrane potential reached a plateau corresponding to the CI- equilibrium potential. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the response evoked by GABA. The effect of GABA was abolished by the chloride channel blocker picrotoxin, and by antagonists of GABAA receptors (bicuculline and SR 95531). The depolarizing action of GABA was mimicked by muscimol, an agonist of GABA A receptors. Taken together, our results indicate that the rapid and transient stimulation of a-MSH release induced by GABA can be accounted for by activation of a chloride conductance which causes membrane depolarization. These data support the notion that the transient stimulation of a-MSH secretion induced by GABA can be accounted for by membrane depolarization which provokes activation of voltage-operated calcium channels. Since no evidence was found for GABA-induced hyperpolarization, the intracellular mechanisms leading to the strong inhibitory effect of GABA on a-MSH secretion remain to be elucidated.
INTRODUCTION The pars intermedia of the pituitary is supplied by a dense network of nerve fibers originating from the hypothalamus ~3'27. In mammals, the axon terminals, which make intimate contacts with the endocrine cells of the intermediate lobe, contain various classical neurotransmitters including catecholamines (mainly dopamine) g, serotonin 22 and y-aminobutyric acid ( G A B A ) 2°. In non-mammalian vertebrates, the pars intermedia is also innervated by fibers containing neuropeptides such as thyrotropin-releasing hormone 17A9'24, corticotropin-releasing factor 21'3°, mesotocinn,17 and neuropeptide y7,35, as well as classical neurotransmitters including dopamine 31 and G A B A ~,33. Most of the neuroendocrine signals contained in these nerve terminals influence the secretory activity of amphibian pituitary melanotrophs 16,29'3°. Since the secretion of melanotropic peptides is globally under negative hypothalamic control, the inhibitory factors (i.e. dopamine, G A B A and neuropeptide Y) likely play a pivotal role in the regulation of melanotropin secretion.
The involvement of G A B A in the regulation of the pars intermedia activity was first demonstrated by Davis and Hadley 8. In mammals, G A B A induces a biphasic effect on a-melanocyte-stimulating h o r m o n e (a-MSH) secretion: a transient stimulation followed by sustained inhibition 9'28. Electrophysiological studies on rat or porcine melanotrophs in primary culture, revealed that G A B A increases m e m b r a n e CI- permeability which causes a depolarization 1°'25'26. The effect of G A B A on chloride conductance is mimicked by isoguvacine 1° and muscimo126 and antagonized by bicuculline 1°'26, indicating that the response of pituitary cells is mediated through activation of G A B A A receptors. We have recently investigated the effect of G A B A on a - M S H release from the amphibian pars intermedia. In the toad X e n o p u s laevis, G A B A produces only inhibition of the secretory process 33'34 and the effect of G A B A appears to be mediated primarily through a G A B A A receptor mechanism 37. In contrast, in the frog Rana ridibunda, G A B A and G A B A A agonists induce a biphasic action on a - M S H secretion l'z. The question arises: are
Correspondence: L. Cazin, Laboratoire d'Endocrinologie Mol6culaire, CNRS URA 650, Unit6 Affili6e h I'INSERM, Facult6 des Sciences, Universit6 de Rouen, BP 118, F-76134 Mont-Saint-Aignan Cedex, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
152 both phases of biphasic phenomena a reflection of the action of a classical GABAA receptor? Electrophysiological techniques on cultured pars intermedia cells represent an ideal approach to answer this question. Using the patch-clamp technique, we have recently shown that frog melanotrophs spontaneously generate action potentials and we have identified the ionic currents underlying spontaneous electrical activity TM. The aim of the present study was to demonstrate the existence of GABA receptors on these cells and to determine the characteristics of the ionic conductances involved in the mechanism of action of GABA. MATERIALS AND METHODS Neurointermediate lobes were removed from 8 freshly decapitated male frogs Rana ridibunda and the pars intermedia cells were enzymatically dispersed, as previously described TM. The cells were plated at a density of 250,000 cells/ml on microwell Terasaki dishes containing Leibovitz medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (0.1 mg/ml kanamycine and 1% antibiotic-antimycotic solution). The cells were allowed to attach to the culture microwell for 24 h at 26 °C in a humidified atmosphere. The incubation medium was replaced by fresh medium every day. The cells were used for electrophysiologicai studies 2-7 days after plating. During electrophysiological recording, the cells were continuously superfused with standard Ringer's solution (in
