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Endocrinology Vol. 139, No. 8 3525-3533
Copyright © 1998 by The Endocrine Society

ARTICLES

Pharmacological and Functional Characterization of Muscarinic Receptors in the Frog Pars Intermedia1

Marianne Garnier2, Marek Lamacz, Ludovic Galas2, Sebastien Lenglet, Marie-Christine Tonon and Hubert Vaudry

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. H. Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The secretion of {alpha}MSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates {alpha}MSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated {alpha}MSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced {alpha}MSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and {alpha}MSH release. In contrast, {omega}-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and {alpha}MSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked {alpha}MSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on {alpha}MSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10-4 M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of {alpha}MSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN AMPHIBIANS, the melanotropic peptide {alpha}MSH, secreted by melanotrope cells of the pars intermedia, plays a pivotal role in the process of skin color adaptation (1). This camouflage aptitude allows the animals to escape to their predators and thus is essential for the survival of endangered amphibian species. The neuroendocrine mechanisms regulating the activity of melanotrope cells in amphibians has been mainly studied in two representative species, the African clawed toad Xenopus laevis (2, 3) and the European green frog Rana ridibunda (4, 5). Using these animal models, it has been demonstrated that {alpha}MSH secretion is controlled by multiple factors, including classical neurotransmitters and neuropeptides. In the frog Rana ridibunda, the activity of melanotrope cells is inhibited by dopamine (6), {gamma}-aminobutyric acid (7, 8), serotonin (9), adenosine (10, 11), {alpha}-adrenergic agonists (12), and neuropeptide Y (13, 14) and is stimulated by ß-adrenergic agonists (12) and TRH (5, 15, 16, 17).

The intermediate lobe of the mammalian pituitary contains both melanotrope cells and corticotrope cells (18). The pars intermedia of amphibians, which is composed of a homogeneous population of melanotrope cells (19), represents a very appropriate model in which to investigate the transduction pathways involved in the mechanism of action of the neuroendocrine messengers regulating {alpha}MSH secretion.

Acetylcholine (ACh) is recognized as an important modulator of the activity of various types of pituitary cells (20, 21, 22, 23, 24). In particular, ACh stimulates the activity of melanotrope cells in mammals (25, 26) and amphibians (27, 28, 29). In both toads and frogs, the effect of ACh on {alpha}MSH secretion is mediated by muscarinic receptors (26, 28). Local synthesis of ACh has been demonstrated in the porcine (30) and toad (29) pars intermedia, indicating that ACh may exert an autocrine control on the activity of melanotrope cells.

The aim of the present study was to characterize the type of muscarinic receptor and the transduction mechanisms mediating the action of ACh in melanotrope cells of the frog pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda) of about 30 g body weight were purchased from a commercial supplier (Couétard, St. Hilaire de Riez, France). The animals were maintained under artificial illumination (lights on from 0600–1800 h) in a temperature-controlled room (8 ± 0.5 C). The animals were killed by decapitation, and the neuroin-termediate lobes (NILs) were immediately dissected under a microscope. All animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Reagents
Carbachol, muscarine, pirenzepine, isoproterenol, genistein, forskolin, isobutylmethylxanthine, pertussis toxin (PTX), nifedipine, {omega}-conotoxin GVIA ({omega}-CgTx), 7,7'-dimethyl eicosadienoic acid, EGTA, HEPES, Leibovitz culture medium (L15), collagenase type IA, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and McN-A-343 were supplied by Research Biochemicals International (Natick, MA). Indo-1 acetoxymethylester was obtained from Molecular Probes (Eugene, OR). Bio-Gel P-2 and the anion exchange resin AG1-X8 (100–200 mesh; formate form) were obtained from Bio-Rad Laboratories (Hercules, CA). BSA (fraction V) was purchased from Boehringer Mannheim (Paris, France). Myo-[3H]inositol was obtained from Amersham (Aylesbury, UK). Tyrphostin 25 (Tyr-A25, AG 82) was obtained from Calbiochem (San Diego, CA). Lavendustin A was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Kanamycin was purchased from Life Technologies (Grand Island, NY). The antibiotic-antimycotic solution and FBS were obtained from BioWhittaker (Gagny, France). Other chemicals were purchased from Sigma.

