effect of acetylcholine on the electrical and secretory activities of frog pituitary melanotrophs

300

Brain Research, 533 (1990) 300-308 Elsevier

BRES 16056

Effect of acetylcholine on the electrical and secretory activities of frog pituitary melanotrophs E. Louiset, L. Cazin, O. Duval, M. Lamacz, M.C. Tonon and H. Vaudry Laboratoire d'Endocrinologie Mol~culaire, CNRS URA 650, Unit~Affili~e g~rlNSERM, Universit~ de Rouen, Mont-Saint-Aignan (France) (Accepted 5 June 1990) Key words: Acetylcholine; Muscarine; Nicotine; Melanocyte-stimulating hormone; Patch-clamp technique; Perifusion technique; Immunocytochemistry

The activity of melanotroph cells of the amphibian pars intermedia is regulated by multiple factors including classical neurotransmitters and neuropeptides. In this study, we have examined the possible involvement of acetylcholine (ACh) in the regulation of electrical and secretory activities of frog pituitary melanotrophs. Electrophysiological recordings were conducted on cultured cells by using the patch-clamp technique in the whole-ceUconfiguration. In parallel, a-MSH release from acutely dispersed pars intermedia cells was studied by means of the perifusion technique. In all cells tested in the current-clamp mode, superfusion with A C h (10 -6 M) gave rise to a depolarization associated with an enhanced frequency of action potentials. Administration of ACh (1(I 6 M) to perifused cells also induced stimulation of a-MSH release. These results indicate that the neurotransmitter ACh exerts a direct stimulatory effect on pituitary melanotrophs. The action of ACh on electrical and secretory activities was mimicked by muscarine (10-5 M), while ACh-induced a-MSH secretion was completely abolished by the muscarinic antagonist atropine (1(16 M). The depolarizing effect of muscarine was suppressed by the specific M1 muscarinic antagonist pirenzepine (10-5 M), indicating the existence of a M1 subtype muscarinic receptor in frog pars intermedia cells. In addition, using a monoclonal antibody against calf muscarinic receptors, we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in cultured intermediate lobe cells. Eiectrophysiological recordings showed that nicotine (10-5 M) induces membrane depolarization associated with an increase of the frequency of action potentials. Nicotine (10-7 - 10-4 M) also caused a dose-related stimulation of a-MSH release from perifused pars intermedia cells. Both electrophysiological recordings and perifusion experiments showed that nicotine-induced stimulation of pituitary melanotrophs was not sensitive to various classical nicotinic antagonists including hexamethonium (10-4 M), a-bungarotoxin (10-5 M), d-tubocurarine (10-5 M), dihydro- fl-erythroi'dine (10-5 M) and toxin F (10-6 M). In addition, the nicotinic agonists cytisine (10-5 M) and 1,1-dimethyl-4-phenylpiperazinium (10-5 M) did not stimulate a-MSH release. In conclusion, the present results show that ACh acts as a neurohormone to stimulate the electrical and secretory activities of frog pars intermedia cells through M~-muscarinicreceptors. Our data also suggest the existence, in our model, of a subtype of nicotinic receptors unrelated to identified nicotinic binding sites.

INTRODUCTION The intermediate lobe of the pituitary produces a multifunctional protein, pro-opiomelanocortin (POMC), which generates, through proteolytic cleavage, a n u m b e r of bioactive peptides including a-melanocyte-stimulating h o r m o n e ( a - M S H ) and fl-endorphins'43. In amphibians, the release of a - M S H is stimulated when the animals are placed on a dark background and thus, the intermediate lobe plays a major role in the neuroendocrine reflex leading to skin color adaptation 4. The intermediate lobe of the frog pituitary is innervated by axons containing a variety of aminergic or peptidergic neurotransmitters, including catecholamines 34"41, serotonin 21, G A B A 2, corticotropin-releasing factor 4°, mesotocin "~'2°, thyrotropin-releasing h o r m o n e

( T R H ) 2°'37" and neuropeptide y9.