mM: 112 NaCI, 2 KCI, 2 CaCI2, 15 HEPES) containing 11 mM glucose (pH adjusted to 7.4 with NaOH). The intracellular solution contained (in mM) 100 KCI, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES (pH adjusted to 7.4 with KOH). All recordings were obtained in the whole-cell configuration of the patch-clamp technique TM. In the present experimental conditions, the CIequilibrium potential was found to be 0 mV according to the Nernst equation. The current and voltage outputs were recorded from the patch-clamp amplifier (EPC 7, List-electronic, Darmstadt, ER.G.) on a digital video recorder (Sony, Japan), and later replayed for analysis on a Gould recorder (type 2200 S). OABA, muscimol or baclofen were administered for 3-10 s via pressure ejection from a glass pipette. For longer applications of agonists or antagonists, the drugs were added to the superfusion medium. Leibovitz medium, GABA, muscimol, bicuculline and picrotoxin were purchased from Sigma Chemical (St. Louis, MO), and baclofen from Ciba-Geigy (Basel). SR 95531 was obtained through the courtesy of Dr. K. Bizi6re (Sanofi, MontpeUier, France). Kanamycine and the antibiotic-antimycotic solution were supplied by Boehringer (Meylan, France).
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Fig. 1. Effect of G A B A on membrane potential in the whole-cell current clamp mode. GABA (10 .4 M) was ejected for 3 s (arrows). The resting potential was -40 inV. A: GABA induced a potential jump towards 0 mV which corresponds to the CI- equilibrium potential in the present experimental conditions. Hyperpolarizing pulses (100 pA; 400 ms) applied at 5-s intervals showed that GABA induced a dramatic increase of membrane conductance. B: dependence of GABA-evoked responses on the imposed levels of membrane potential. C: relationship between membrane potential and the amplitude of the response showing that the reversal potential was 0 mV.
F~ ' ~ , ~ ~ t Fig. 2. Effects of GABA and picrotoxin on membrane current (A-C) and membrane potential (D-F) in the whole-cell configuration. The membrane currents were measured from one cell and the membrane potentials from another cell. A: inward current activated by a 3-s ejection of GABA (10 -4 M; arrow) on a cell held at -40 inV. B: superfusion with picrotoxin (10 4 M) for 50 s (horizontal bar) totally abolished this current. C: during the washout, the effect of GABA was totally recovered. D: depolarization induced by a 3-s ejection of GABA. E: during superfusion with picrotoxin, GABA was ineffective. F: GABA-evoked depolarization during the washout.
153 RESULTS The effect of G A B A on pituitary melanotrophs has been studied in 54 cultured cells. At the resting potential, all of them responded to G A B A application with a depolarization which was rapid in onset and reversible. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the depolarization phase induced by G A B A (Fig. 1A). Typically, G A B A induced a membrane potential jump towards 0 mV, a value corresponding to the C1- equilibrium potential (ECI-) in the present experimental conditions. When the membrane potential was held at this value, the application of G A B A led to an increase of membrane conductance without any detectable membrane potential variation (data not shown). To further demonstrate the chloride dependency of G A B A evoked depolarization, the relationship between the membrane potential and the amplitude of the response was studied. As illustrated in Fig. 1B, for imposed negative potentials, G A B A gave rise to depolarization while for positive potentials, G A B A induced hyperpolarization. The response amplitude-membrane potential relationship was linear and the deduced reversal potential value corresponded to the EC1- (0 mV). The involvement of chloride ions was also demonstrated by current and voltage recordings performed in the presence of the chloride channel blocker picrotoxin (10-4 M). AS shown in Fig. 2, G A B A induced an inward current which was totally suppressed by picrotoxin. Thus, it appeared that the inward current evoked by G A B A was carried by chloride ions leaving the cell. Picrotoxin sensitivity was also found in voltage recordings, although a slight depolarizing effect of G A B A was still observed in the presence of the chloride channel blocker (Fig. 2E). G A B A A receptor antagonists (bicuculline or SR 95531) and agonist (muscimol) were used to study the