Perifusion technique
The perifusion system used in this study has been previously described in detail (15). Briefly, NILs were incubated for 15 min in a Ringer’s solution consisting of 15 mM HEPES, 112 mM NaCl, 2 mM KCl, and 2 mM CaCl2 supplemented with 2 g/liter glucose and 0.3 g/liter BSA. The solution was gassed with O2-CO2, (95:5, vol/vol) before use, and the pH was adjusted to 7.35. Then NILs were layered between two beds of Bio-Gel P2 into a plastic column (id, 0.9 cm) delimited by two Teflon pestles (four NILs per perifusion chamber). The tissues were perifused with the Ringer’s solution at a constant flow rate (0.25 ml/min) and temperature (24 C). The effluent perifusate was collected as 7.5-min fractions during stabilization periods and 2.5-min fractions during infusion of the secretagogues. The fractions were immediately chilled at 4 C, and the concentration of {alpha}MSH was measured in each fraction on the same day as the perifusion experiment using a double antibody RIA procedure (31). The perifusion profiles are expressed as percentages of the basal secretory level, calculated as the mean of four consecutive fractions collected just before the infusion of the secretagogue.

Cell culture
NILs were collected in Ca2+-free Ringer’s solution (15 mM HEPES, 112 mM NaCl, 2 mM KCl, 1 mM EGTA, 2 g/liter glucose, and 0.3 g/liter BSA). The tissues were enzymatically dispersed at 22 C for 20 min with collagenase (1.5 mg/ml) in the same solution. Nondissociated neural lobes were allowed to settle, and the supernatant containing dissociated pars intermedia tissue was sampled and centrifuged (30 x g, 5 min). After three rinses with Ca2+-free Ringer’s solution, the cells were dispersed by gentle aspiration through a siliconized Pasteur pipette with a flame-polished tip. The cells were resuspended in L15 culture medium adjusted to frog osmolality (L15/water = 1:0.4) supplemented with 0.2 g/liter glucose, 63 mg/liter CaCl2, and 1% of the kanamycin and antibiotic-antimycotic solutions (fL15). Finally, cells were plated on 35-mm glass coverslips previously coated with poly-L-lysine (10 µg/ml) at a density of 15,000 cells/coverslip. When the cells had settled, coverslips were covered with 2 ml fL15 medium supplemented with 10% FBS. Cells were cultured in a humid atmosphere incubator at 24 C, and the culture medium was renewed every 48 h. Microfluorometric measurements were performed on 3- to 5-day-old cultured cells.

Intracellular calcium measurements
Cultured melanotrope cells were incubated at room temperature with 5 µM indo-1/acetoxymethylester in fL15 medium for 30 min and washed twice with Ringer’s solution. The cytosolic calcium concentration ([Ca2+]i) was monitored by a dual emission microfluorometric system constructed from a Nikon Diaphot inverted microscope equipped for epifluorescence with an oil immersion objective (x100; CF Fluor series; numerical aperture, 1.3) as previously described (32). The fluorescence emission of indo-1, induced by excitation at 355 nm, was recorded at two wavelengths (405 nm, corresponding to the complexed form, and 480 nm, corresponding to the free form) by separate photometers (P1; Nikon, Melville, NY). The 405/480 ratio (R) was determined using an AS1-type acquisition card (Notocord Systems, Croissy-sur-Seine, France). All three signals (405 nm, 480 nm, and R) were continuously recorded with the JAD-FLUO program (version 1.2; Notocord Systems, Croissy-sur-Seine, France). [Ca2+]i was calculated according to the formula established by Grynkiewicz et al. (33): [Ca2+]i = Kd x ß (R - Rmin)/(Rmax - R), where Rmin represents the minimum fluorescence ratio obtained after incubation of cells in Ringer’s solution containing 10 mM EGTA and 10 µM ionomycin, Rmax is the maximum fluorescence ratio obtained after incubation of cells in Ringer’s solution containing 10 mM CaCl2 and 10 µM ionomycin, and ß is the ratio of fluorescence yield from the Ca2+min/Ca2+max indicator at 480 nm. The values obtained for Rmin, Rmax, and ß were 0.16, 1.82, and 1.62, respectively. The dissociation constant for indo-1 (Kd = 250 nM) has been previously determined (34).