Several of these

neurotransmitters are clearly involved in the regulation of a - M S H release from the frog pars intermedia. For instance, dopamine 1, serotonin 21, G A B A 2 and neuropeptide y9 inhibit melanotropin secretion while T R H 38 and fl-adrenergic agonists 39 exert a stimulatory effect on a - M S H release. The neurotransmitter acetylcholine (ACh) modulates the secretory activity of various endocrine 19'29 and exocrine glands 33. In particular, muscarinic agonists inhibit prolactin 36 and adrenocorticotropin release 16, and regulate thyrotropin 46 and growth h o r m o n e secretion 5'6'48 from anterior pituitary cells. In addition, A C h has been shown to regulate the electrical activity of a n u m b e r of secretory cells such as lacrimal cells 18 or r-cells of the endocrine pancreas 28.

Correspondence: L. Cazin, Laboratoire d'Endocrinologie Mol6culaire, CNRS URA 650, Unit6 Affili6e h I'INSERM, 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)

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tion of pituitary melanotrophs are scarce and controversial 14'44. We have recently decided to re-examine this issue and we have found that, in the frog, ACh stimulates a-MSH release from whole neurointermediate lobes 22. The aim of the present study was to characterize the pharmacological profile of cholinergic receptors by examining the electrical and secretory activities of frog pituitary melanotrophs in primary culture. We demonstrate that ACh increases the action potential frequency and the secretory activity of isolated pituitary cells through recruitment of M 1 muscarinic receptors.

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Frog pars intermedia cells were dissociated by enzymatic digestion 25 and plated at a density of 250 000 cells/ml in tissue culture dishes. The cells were cultured at 26 °C in a humidified atmosphere during 2-7 days in Leibowitz medium supplemented with 10% heat-inactivated foetal calf serum and antibiotics (0.1 mg/ml kanamycine and 1% antibiotic-antimycotic solution). The incubation medium was replaced by fresh medium every day. Cells prepared in this way have been shown to be viable by a number of criteria including the ability to generate Na+-Ca 2+ action potentials 25, to respond to a variety of agents z6"27 and to release and synthetize a-MSH 26. During the recordings, the cells were continuously superfused with standard Ringer solution (112 mM NaCl, 2 mM KCI, 2 mM CaCi2, 11 mM glucose, 15 mM HEPES-NaOH ; pH 7.4). The patch pipette solution contained 100 mM KCI, 2 mM MgC12, 1 mM CaCl z, 10 mM EGTA, 10 mM HEPES-KOH, pH 7.4. Voltage signals were recorded with a List Electronic EPC 7 patch-clamp amplifier (Darmstadt, F.R.G.), using the tight-seal whole-cell recording technique 15. Electrical recordings were stored on a digital video recorder (Sony, Japan) and replayed later for analysis on a Type 2200 S Gould recorder (U.S.A.). Cholinergic agents (acetyicholine, muscarine or nicotine) were diluted in the same solution that bathed the cells, and applied for 1-10 s by pneumatic pressure ejection from micropipettes. Concentrations were 10-6 M or 10-5 M in the application pipette and, therefore, were

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Fig. 1. Action of ACh on electrical and secretory activities of frog pars intermedia cells. A: depolarization and action potential discharge recorded in the whole-cell current-clamp mode. The response was induced by ACh (10-6 M) applied by pressure ejection for 1 s (arrow). Resting potential was -40 mV. B: sustained depolarization and firing induced, in the same cell, by prolonged (30 s) exposure to ACh. The horizontal bar below the record indicates the duration of ACh application. C: effect of ACh on a-MSH secretion by dispersed frog pars intermedia cells. After a 30-rain equilibration period, ACh (10 -6 M) was infused for 10 min. The data represent the mean + S.E.M. of 4 independent perifusion experiments.

Although significant amounts of ACh are present in the intermediate lobe of the pituitary ~2, the data concerning the possible involvement of ACh in the regula-

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Fig. 2. Effect of ACh in the presence or absence of atropine on a-MSH secretion by dispersed frog pars intermedia cells. After a 30-min equilibration period, atropine (10-6 M) was infused for 70 min. During atropine administration, a pulse of ACh (10-4 M) was applied for 10 rain. After atropine withdrawal, the cells were exposed to a second pulse of ACh (10-4 M; 10 min). The data represent the mean + S.E.M. of 4 independent experiments.