pharmacological profile of the G A B A receptors in our cell model. Fig. 3 illustrates the inhibitory effect of bicuculline (10 -4 M) on GABA-induced depolarization. The onset of bicuculline exposure was followed by transient fluctuations of the membrane potential and afterwards, the voltage trace became more stable. The specific G A B A A antagonist SR 95531 (10 -4 M) did not modify either the membrane potential or the membrane conductance but blocked the effect of G A B A ; this blocking effect was markedly reduced if the cell had been previously exposed to G A B A (Fig. 4). The depolarization produced by G A B A was mimicked by the selective G A B A A agonist muscimol (Fig. 5). The profile of the response to muscimol was identical to that obtained with
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Fig. 4. Effect of GABA in the presence or absence of the GABAA antagonist SR 95531 on membrane potential (A-C) and membrane current (D,E). A: during superfusion with SR 95531 (10-4 M; horizontal bar), GABA (3.3 x 10-4 M; arrow) did not induce any effect. Hyperpolarizing pulses (100 pA; 400 ms) applied at 5-s intervals showed no change in membrane conductance. B: effect of GABA during washout showing both depolarization and increased membrane conductance. C: when the cell was perifused again with SR 95531, the depolarizing effect of GABA was only partially reduced. D: voltage-clamp recording from another cell held at -40 mV. GABA (10-4 M) had no effect during superfusion with SR 95531. E: during the washout, GABA evoked an inward current.
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G A B A , i.e. a voltage jump from the resting potential to 0 mV (EC1-), followed by arrest of action potentials. After washing with the standard saline solution, spontaneous action potentials reappeared progressively. The depolarizing effect of muscimol was also blocked by picrotoxin (Fig. 5B). However, when the cells were pre-exposed to muscimol, picrotoxin could not block muscimol-induced depolarization (Fig. 5C,D). DISCUSSION We have previously shown that G A B A modulates the secretory activity of the amphibian pars intermedia in vitro 1"2. In the present study, we provide evidence for a direct effect of G A B A on the electrophysiological activity of frog melanotrophs in primary culture. In concert with previous studies conducted with mammalian pars intermedia cells 1°'25'26, our data show that G A B A induces membrane depolarization associated with a decrease of membrane resistance. The effect of G A B A on membrane potential was mimicked by the G A B A A receptor agonist muscimol, and suppressed by the clas-
sical G A B A A antagonist bicuculline as well as by SR 95531; this latter substance being a member of a new class of G A B A g receptor antagonists 38. These observations are consistent with previous studies on a-MSH secretion which demonstrated the existence of typical G A B A A receptors in frog melanotrophs 1. Pharmacological and electrophysiological studies have clearly demonstrated that, in rat 26 and porcine pars intermedia cells 1°, as well as in rat lactotrophs 15, the action of G A B A is predominantly mediated through activation of G A B A A receptors. However, in rat melanotrophs, Taraskevich and Douglas 26 also reported an inhibitory effect of baclofen, a specific G A B A B agonist, on spontaneous action potentials. Recent studies have shown that, in the toad Xenopus laevis, G A B A B receptors play a major role in the mechanism of action of G A B A on melanotropin secretion 37. In this latter species, baclofen caused an inhibition of a-MSH release which was ascribed to a depression of the adenylate cyclase activity36. Although baclofen did not induce any change of membrane voltage or current in our cell preparation (data not shown), we cannot exclude a possible involvement of G A B A B receptors in these cells. In fact, perifusion experiments conducted with frog pars intermedia reveal that baclofen exhibits a weak inhibitory effect on a-MSH secretion 1. The apparent refractoriness of frog melanotrophs to baclofen, observed in the whole-cell configuration, could be accounted for by the dialysis phenomenon which is known to occur during whole-cell recording ~2. Altogether we conclude that the electrophysiological effects of G A B A observed in the present study can be ascribed to its action on G A B A A receptors. Electrophysiological studies on cultured neurons 6 and adrenal chromaffin cells 5 have established that the effect of G A B A on G A B A A receptors is predominantly mediated by a gated chloride channel which is integrally associated with the receptor 3'23. The chloride dependency of GABA-induced depolarization in our model was supported by the observation that G A B A caused a potential jump toward the calculated ECI-, regardless of the value of the holding potential. In addition, at holding potentials corresponding to ECI-, G A B A increased the membrane conductance without changing the membrane potential. The fact that the action of G A B A was suppressed by the Cl- channel blocker picrotoxin was also consistent with the view that the depolarizing action of G A B A resulted from an efflux of chloride ions. The voltage jump towards positive values induced by G A B A , generated a brief discharge of action potentials. Typically, the transient increase of firing was followed by a plateau response characterized by complete disappearance of electrical activity. Such a depolarization, which totally inactivates Na + channels and significantly reduces
