Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder

Previous studies have suggested the presence of multiple muscarinic recepto r subtypes in guinea pig gallbladder smooth muscle, although the relative a bundance and functional role of these subtypes remains an area of significa nt research efforts. The present study utilized both radioligand kinetic an d functional experiments to further probe the nature of the muscarinic rece ptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [H-3] N-me thylscopolamine ([H-3]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel expe riments conducted on the cloned muscarinic M-1-M-5 subtypes in Chinese hams ter ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [H-3]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladd er, but not the gallbladder, demonstrating biphasic radioligand dissociatio n kinetics. In functional experiments, carbachol caused a concentration-dep endent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-f ree or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplit udes of the maximal contractions corresponding to 45.8 +/- 8.0% and 33.2 +/ - 6.6% of control responses in normal PSS, respectively. Furthermore, the s timulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substitu ted PSS. The antagonist, methoctramine (1 x 10(-7)-3 x 10(-5) M), exerted o nly a slight inhibition of carbachol (10(-5) M)-induced contractions in 5 m M Sr2+-substituted medium, whereas it was significantly more potent in anta gonizing gallbladder contractions in response to 10(-5) M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipote nt in antagonizing carbachol-induced contractions in Ca2+-free (pIC(50): 6. 85 +/- 0.11 for atropine and 5.75 +/- 0.32 for tripitramine) and Sr2+-subst ituted media (pIC(50): 6.88 +/- 0.25 for atropine and 5.70 +/- 0.16 for tri pitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC(50): 5.66 +/- 0.23) than in Sr2+-substituted PSS (pIC(50): 5.33 +/- 0. 21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two sub types may represent the muscarinic M-3 and M-4 receptors, although the pres ence of the muscarinic M-2 receptor subtype is also suggested from the bind ing data. (C) 2000 Elsevier Science B.V. All rights reserved.