SIGNAL-TRANSDUCTION PATHWAYS OF MUSCARINIC RECEPTORS IN CIRCULAR SMOOTH-MUSCLE FROM THE RABBIT CECUM

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. In Myo-[H-3]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [H-3]inositol phosphates ([H-3]IPs) accumulation (EC(50) of 3 +/- 1 mu M). The M(1)-selective antagonist pirenzepine (PRZ), the M(2)-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M(3)-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [H-3]inositol phosphates accumulation with the following order of potency: p-F-HHSiD > PRZ > AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP gamma[S] elicited a concentration-dependent increase in [H-3]inositol phosphates accumulation. The concentration-response curve for GTP gamma[S] was shifted to the left when cells were incubated with 1 mu M carbachol. The [H-3]inositol phosphates accumulation elicited by simultaneous addition of 0.1 mu M GTP gamma[S] and 1 mu M carbachol to permeabilized cells was significantly decreased (78.28 +/- 18.23 % inhibition) when cells were preincubated for 5 min with 0.1 mM GDP beta[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [H-3]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 mu M final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 mu M isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [H-3]inositol phosphates accumulation is mediated by M(3) subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M(2) subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein G(i).