155 Ca 2+ conductance TMcaused the arrest of firing during the p l a t e a u response. A n interesting p h e n o m e n o n with respect to the action of picrotoxin and, to a lesser extent that o f S R 95531, is that p r e - e x p o s u r e of the r e c e p t o r to agonists (muscimol and G A B A , respectively) r e d u c e d the ability of these antagonists to block agonist e v o k e d responses. A p p a r e n t l y , this p r e - e x p o s u r e induced an antagonist-insensitive state, possibly by changing the c o n f o r m a t i o n a l state of the receptor. Pharmacological studies using the in vitro perifusion m o d e l have shown that, in rat 28 and frog pars i n t e r m e d i a L32, G A B A induces a biphasic effect on a - M S H secretion: a brief stimulation of m e l a n o t r o p i n release followed by a sustained inhibition. In the frog, the transient stimulatory phase is suppressed by both G A B A A antagonists and chloride channel blockers ~. These d a t a strongly suggest that the first phase of the action of G A B A (transitory stimulation) can be attributed to activation of Cl- channels, leading to chloride efflux. Since b o t h nifedipine (a d i h y d r o p y r i d i n e Ca 2÷ channel b l o c k e r ) and t e t r o d o t o x i n (a blocker of voltage-sensitive N a ÷ channels) suppressed G A B A - i n d u c e d stimulation of a - M S H release 32, it a p p e a r s that the chloride conductance must cause activation of fast N a + channels, which
in turn induces action potentials and activates voltageo p e r a t e d calcium channels TM. A l t h o u g h t e t r o d o t o x i n abolished the electrical activity of these cells TM, it had no effect on G A B A - e v o k e d inhibition of a - M S H release (second phase of the response). Thus, the inhibitory phase of the secretory response cannot be r e g a r d e d as a direct consequence of the cessation of firing. In the present study, no evidence was found for G A B A - i n d u c e d hyperpolarization, even when G A B A was perfused for 1.5 min (data not shown). T h e r e f o r e , the inhibitory response could very well be m e d i a t e d through intracellular mechanisms, which in our studies is not reflected in any electrophysiological m e m b r a n e changes, possibly due to a dialysis p h e n o m e n o n 12.
REFERENCES
Neuroendocrinology, 26 (1978) 277-282. 9 Demeneix, B.A., DesauUes, E., Feltz, P. and Loeffler, J.P., Dual population of GABA A and GABA n receptors in rat pars intermediate demonstrated by release of a-MSH caused by barium ions, Br. J. Pharmacol., 82 (1984) 183-190. 10 Demeneix, B.A., Taleb, O., Loeffler, J.P. and Feltz, P., GABA g receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release, Neuroscience, 17 (1986) 1275-1285. 11 Dierickx, K. and Vandesande, E, Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibers in the pars intermedia of the amphibian hypophysis, Cell Tissue Res., 174 (1976) 25-53. 12 Dufy, B., Jaken, S. and Barker, J.L., Intracellular Ca 2÷dependent protein kinase C activation mimics delayed effects of thyrotropin-releasing hormone on clonal pituitary cell excitability, Endocrinology, 121 (1987) 793-802. 13 Fuxe, K., Cellular localization of monoamines in the median eminence and infundibular stem of some mammals, Z. Zellforsch., 61 (1964) 710-724. 14 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, EG., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pflagers Arch., 39 (1981) 85-100. 15 Inenaga, K. and Mason, W.T., y-Aminobutyric acid modulates chloride channel activity in cultured primary bovine lactotrophs, Neuroscience, 23 (1987) 649-660. 16 Jenks, B.G., Verburg-van Kemenade, B.M.L. and Martens, G.M.J., Proopiomelanocortin the amphibian pars intermedia: a neuroendocrine model system. In M.E. Hadley, The Melanotropic Peptides, C.R.C. Press New York, 1988, pp. 103-126. 17 Lamacz, M., Hindelang, C., Tonon, M.C., Vaudry, H. and Stoeckel, M.E., Three distinct TRH-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for colocalization of TRH and mesotocin in fibers innervating pars intermedia cells, Neuroscience, 32 (1989) 451-462.