Inositol phospholipid turnover
Measurement of membrane phospholipid metabolites was performed as previously described (16). Whole NILs were incubated in fL15 medium with myo-[3H]inositol (100 µCi/ml) for 18 h. The pulse medium was then discarded, and the NILs were washed six times with Ringer’s solution supplemented with 1 mM inositol. The NILs were preincubated for 10 min with 10 mM LiCl and exposed for various durations to muscarine in the presence of 10 mM LiCl. The reaction was stopped by addition of ice-cold 20% trichloroacetic acid. The NILs were homogenized, and the membrane fraction was removed by centrifugation. Inositol phosphates contained in the supernatant were analyzed by anion exchange chromatography on AG1-X8 resin, as previously described (16). Free [3H]inositol was eluted by water, whereas inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were sequentially eluted by a step gradient of ammonium formate (0.2, 0.45, and 0.8 M, respectively) in 0.1 M formic acid. For each sample, 38 fractions (4 ml each) were collected, and the radioactivity was determined in a 1217 Rackbeta counter (Wallac, Eury, France).

cAMP measurement
Whole NILs were preincubated for 30 min at room temperature with 0.1 mM isobuthylmethylxanthine. The NILs were then incubated for 20 min with muscarine, isoproterenol, or forskolin. The reaction was stopped by addition of ice-cold 20% trichloroacetic acid. NILs were homogenized and centrifuged (10,000 x g for 10 min). Trichloroacetic acid was eliminated from the supernatant by three successive rinses with 1 ml water-saturated diethyl ether. After evaporation of the ether phase, the supernatant was lyophilized, and the cAMP content in the dried extract was measured by RIA, following the procedure recommended in the cAMP RIA kit (Amersham).

Statistical analysis
Values are expressed as the mean ± SEM. Statistical comparisons between groups were made using ANOVA, followed by Student’s t test. Differences were taken to be statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacological characterization of muscarinic receptors
Administration of graded concentrations of muscarine to perifused frog NILs induced a dose-related stimulation of {alpha}MSH release (Fig. 1Go, A and B). The minimum effective concentration was 3 x 10-6 M. At a concentration of 10-4 M, muscarine caused a 3-fold increase in {alpha}MSH secretion (Fig. 1AGo). The dose-response curves obtained with various muscarinic agonists are compared in Fig. 1BGo. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated {alpha}MSH release in a concentration-dependent manner, and the ED50 values were, respectively, 1.2 x 10-5 and 3.2 x 10-6 M. In contrast, the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect on {alpha}MSH release from perifused frog NILs (Fig. 1BGo).



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  		Figure 1. Effect of muscarinic receptor agonists on {alpha}MSH secretion from perifused frog NILs. A, Perifusion profiles showing effect of graded concentrations of muscarine (Musc; 10 min) on {alpha}MSH release. Profiles represent the mean (±SEM) secretion pattern of four to seven independent perifusion experiments. The mean basal level of {alpha}MSH release (100% basal level) was calculated as the mean {alpha}MSH concentration in the four consecutive fractions (30 min; {circ}) collected before administration of the muscarinic agonist. B, Semilogarithmic plot showing the effects of increasing concentrations of the nonselective muscarinic receptor agonists muscarine (Musc) and carbachol (Carb) and the M1-selective agonist McN-A-343 (McN) on {alpha}MSH release from perifused frog NILs. All experimental values were calculated from data similar to those presented in A. Each point represents the mean (±SEM) of three to seven experiments. The mean basal level of {alpha}MSH release in these experiments was 77 ± 11 pg/min·NIL.

 
Administration of the muscarinic receptor antagonist 4-DAMP (10-6 M) totally abolished the stimulatory effect of muscarine (10-4 M) on {alpha}MSH release (Fig. 2AGo). A series of experiments similar to those presented in Fig. 2AGo was conducted with different concentrations of muscarinic antagonists. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-DAMP inhibited the muscarine-induced {alpha}MSH release in a dose-dependent manner (Fig. 2BGo). The respective ED50 were 1.1 x 10-7 and 2.1 x 10-8 M.



