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

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

The secretion of 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 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 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 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 MSH release. In contrast, -conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca²⁺]_i and 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 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 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 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.