1 Adjeroud, S., Tonon, M.C., Lamacz, M., Leneveu, E., Stoeckel, M.E., Tappaz, M.L., Cazin, L., Danger, J.M., Bernard, C. and Vaudry, H., GABAergic control of a-melanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro, Brain Res. Bull., 17 (1986) 717-723. 2 Adjeroud, S., Tonon, M.C., Leneveu, E., Lamacz, M., Danger, J.M., Gouteux, L., Cazin, L. and Vaudry, H., The benzodiazepine agonist clonazepam potentiates the effects of y-aminobutyric acid on a-MSH release from neurointermediate lobes in vitro, Life Sci., 40 (1987) 1881-1887. 3 Barker, J.L. and Owen, D.G., Electrophysiological pharmacology of GABA and diazepam in cultured CNS neurons. In R.W. Olsen and J.C. Venter (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Alan R. Liss, New York, 1986, pp. 135-156. 4 Bjfrklund, A., Falck, B., Hromek, E, Owman, C. and West, K.A., Identification and terminal distribution of the tuberohypophyseal monoamine fiber system in the rat by means of stereotaxic and microspectrofluorimetric techniques, Brain Research, 17 (1970) 1-23. 5 Bormann, J. and Clapham, D.E., y-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2168-2172. 6 Bormann, J., Hamill, O.P. and Sakmann, B., Mechanism of anion permeation through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones, J. Physiol. (Lond.), 385 (1987) 243-286. 7 Danger, J.M., Guy, J., Benyamina, M., J6gou, S., Leboulenger, E, C6t6, J., Tonon, M.C., Pelletier, G. and Vaudry, H., Localization and identification of neuropeptide Y (NPY)-like immunoreactivity in the frog brain, Peptides, 6 (1985) 12251236. 8 Davis, M.D. and Hadley, M.E., Pars intermedia electrical potentials: changes in spike frequency induced by regulatory factors of melanocyte-stimulating hormone (MSH) secretion,
Acknowledgements. This research was supported by grants from INSERM (86-4016), the Commission of the European Communities (87 300 445), the Minist~re des Affaires Etrang~res and the Minist6re de la Recherche et de la Technologie (R6seau Europ6an de Laboratoires) and the Conseil R6gional de Haute-Normandie. We are indebted to Dr. K. Bizi6re, SANOFI-Research, Montpellier, France for the kind gift of SR 95531. EH.M.M.V.D.E was a recipient of a fellowship from the Commission of the European Communities (ERASMUS program no. ICP-88-NL-0122/actie 2). We thank Mrs. Catherine Buquet and Hel6ne Dudouit for expert technical assistance and Mrs. Sabrina Moreau for typing the manuscript.
156 18 Louiset, E., Cazin, L., Lamacz, M., Tonon, M.C. and Vaudry, H., Patch-clamp study of the ionic currents underlying action potentials in cultured frog pituitary melanotrophs, Neuroendocrinology, 48 (1988) 507-515. 19 Mimnagh, K.M., Bolaffi, J.L., Montgomery, N.M. and Kaltenbach, J.C., Thyrotopin-releasing hormone (TRH): immunohistochemical distribution in tadpole and frog brain, Gen. Comp. EndocrinoL, 66 (1987) 394-404. 20 Oertel, W.H., Mugnaini, E., Tappaz, M.L., Weise, V.K., Dahl, A.L., Schmechel, D.E. and Kopin, I.J., Central GABAergic innervation of neurointermediate pituitary lobe: biochemical and immunocytochemical study in the rat, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 675-679. 21 Oliverau, M. and Olivereau, J., Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish, Peptides, 9 (1988) 13-21. 22 Saland, L.C., Wallace, J.A. and Comunas, F., Serotoninimmunoreactive nerve fibers of the rat pituitary: effects of anticatecholamine and antiserotonin drugs on staining patterns, Brain Research, 368 (1986) 310-318. 