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  		Figure 2. Effect of muscarine in the absence or presence of muscarinic receptor antagonists on {alpha}MSH release from perifused frog NILs. A, Perifusion profiles showing effect of muscarine (Musc; 10-4 M; 10 min) on {alpha}MSH release in control conditions (left panel) or during prolonged administration of the M3>M1 antagonist 4-DAMP (10-6 M; right panel). The antagonist was administered 45 min before the pulse of muscarine. Profiles represent the mean (±SEM) secretion pattern of four independent perifusion experiments. See Fig. 1Go for other designations. B, Semilogarithmic plot showing the effects of increasing concentrations of the M1>M3 antagonist pirenzepine (Pir) and the M3>M1 antagonist 4-DAMP on {alpha}MSH release induced by muscarine (10-4 M). All experimental values were calculated from data similar to those presented in A. Each point represents the mean (±SEM) of three or four independent experiments. Results are expressed as a percentage of the response induced by muscarine in the absence of antagonist. The mean basal level of {alpha}MSH release in these experiments was 200 ± 19 pg/min·NIL. **, P < 0.01; ***, P < 0.001.

 
Effect of muscarine on Ca2+mobilization
Under resting conditions, the mean [Ca2+]i in cultured frog melanotrope cells was 33 ± 4 nM (n = 26). Ejection of muscarine (10-4 M; 10 sec) in the vicinity of the cells elicited a rapid and significant (P < 0.001) increase in [Ca2+]i to 120 ± 11 nM (Fig. 3Go, trace A). Thereafter, [Ca2+]i returned gradually to the basal level within 1.5 min. When the cells were incubated in calcium-free medium supplemented with 3 mM EGTA, the effect of muscarine on [Ca2+]i was totally abolished (Fig. 3Go, trace B).



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  		Figure 3. Effect of muscarine (10-4 M; 10 sec) on [Ca2+]i in cultured frog melanotrope cells. A, In the presence of 2 mM Ca2+, muscarine induced an immediate increase in [Ca2+]i. The profile represents the mean (±SEM) response of 26 cells. B, In calcium-free medium containing 3 mM EGTA, muscarine did not modify [Ca2+]i. The profile represents the mean (±SEM) response of 17 cells. The arrow indicates the onset of muscarine administration.

 
In the presence of 2 mM Ca2+, the mean rate of secretion of {alpha}MSH from perifused frog NILs was 69 ± 10 pg/min·NIL. Suppression of calcium in the perifusion medium markedly reduced the basal rate of {alpha}MSH release and completely abolished the stimulatory effect of muscarine on {alpha}MSH secretion (Fig. 4AGo). Similarly, addition of NiCl2 (3 mM) to a Ringer’s buffer containing 2 mM Ca2+ inhibited both the spontaneous and the muscarine-evoked {alpha}MSH secretion (Fig. 4BGo).



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  		Figure 4. Effect of calcium suppression (Ca2+, 0; EGTA, 3 mM; A) or perifusion with NiCl2 (Ca2+, 2 mM; Ni2+, 3 mM; B) on basal and muscarine-induced {alpha}MSH release. The pulses of muscarine (Musc; 10-4 M; 10 min) were administered 45 min after the onset of perifusion with Ca2+-free or Ni2+-containing medium. Profiles represent the mean (±SEM) secretion pattern of 4 (A) and 5 (B) independent perifusion experiments. The mean basal level of {alpha}MSH release in these experiments was 69 ± 10 pg/min·NIL. See Fig. 1Go for other designations.

 
Preincubation of the cells for 20–60 min with the N-type calcium channel blocker {omega}-CgTx (10-6 M) did not significantly modify the [Ca2+]i rise evoked by 10-4 M muscarine (Fig. 5AGo). Likewise, addition of the L-type calcium channel blocker nifedipine (10-5 M) to the incubation medium did not significantly affect the [Ca2+]i response to muscarine (Fig. 5BGo).