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Fig. 4. Immunofluorescence microphotograph of frog pars intermedia cells after 2 days of culture, illustrating the existence of muscarinic receptor-like immunoreactivity. Intense labeling was observed in melanotrophs (m) which exhibit a spherical or bipolar shape while fibroblasts (f) were less stained. Scale bar -~ 20/~m.

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Fig. 3. Action of muscarine on electrical and secretory activities of frog pars intermedia cells. A: effects of muscarine in the absence or presence of pirenzepine on the membrane potential of a cultured cell studied in the whole-cell current-clamp mode. The resting potential was -35 mV. Muscarine (10-5 M) was ejected during 3 s (arrows). Hyperpolarizing pulses (100 pA, 400 ms) were applied at 5-s intervals to examine possible changes in membrane conductance during the recordings. Upper trace: muscarine induced a depolarization and a dramatic increase of membrane conductance. Lower trace: superfusion with pirenzepine ( l f f 5 M) during 1.6 min (horizontal bar below the record) totally abolished the effects of muscarine. B: effect of muscarine on a-MSH secretion from dispersed frog pars intermedia cells. After a 20-min equilibration period, muscarine (10 5 M) was infused for 10 min. The data represent the mean + S.E.M. of 4 independent perifusion experiments.

necessarily lower at the cell surface. Muscarinic and nicotinic antagonists were superfused with standard Ringer solution. To study the effect of acetylcholine during longer periods (exceeding 10 s), the superfusion system was employed to deliver the acetylcholine solution at a well-defined concentration (10-6 M). Perifusion o f pars intermedia cells The perifusion system used in this study has previously been described in detail 26. Briefly, 50 000 freshly dissociated pars intermedia cells were gently mixed with 1 ml preswollen Bio-Gel P2 and packed in a siliconized glass tube. The cells were perifused at a constant flow rate of 0.6 ml/min. The perifusion medium consisted of a standard Ringer solution supplemented with bovine serum albumin (0.3 g/l). The solution was gassed for 15 min with Oz/CO 2 (95:5) and the pH was adjusted to 7.4 with NaOH. The temperature of the medium was maintained at 28 °C. The cells were allowed to stabilize for 30 min before the experimental manipulations commenced. The substances tested in the system were directly dissolved in the perifusion medium, just before administration. The effluent medium was collected as 7.5-rain fractions during the stabilization

period and 1- or 2.5-min fractions during infusion of the secretagogues in order to get detailed information concerning the timecourse of the response of pars intermedia cells. The fractions were immediately chilled on ice and radioimmunoassayed for a-MSH in duplicate on the same day, using a double-antibody radioimmunoassay procedure 42. lmmunofluorescence procedure Immunocytochemical studies of cell cultures were carried out on 1- to 7-day-old cells. The culture medium was aspirated and the cells were rinsed 3 times with PBS before and after fixation in methanol (10 min, -20 °C). All antisera were diluted in PBS supplemented with 1 % human serum albumin and 0.3 % Triton X-100. The cells were preincubated with normal goat serum (diluted 1:25) for 30 min at room temperature, and rinsed 3 times with PBS. The cells were then incubated overnight in a moistened chamber at 4 °C with monoclonal antibodies M-35 raised in mouse against muscarinic receptor 23. The antibodies were diluted 1:100 in PBS containing 0.3 % Triton X-100 and 0.1% human serum albumin. After rinsing with PBS, the cells were incubated for 1 h in a dark moistened chamber at 20 °C with fluorescein isothiocyanate-conjugated goat 7-globulins directed against mouse lgM (GAM/FITC, Nordic Immunology) diluted 1:60. The cells were counterstained for 3 min with Evan's blue (diluted 1:10 000 in distilled water), mounted with PBS-glycerol (1:1) and examined under a Leitz Orthoplan fluorescence microscope. The specificity of the immunoreaction was controlled by using monoclonal antibodies against Factor H, a plasma protein which is not related to membrane receptors. Reagents Acetylcholine, atropine, cytisine, 1,1-dimethyl-4-phenyl-piperazinium, hexamethonium, muscarine and D-tubocurarine were purchased from Sigma Chemical Company (St. Louis, MO). Nicotine was obtained from Merck (ER.G.). Pirenzepine was obtained from Boehringer Ingelheim Laboratories (Reims, France). Dihydrofl-erythroidine was donated by Merck, Sharp and Dohme. Toxin F (also referred to as neuronal bungarotoxin, bungarotoxin 3.1 or kappa-bungarotoxin) was purchased from Biotoxins, Inc. (St. Cloud, U.S.A.). The mouse monoclonal antibody (M-35) raised against calf brain muscarinic receptor was a generous gift from Dr. D. Strosberg (Institut Pasteur, Paris).