23 Schofield, P.R., Davelizon, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, M., Rhee, L.M., Ramachandran, J., Reale, V., Glencorese, T.A., Seeburg, EM. and Barnard, E.A., Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family, Nature (Lond.), 328 (1987) 221-227. 24 Seki, T., Nakai, Y., Shioda, S., Mitsuma, T. and Kikuyama, S., Distribution of immunoreactive thyrotropin-releasing hormone in the forebrain and hypophysis of the bullfrog, Rana catesbeiana, Cell Tissue Res., 233 (1983) 507-516. 25 Taraskevich, P.S. and Douglas, W.W., GABA directly affects electrophysiological properties of pituitary pars intermedia cells, Nature (Lond.), 299 (1982) 733-734. 26 Taraskevich, P.J. and Douglas, W.W., Pharmacological and ionic features of y-aminobutyric acid receptors influencing electrical properties of melanotrophs isolated from the rat pars intermedia, Neuroscience, 14 (1985) 301-308. 27 Terlou, M. and Ploemacher, R.E., The distribution of monoamines in the tel-, di- and mesencephalon of Xenopus laevis tadpoles, with special reference to the hypothalamo-hypophysial system, Z. Zellforsch., 137 (1973) 521-540. 28 Tomiko, S.A., Taraskevich, P.S. and Douglas, W.W., GABA acts directly on cells of pituitary pars intermedia to alter hormone output, Nature (Lond.), 301 (1983) 706-707. 29 Tonon, M.C., Leroux, P., Stoeckel, M.E., J6gou, S., Pelletier, G. and Vaudry, H., Catecholaminergic control of a-melanocyte-
stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro: evidence for direct stimulation of a-MSH release by thyrotropin-releasing hormone, Endocrinology, 112 (1983) 133-141. 30 Tonon, M.C., Burlet, A., Lauber, M., Cuet, P., J6gou, S., Gouteux, L., Ling, N. and Vaudry, H., Immunohistochemicai localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda, Neuroendocrinology , 40 (1985) 109-119. 31 Tonon M.C., Danger, J.M., Lamacz, M., Leroux, P., Adjeroud, S., Andersen, A.C., Verburg-van Kemenade, B.M.L., Jenks, B.G., Pelletier, G., Stoeckel, M.E., Burlet, A., Kupryszewski, G. and Vaudry, H., Multihormonal control of melanotropin secretion in cold-blooded vertebrates. In M.E. Hadley (Eds.), The Melanotropic Peptides, C.R.C. Press, New York, 1988, pp. 127-171. 32 Tonon, M.C., Adjeroud, S., Lamacz, M., Louiset, E., Danger, J.M., Desrues, L., Cazin, L., Nicolas, P. and Vandry, H., Central-type benzodiazepines and the octadecaneuropeptide (ODN) modulate the effects of y-aminobutyric acid on the release of a-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro, Neuroscience, 31 (1989) 485-493. 33 Verburg-van Kemenade, B.M.L., Jenks, B.G. and Driessen, A.G.Y., GABA and dopamine act directly on melanotrophs of Xenopus to inhibit MSH secretion, Brain Res. Bull., 17 (1986) 697-704. 34 Verburg-van Kemenade, B.M.L., Tappaz, M., Paut, L. and Jenks, B.G., GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence, Endocrinology, 118 (1986) 260-267. 35 Verburg-van Kemenade, B.M.L., Jenks, B.G., Danger, J.M., Vaudry, H., Pelletier, G. and Saint-Pierre, S., A NPY-like peptide may function as MSH-release inhibiting factor in Xenopus laevis, Peptides, 8 (1987) 61-67. 36 Verburg-van Kemenade, B.M.L., Jenks, B.G. and Houben, A.J.H.M., Regulation of cyclic AMP synthesis in amphibian melanotrope cells through catecholamine and GABA receptors, Life Sci., 40 (1987) 1859-1968. 37 Verburg-van Kemenade, B.M.L., Jenks, B.G., Lenssen, F.G.A. and Vaudry, H., Characterization of GABA receptors in the neurointermediate lobe of the amphibian Xenopus laevis, Endocrinology, 120 (1987) 622-628. 38 Wermuth, C.G. and Bizi~re, K., Pyridazinyl-GABA derivates: a new class of synthetic GABAA antagonists, Trends Pharmacol. Sci., 7 (1986) 421-424.