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  		Figure 5. Effect of muscarine (10-4 M; 10 sec) on [Ca2+]i in cultured frog melanotrope cells in the presence of calcium channel blockers. A, Effect of muscarine on [Ca2+]i in the presence of the L-type calcium channel blocker, nifedipine (10-5 M). B, Effect of muscarine on [Ca2+]i in the presence of the N-type calcium channel blocker, {omega}-conotoxin GVIA (10-6 M). Cells were incubated with nifedipine or {omega}-conotoxin GVIA for 20–60 min before the administration of the pulse of muscarine. Profiles represent the mean (±SEM) responses of 14 (A) and 15 cells (B). The arrow indicates the onset of muscarine administration.

 
Prolonged exposure of perifused frog NILs to {omega}-CgTx (10-6 M) did not impair the stimulatory effect of muscarine (10-4 M) on {alpha}MSH secretion (Fig. 6Go, A and B). Similarly, perifusion of NILs in the presence of nifedipine (up to 10-4 M) did not significantly reduce the muscarine-evoked {alpha}MSH release (Fig. 6Go, A, C, and D).



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  		Figure 6. Effect of muscarine in the absence or presence of calcium channel blockers on {alpha}MSH release from perifused frog NILs. A single pulse of muscarine (Musc; 10-4 M; 10 min) was administered under control conditions (A) or during prolonged infusion of 1 µM {omega}-CgTx (B), 10 µM nifedipine (C), or 100 µM nifedipine (D). Profiles represent the mean (±SEM) secretion pattern of three to six independent perifusion experiments. The mean basal level of {alpha}MSH release in these experiments was 126 ± 22 pg/min·NIL. See Fig. 1Go for other designations.

 
Effect of muscarine on inositol phospholipid turnover
The effect of muscarine (10-4 M) on phosphoinositide (PI) hydrolysis was investigated by measuring inositol phosphate levels after incorporation of myo-[3H]inositol by frog NILs. Exposure of NILs to 10-4 M muscarine provoked a modest, but significant, increase in IP3 formation. The enhancement of IP3 production occurred within the first minute of administration of muscarine and reached a maximum at 5 min. Thereafter, the IP3 level gradually declined and returned to basal values 15 min after the onset of muscarine administration (Fig. 7Go). Muscarine also caused a marked increase in IP2 levels; the maximum effect was observed after 10 min of incubation with muscarine. A significant increase in IP1 was also observed after 5 min of incubation with muscarine.



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  		Figure 7. Effects of muscarine on IP3, IP2, and IP1 formation in myo-[3H]inositol-prelabeled frog NILs. After a 10-min preincubation with 10 mM LiCl, NILs were incubated in the presence of 10-4 M muscarine for the times indicated. Results are expressed as a percentage of the muscarine-induced inositol phosphate (IPx) level in the control. Each value represents the mean (±SEM) of 5–14 independent experiments. Mean inositol phosphate levels in controls were 292 ± 26, 1,160 ± 104, and 16,332 ± 1,490 cpm/NIL, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Incubation of NILs with HEPES buffer containing 6 mM EGTA for 20 min did not affect the basal production of total inositol phosphates, but totally abolished the stimulatory effect of muscarine (10-4 M; 20 min) on inositol phosphate formation (Fig. 8Go).



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  		Figure 8. Effect of EGTA on muscarine-induced inositol phosphate (IPx) formation. Myo-[3H]inositol-labeled NILs were preincubated for 20 min in HEPES buffer in the absence or presence of 6 mM EGTA. NILs were then incubated for 20 min in the same medium in the absence or presence of muscarine (10-4 M). Results are expressed as a percentage of the IPx level in the absence of EGTA and muscarine. Data are the mean (±SEM) values from five independent experiments. *, P < 0.05; **, P < 0.01.

 
Prolonged administration of the PI turnover blocker neomycin (3 mM; 110 min) to perifused frog NILs significantly (P < 0.01) inhibited the stimulatory effect of muscarine (10-4 M; 10 min) on {alpha}MSH release (Fig. 9Go). In addition, pretreatment of frog NILs with PMA (10-6 M; 24 h) markedly reduced the effect of muscarine on {alpha}MSH secretion (Fig. 10Go). In contrast, the phospholipase A2 (PLA2) inhibitor 7,7'-dimethyl eicosadienoic acid did not affect muscarine-induced {alpha}MSH release (data not shown).



