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

The effects of muscarinic acetylcholine receptor stimulation on phosph oinositides 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) accumulatio n (EC(50) of 3 +/- 1 mu M). The M(1)-selective antagonist pirenzepine (PRZ), the M(2)-selective AF-DX 116 2-[(diethyl-amino)methyl]-1-piperi dinyl]acetyl]-5, 1-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) an d the M(3)-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) in hibited the carbachol-induced [H-3]inositol phosphates accumulation wi th the following order of potency: p-F-HHSiD > PRZ > AF-DX 116. In sap onin-permeabilized circular smooth muscle cells, carbachol and GTP gam ma[S] elicited a concentration-dependent increase in [H-3]inositol pho sphates accumulation. The concentration-response curve for GTP gamma[S ] was shifted to the left when cells were incubated with 1 mu M carbac hol. The [H-3]inositol phosphates accumulation elicited by simultaneou s addition of 0.1 mu M GTP gamma[S] and 1 mu M carbachol to permeabili zed cells was significantly decreased (78.28 +/- 18.23 % inhibition) w hen cells were preincubated for 5 min with 0.1 mM GDP beta[S]. In nonp ermeabilized cells, pertussis toxin did not alter the carbachol-induce d increase in [H-3]inositol phosphates accumulation. On the other hand , the 0.1 mM carbachol-induced inhibition of forskolin-stimulated aden ylate cyclase activity in circular smooth muscle homogenates was signi ficantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 mu M final co ncentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 mu M isoproterenol was abolished by p ertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabb it caecum, the muscarinic receptor stimulation of [H-3]inositol phosph ates accumulation is mediated by M(3) subtype receptors coupled to a p ertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M(2) subtype receptors coupled to a pe rtussis toxin-sensitive GTP-binding protein G(i).