Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum.

Abstract

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)