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  		Figure 9. Effect of muscarine in the absence or presence of the phosphatidylinositol turnover blocker neomycin on {alpha}MSH release from perifused frog NILs. A single pulse of muscarine (Musc; 10-4 M; 10 min) was administered in control conditions (left panel) or during prolonged infusion of neomycin (3 mM; 110 min; right panel). Profiles represent the mean (±SEM) secretion pattern of six independent perifusion experiments. The mean basal level of {alpha}MSH release in these experiments was 119 ± 7 pg/min·NIL. See Fig. 1Go for other designations.

 


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  		Figure 10. Effect of long term treatment with PMA on muscarine-induced {alpha}MSH release from perifused frog NILs. Intact NILs were incubated for 24 h in fL15 alone (left panel) or in fL15 supplemented with PMA (10-6 M; right panel). Then, a single pulse of muscarine (Musc; 10-4 M) was administered for 10 min. Profiles represent the mean (±SEM) secretion pattern of four independent perifusion experiments. The mean basal level of {alpha}MSH release in these experiments was 98 ± 12 pg/min·NIL. See Fig. 1Go for other designations.

 
Effect of muscarine on cAMP formation
Incubation of frog NILs with muscarine (10-4 M) produced an elevation of the cAMP content in the tissue (+40%; P < 0.01; Fig. 11Go). However, this effect was modest compared with those of the ß-adrenergic agonist isoproterenol (10-5 M) and forskolin [5 x 10-5 M; +85% (P < 0.001) and +260% (P < 0.001), respectively].



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  		Figure 11. Effects of muscarine, isoproterenol, and forskolin on cAMP content in frog NILs. Intact NILs were incubated in HEPES buffer alone (C, control) or supplemented with 100 µM muscarine (Musc), 10 µM isoproterenol (Iso), or 50 µM forskolin (FK). Data are the mean (±SEM) values from 5–14 independent experiments. **, P < 0.01; ***, P < 0.001 (vs. control).

 
Effect of PTX on muscarine-induced {alpha}MSH secretion
To test the possible involvement of a PTX-sensitive G protein in the mechanism of action of muscarine, frog NILs were incubated with PTX (1 µg/ml) for 18 h. Pretreatment of the tissue with PTX did not inhibit the stimulatory effect of muscarine (10-4 M; 10 min) on {alpha}MSH release (Fig. 12Go).



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  		Figure 12. Effect of PTX pretreatment on muscarine-induced {alpha}MSH release from perifused frog NILs. Intact NILs were incubated for 18 h in fL15 alone (left panel) or in fL15 supplemented with PTX (1 µg/ml; right panel). Then, a single pulse of muscarine (Musc; 10-4 M) was administered for 10 min. Profiles represent the mean (±SEM) secretion pattern of four independent perifusion experiments. The mean basal level of {alpha}MSH release in these experiments was 90 ± 6 pg/min·NIL. See Fig. 1Go for other designations.

 
Effect of tyrosine kinase inhibitors on muscarine-induced {alpha}MSH secretion
The effect of a series of tyrosine kinase inhibitors on the secretory response of frog NILs to muscarine was investigated (Fig. 13Go). Lavendustin A (10-7 M; Fig. 13Go, A and B), genistein (10-5 M; Fig. 13Go, A and C), and Tyr-A25 (10-4 M; Fig. 13Go, A and D) did not inhibit muscarine-induced {alpha}MSH release.