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Fig. 5. Action of nicotine on electrical and secretory activities of frog pars intermedia cells. A: depolarization and action potential discharge were recorded in 2 cultured frog melanotrophs in the whole-cell current-clamp mode. Nicotine (10-5 M) was applied by pressure ejection for 3 s (arrows) to a cell exhibiting no spontaneous action potentials (left) and to a cell spontaneously active (right). In both cells, the resting potential was -40 mV. B: effect of nicotine on a-MSH release by dispersed frog pars intermedia cells. After a 30-min equilibration period, increasing concentrations of nicotine (ranging from 10- 7 M to 10-4 M) were infused for 10 rain at 90-min intervals. The data represent the mean + S.E.M. of 4 independent perifusion experiments. C: semi-logarithmic plot showing the dose-response curve of nicotine. The amount of a-MSH released during the 10-min nicotine infusion period is expressed as the net area under the peaks observed on the perifusion profiles. Each point represents the mean + S.E.M. of 4 independent experiments. RESULTS

Effect of acetylcholine on electrical and secretory activities of pituitary melanotrophs. T h e effect of A C h on the electrical activity was studied on 47 cultured m e l a n o t r o p h s , in the whole-cell configuration. A l l of t h e m r e s p o n d e d to A C h application by a depolarization leading to an increased frequency of action potentials (Fig. 1A). W h e n the cells were continuously exposed to A C h (10- 6 M) for a p e r i o d of 30 s, they exhibited a sustained enhanced firing of action potentials which gradually r e t u r n e d to the resting level after r e m o v a l of the n e u r o t r a n s m i t t e r (Fig. 1B). A s shown in Fig. 1C, infusion of A C h (10 -6 M) during 10 min caused a m a r k e d biphasic stimulation of a - M S H release from acutely dispersed pars intermedia cells. During the first phase, the secretion of a - M S H rapidly increased,

reaching a m a x i m u m within 2 min (approximatively 150% of the basal level). Despite continued A C h infusion, the secretion rate of a - M S H then decreased dramatically to about 130 % of basal level and a plateau was observed in the release profile. W h e n A C h was removed, the secretory rate gradually returned to basal level.

Characterization of muscarinic receptors. The cholinergic agonist, muscarine, and the muscarinic antagonists, atropine and p i r e n z e p i n e , were used to study the pharmacological characteristics of muscarinic receptors in our cell model. A s illustrated in Fig. 2, the A C h - i n d u c e d a - M S H secretion was c o m p l e t e l y abolished by atropine (10 -6 M). T h e electrophysiological recordings o b t a i n e d in the current-clamp m o d e of the whole-cell configuration showed that micro-ejection of muscarine (10 -5 M) at the vicinity of the cell gave rise to a

