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-[3H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [3H]inositol phosphates ([3H]IPs) accumulation (EC50 of 3 +/- 1 microM). The M1-selective antagonist pirenzepine (PRZ), the M2-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hypyrido[2,3-b][1,4]benzodiazepin-6-one) and the M3-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [3H]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 [3H]inositol phosphates accumulation. The concentration-response curve for GTP gamma [S] was shifted to the left when cells were incubated with 1 microM carbachol. The [3H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 microM GTP gamma [S] and 1 microM 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 [3H]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 microM final concentration).(ABSTRACT TRUNCATED AT 250 WORDS)