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  		Figure 13. Effect of tyrosine kinase inhibitors on muscarine-induced {alpha}MSH release from perifused frog NILs. A single pulse of muscarine (Musc; 10-4 M; 10 min) was administered under control conditions (A) or during prolonged infusion of 0.1 µM lavendustin A (B), 10 µM genistein (C), or 100 µM tyrphostin 25 (C). Profiles represent the mean (±SEM) secretion pattern of three to six independent perifusion experiments. The mean basal level of {alpha}MSH in these experiments was 104 ± 14 pg/min·NIL. See Fig. 1Go for other designations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pharmacological profile of muscarinic receptors in frog melanotrope cells
Previous studies have shown that ACh stimulates the electrical (28) and secretory (27) activities of frog melanotrope cells through activation of muscarinic receptors. As the effect of ACh was blocked by the M1-preferring muscarinic receptor antagonist pirenzepine, but not by the selective M2 receptor antagonist gallamine, it was suggested that the action of ACh was mediated through an M1-like muscarinic receptor.

However, five subtypes of muscarinic receptors have now been identified (35), allowing a more rigorous pharmacological characterization of the receptor subtypes. In particular, it was found that pirenzepine does not clearly discriminate between the M1 and M3 receptor subtypes (36).

The present study has shown that the nonspecific muscarinic agonists muscarine and carbachol both induced a dose-dependent stimulation of {alpha}MSH release, whereas the selective M1-receptor agonist McN-A-343 did not affect the secretory activity of frog NILs, indicating that M1 receptors are not involved in the stimulatory effect of ACh on {alpha}MSH secretion. The fact that the M3>M1 antagonist 4-DAMP was 5 times more potent than the M1>M3 antagonist pirenzepine in inhibiting the muscarine-evoked stimulation of {alpha}MSH secretion suggests that the effect of ACh on frog NIL is mediated by M3, rather than M1, receptors. Consistent with this observation, it has been reported that M3 receptors are expressed in various types of endocrine cells (37, 38, 39).

Transduction mechanisms associated with activation of muscarinic receptors in frog melanotrope cells
Stimulation of M1, M3, and M5 muscarinic receptors can activate a number of signaling pathways (see Ref. 40 for review). Although these receptor subtypes are generally associated with activation of polyphosphoinositide turnover, the involvement of PLA2 and phospholipase D, adenylyl cyclase, and tyrosine kinases has also been described. In the present study, we have investigated the transduction mechanisms involved in the muscarine-induced stimulation of {alpha}MSH release in frog NILs.

Administration of muscarine to cultured frog melanotrope cells provoked a rapid, monophasic elevation of [Ca2+]i. Muscarinic agonists usually induce an immediate transient [Ca2+]i rise due to mobilization of intracellular Ca2+ stores, followed by a sustained plateau phase resulting from Ca2+ influx (36). When frog melanotrope cells were incubated under Ca2+-free conditions, the [Ca2+]i increase evoked by muscarine was totally abolished. Similarly, the stimulatory effect of muscarine on {alpha}MSH release was abrogated when frog NILs were perifused with Ca2+-free or Ni2+-supplemented medium. These results indicate that the response of melanotrope cells to muscarine requires calcium influx. However, neither the L-type Ca2+ channel blocker nifedipine nor the N-type Ca2+ channel blocker {omega}-CgTx could prevent the elevation of [Ca2+]i provoked by muscarine. Likewise, the stimulatory effect of muscarine on {alpha}MSH release was not significantly affected by addition of nifedipine or {omega}-CgTx in the perifusion medium. Administration of a high concentration of nifedipine (100 µM), which was supposed to block low voltage activated Ca2+ channels (T channels) (41), did not inhibit the stimulatory effect of muscarine on {alpha}MSH release either. These data suggest that the Ca2+ influx involved in the secretory response to muscarine may be accounted for by activation of P- or Q-type channels. Consistent with this hypothesis, the occurrence of P- and Q-type Ca2+ channels has been recently described in rat melanotrope cells (42). Alternatively, muscarinic receptors might activate receptor-operated Ca2+ channels (43). Electrophysiological studies are clearly required to investigate the type of Ca2+ channels implicated in the muscarine-induced Ca2+ entry.

