EVIDENCE THAT M3-MUSCARINIC-RECEPTORS IN RAT PAROTID-GLAND COUPLE TO 2 2ND MESSENGER SYSTEMS

The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [H-3]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) almost-equal-to 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) >> pirenzepine (136) > 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (K(i)) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed K(i) values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and G(i)-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.