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Fig. 6. Action of nicotinic agonists on a-MSH secretion by dispersed pars intermedia cells. After a 30-min equilibration period, a pulse of nicotine (10-5 M), cytisine (10-5 M) or 1,1-dimethyl-4-phenylpiperazinium(DMPP 10-5 M) was administered for 10 min. Cytisine and DMPP failed to mimic the stimulatory effect of nicotine on a-MSH secretion. The data represent the mean + S.E.M. of 4 independent perifusion experiments. depolarization (Fig. 3A). When repeated hyperpolarizing pulses (100 pA, 400 ms) were applied at 5-s intervals, an increase of the membrane conductance was observed throughout the depolarization phase induced by muscarine. The effects of muscarine on membrane potential and membrane conductance were totally abolished by the specific M 1 muscarinic receptor antagonist pirenzepine (10- 5 M). Muscarine also induced a marked stimulation of a-MSH release from perifused pituitary cells (Fig. 3B). The time-course of the secretory response to muscarine was characterized by a rapid and transient stimulation, which occurred during the first 2 min, followed by stabilization of the secretory rate at an intermediate value during the last 5 min of infusion of the secretagogue. The indirect immunofluorescence technique, using monoclonal antibodies to muscarinic receptors, revealed the occurrence of an intense immunoreaction in all cells considered as melanotrophs according to their morphological characteristics previously described 26 (Fig. 4). Fibroblasts, which are easily recognizable, due to their bigger size and multipolar shape 26, were also immunopositive, but the intensity of labeling was much lower in these cells than in melanotroph cells. The existence of unstained cells was never noticed in the culture dishes.

release (Fig. 5B). Again, the kinetics of the secretory response of the cells to nicotine was similar to that observed with ACh or muscarine. The minimum effective dose was 10 -7 M and the maximum stimulation occurred at a dose of 10-5 M nicotine (Fig. 5C). The response was not mimicked by cytisine (10-5 M) or 1,1-dimethyl4-phenylpiperazinium (DMPP, 10- 5 M), 2 nicotinic agonists (Fig. 6). The effect of nicotine (10 -6 o r 10-5 M) on a-MSH release was investigated during prolonged administrations of 4 different nicotinic antagonists: hexamethonium (10 -4 M), d-tubocurarine (10-5 M), toxin F (10 -6 M) or dihydro-fl-erythro~dine (10-5 M). None of these nicotinic antagonists had any effect on nicotineevoked a-MSH secretion (Fig. 7). In addition, the electrophysiological effect of nicotine was not affected by infusion of hexamethonium (10 -4 M) or a-bungaratoxin (10-5 M), 2 highly potent nicotinic antagonists (Fig. 8). In order to verify that the stimulatory effect of nicotine on a-MSH release could not be accounted for by activation of muscarinic receptors, the effect of nicotine (10 -6 M) was tested in the presence of atropine (10 -6 M). The results indicated that the muscarinic antagonist did not modify the response of pituitary melanotrophs to nicotine (Fig. 9).

Characterization of nicotinic receptors.

DISCUSSION

Application of nicotine (10 -5 M) via the micro-ejection system caused both membrane depolarization and an increase of the frequency of the action potential discharge (Fig. 5A). Administration of nicotine, at doses ranging between 10 -7 and 10 -4 M, to perifused pituitary cells gave rise to a dose-dependent stimulation of a-MSH

In the early study of Dierst-Davis et al. 11, it was reported that in vivo injection of ACh in the frog hypothalamus causes pigment dispersion in skin melanophores. Paradoxically enough, however, these authors observed that the cholinergic antagonist atropine also

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Fig. 7. Action of nicotine, in the absence or in the presence of nicotinic antagonists, on a-MSH secretion by dispersed pars intermedia cells. After a 30-min equilibration period, hexamethonium (10 -4 M; 70 min) or d-tubocurarine (10-5 M; 70 min) or toxin F (10-6 M; 40 min) or dihydro-fl-erythroi'dine (DHBE 10-5 M; 70 min) were administered. During the infusion of each antagonist, a pulse of nicotine (10- 5 or 10~ M) was added for 10 min. The cells were allowed to stabilize for 75 min, then a second pulse of nicotine (10-5 or 10-6 M) was perifused for 10 min (upper and lower profiles). The data represent the mean + S.E.M. of 4 independent perifusion experiments.