The present data have shown that muscarine induces a significant increase in inositol phosphate formation in frog NILs. In addition, neomycin, a drug known to block PI turnover by directly binding to PIP2 and PIP (44), inhibited the muscarine-evoked stimulation of {alpha}MSH release. In contrast, pretreatment of NILs with PTX did not affect the secretory response of the tissue to muscarine. These data indicate that the stimulatory effect of muscarine on frog melanotrope cells can be ascribed to activation of a phospholipase C (PLC) through a PTX-insensitive G protein. In agreement with these findings, it has been shown that M2 and M4 muscarinic receptors are coupled to PTX-sensitive G proteins, whereas M1, M3, and M5 muscarinic receptors are generally coupled to PTX-insensitive G proteins (36). Although the implication of a PLA2 in the mechanism of action of ACh has been described in various models (45), including frog adrenocortical cells (46), our data have shown that PLA2 is not involved in the muscarine-evoked stimulation of {alpha}MSH release in the frog pars intermedia. Finally, the fact that suppression of extracellular calcium totally abolished the stimulatory effect of muscarine on inositol lipid turnover indicated that the calcium influx provoked by muscarine was necessary for activation of PLC. In agreement with this idea, it has been reported that PLC activity is regulated by physiologically relevant Ca2+ concentrations (47, 48, 49).

Administration of a short pulse of PMA to perifused frog NILs induced a significant stimulation of {alpha}MSH release (data not shown), indicating the importance of PKC in the regulation of {alpha}MSH secretion. In contrast, prolonged exposure of NILs to PMA, which is known to induce down-regulation of PKC (50), markedly reduced the effect of muscarine on {alpha}MSH release. These data confirmed that the stimulatory action of ACh on frog melanotrope cells is mediated through the PLC/PKC transduction pathway.

It has been previously shown that M2 and M4 muscarinic receptors are negatively coupled to adenylate cyclase, whereas activation of odd-numbered receptors is associated with either a decrease (51) or an increase in the cAMP level. The present study has shown that muscarine causes only a slight increase in cAMP formation in frog NILs. This effect of muscarine could be secondary to the elevation of [Ca2+]i and/or to PKC activation, as described in other models (52, 53, 54). It has also been found that stimulation of muscarinic receptors (43, 55, 56), particularly the M3 receptor subtype (57, 58, 59), can activate a tyrosine kinase. However, the stimulatory effect of muscarine on {alpha}MSH release was not affected by three potent tyrosine kinase inhibitors, which have recently been shown to potentiate the inhibitory effect of {gamma}-aminobutyric acid on frog melanotrope cells (60). These data suggest that tyrosine phosphorylation is not involved in the transduction events promoted by activation of muscarinic receptors in the frog pars intermedia.

Figure 14Go summarizes the proposed mechanism of action of ACh on frog melanotrope cells as revealed by the present study. Activation of an M3-like muscarinic receptor induces Ca2+ influx via Ni2+-sensitive calcium channels. ACh also activates a phospholipase C whose activity strongly depends on Ca2+ influx and causes a modest stimulation of adenylyl cyclase. In addition to Ca2+ mobilization, activation of PKC is implicated in the secretory response of frog melanotrope cells to ACh. In contrast, PLA2 and tyrosine kinases do not appear to play any significant role in the cholinergic stimulation of {alpha}MSH release.



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  		Figure 14. Schematic representation summarizing the intracellular events associated with cholinergic stimulation of frog melanotrope cells. Binding of ACh to a muscarinic receptor pharmacologically related to the mammalian M3 receptor induces Ca2+ entry via Ni2+-sensitive calcium channels. ACh also causes activation of PLC through a PTX-insensitive G protein, generating diacylglycerol (DAG) and IP3. Concurrently, ACh provokes a modest stimulation of adenylyl cyclase (AC). The increase in [Ca2+]i resulting from Ca2+ influx is required for the ACh-induced stimulation of PLC and might be responsible for the activation of adenylyl cyclase (dotted line). The increase in [Ca2+]i and the activation of PKC are both involved in the ACh-induced stimulation of {alpha}MSH release.

 


   Acknowledgments
 
The authors thank Miss C. Buquet for technical support during cell culture.


   Footnotes
 
1 This work was supported by grants from INSERM U-413, the European Union (Human Capital and Mobility Program, Grant ERBCHRXCT920017), and the Conseil Régional de Haute-Normandie. Back

2 Recipient of a fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur, et de la Recherche. Back

Received February 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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