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A C h stimulates m e l a n o t r o p i n secretion 14'22, while others suggest that A C h does not regulate a - M S H release 44. We have therefore decided to re-examine this issue using the patch-clamp technique on cultured pars i n t e r m e d i a cells and the perifusion technique on dispersed cells. The results r e p o r t e d here show that acetylcholine stimulates both the electrical activity and the secretory activity of frog melanotrophs. In concert with this finding, H a d l e y et al. TMalso r e p o r t e d a stimulatory effect of A C h on a - M S H release from the n e u r o i n t e r m e d i a t e lobe of R a n a

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Based on pharmacological and immunocytochemical criteria, the effect of A C h on frog m e l a n o t r o p h s app e a r e d to be m e d i a t e d through activation of M 1 muscarinic receptors: (1) the stimulatory effect of A C h was mimicked by muscarine and totally blocked by the muscarinic antagonist atropine; (2) m e m b r a n e depolarization and the increase of m e m b r a n e conductance e v o k e d by muscarine were both inhibited by the selective M 1 muscarinic antagonist pirenzepine; (3) using a monoclonal antibody against bovine brain muscarinic receptors 23 we have visualized a h o m o l o g o u s protein in cultured frog melanotrophs. The observation that not only pituitary m e l a n o t r o p h s , but also to a certain extent fibroblasts, were i m m u n o s t a i n e d with the muscarinic receptor antibody is consonant with previous findings which showed the existence of muscarinic receptors at the surface of human fibroblast cells 3. In mammals, A C h has been shown to regulate the secretion of several anterior pituitary hormones, including prolactin 36, adrenocorticotropin 16 and growth hormone 5'4~ via a muscarinic mechanism. In concert with these findings, muscarinic receptors have been identified

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Fig. 8. Action of nicotine in the absence or presence of hexamethonium CA) or a-bungarotoxin (B) on membrane potential of 2 cultured frog melanotrophs studied in the whole-cell current-clamp mode. A: nicotine (10-5 M) was ejected during 3 s (arrows) on a cell held at -40 mV. Upper trace: nicotine-induced depolarization to -20 mV. Middle trace: superfusion with hexamethonium (10-4M) during 2.1 min (horizontal bar under the record) did not affect the depolarization induced by nicotine. Lower trace: nicotine-evoked depolarization during the washout. B: nicotine (10 5 M) was ejected during 3 s (arrow) on a cell held at -60 mV. Superfusion with a-bungarotoxin (10 5 M) during 1 min (horizontal bar under the record) did not suppress the stimulatory action of nicotine. induces m e l a n o p h o r e expansion. More recent investigations on isolated amphibian n e u r o i n t e r m e d i a t e lobes have led to conflicting results, with studies indicating that Atropine 10-6 M

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Fig. 9. Effect of nicotine in the presence or absence of atropine on a-MSH secretion by dispersed frog pars intermedia cells. After a 30-min equilibration period, atropine (10 6 M) was infused for 70 min. During atropine administration, a pulse of nicotine (10 6 M) was applied for 10 min. After atropine withdrawal, the cells were exposed to a second pulse of nicotine (10 6 M; 10 min). The data represent the mean + S.E.M. of 4 independent perifusion experiments.

307 in the a n t e r i o r pituitary of the rat 17'3°. The involvement of muscarinic receptors in the stimulatory effect of A C h on frog pars i n t e r m e d i a cells is now clearly d e m o n s t r a t e d . O u r study also suggests that nicotinic receptors are present in isolated pituitary m e l a n o t r o p h s , since both electrophysiological and secretory activities were stimulated by nicotine. Nevertheless, as atropine completely abolished A C h - i n d u c e d a - M S H release, it appears that nicotinic receptors are not involved in the effects of acetyicholine. In addition we o b s e r v e d that (1) four different nicotinic antagonists failed to inhibit nicotinee v o k e d a - M S H release and (2) two nicotinic agonists had no effect on a - M S H secretion. These findings support the concept that frog m e l a n o t r o p h s possess a nicotinic rec e p t o r subtype u n r e l a t e d to identified nicotinic binding sites 24'31"45'47. In this respect it is interesting to mention that, in amphibians, m e l a n o t r o p h cells display dopaminergic receptors which exhibit pharmacological characteristics distinct from those of the m a m m a l i a n D 2 subtype 44. The role and the pharmacological profile of the nicotinic r e c e p t o r s present on frog m e l a n o t r o p h cells, as well as the nature of the e n d o g e n o u s ligand which m a y stimulate these receptors remain to be d e t e r m i n e d . The physiological significance of A C h - i n d u c e d stimulation of electrical and secretory activity of m e l a n o t r o p h cells needs further investigations. In particular, the source of A C h which m a y stimulate muscarinic receptors is currently a m a t t e r of speculation. In the rat, Egozi et al. 12 have recently shown that the n e u r o i n t e r m e d i a t e lobe of the pituitary contains high amounts of A C h . In fact, REFERENCES 1 Adjeroud, S., Tonon, M.C., Gouteux, L., Leneveu, E., Lamacz, M., Cazin, L. and Vaudry, H., In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system, IV. Interaction between dopamine and thyrotropin-releasing hormone on a-melanocyte stimulating hormone secretion, Gen. Comp. Endocrinol., 64 (1986) 428434. 2 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., GABA-ergic control of a-melanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro, Brain Res. Bull., 17 (1986) 717-724. 3 Andr6, C., Marullo, S., Guillet, J.G., Convents, A., Lauwerays, M., Kaveri, S., Hoebeke, J. and Strosberg, A.D., Immunochemical studies of the muscarinic acetylcholine receptor, J. Recept. Res., 7 (1987) 89-103. 4 Bagnara, J.T. and Hadley, M.E., Chromatophores and Color Change; The Comparative Physiology of Animal Pigmentation. Prentice-Hall, Englewood Cliffs, NJ, 1973. 5 Canonico, P.L., Jarvis, W.D., Sortino, M.A., Scarpagnini, U. and MacLeod, R.M., Cholinergic stimulation of inositol phosphate production in cultured anterior pituitary cells, Neuroendocrinology, 46 (1987) 306-311. 6 Carmeliet, P. and Denef, C., Immunocytochemical and pharmacological evidence for intrinsic cholinergic system modulating prolactin and growth hormone release in rat pituitary, Endocrinology, 123 (1988) 1128-1139.

the concentration of A C h is 3 - 6 times higher in the posterior lobe than in the distal lobe of the pituitary. In the n e u r o i n t e r m e d i a t e lobe of amphibians, electronmicroscopic studies have r e v e a l e d the presence of nerve terminals containing small vesicles ( 3 0 0 - 4 0 0 / ~ ) , but the cholinergic nature of these nerve fibers has not been expressly established 32. A l t e r n a t i v e l y , A C h m a y be synthesized locally within the frog n e u r o i n t e r m e d i a t e lobe, since p r o d u c t i o n of A C h has b e e n d e m o n s t r a t e d in non-neuronal tissues 13'35. Consistent with this latter hypothesis are the findings of C a r m e l i e t and D e n e f 6'7 that rat pituitary corticotrophs and the mouse pituitary cell line AtT20 synthesize and release significant amounts of A C h . Since these 2 types of cells, like m e l a n o t r o p h cells of the pars i n t e r m e d i a , synthesize the precursor protein P O M C , possible c o o r d i n a t e expression Of P O M C and choline-acetyltransferase within pituitary cells remains to be investigated. Acknowledgements. We are indebted to Dr. D. Strosberg (Institut Pasteur, Paris) for the mouse monoclonal antibody (M-35) raised against calf brain muscarinic receptors. We would like to thank Dr. M. Fontaine (INSERM U 78, Bois Guillaume, France) for providing monoclonal antibody against Factor H and to Drs. K. Scheibli and R. Andreatta (Ciba-Geigy, Basel, Switzerland) for their generous gift of synthetic a-MSH. We are grateful to Drs. C. Mulle and C. Vidal for helpful suggestions. We thank Mrs. C. Buquet for expert technical assistance. E.L. was a recipient of a post-doctoral fellowship from the Fondation de la Recherche M6dicale Fran~aise. This research was supported in part by the Institut National de la Sant6 et de la Recherche M6dicale (Grants 86-4016 and 89-4015), by the European Economic Community (Grant 87 300 445) and by the Conseil R6gional de Haute-Normandie.

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