PHARMACOLOGICAL COUPLING AND FUNCTIONAL-ROLE FOR THE MUSCARINIC RECEPTOR SUBTYPES IN ISOLATED CELLS FROM THE CIRCULAR SMOOTH-MUSCLE OF THE RABBIT CECUM

The regulation of isolated smooth muscle cells from the circular layer of the rabbit cecum by muscarinic receptors was studied in this paper . Binding of N-[H-3]methylscopolamine was found to be specific, satura ble (maximal binding capacity of about 325,000 sites/cell) and of high affinity (dissociation constant [K-D) of 0.52 +/- 0.12 nM] The muscar inic M(1)-selective antagonist pirenzepine (PRZ), the muscarinic M(2)- selective AF-DX 116 -dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) and the muscarinic M(3)-selective para-fluoro-hexahydro-sila-difenido l (p-F-HHSiD) inhibited N-[H-3]methylscopolamine binding with respecti ve inhibition constants (K-i) of (in nanomolar): 1018 +/- 382, 254 +/- 76 and 916 +/- 305. [H-3]inositol phosphates accumulation was increas ed by carbachol (CCh) (EC(50) of 3 +/- 1 mu M). Antagonists competitiv ely inhibited the CCh-induced [H-3]inositol phosphates accumulation wi th the following order of potency: atropine > p-F-HHSiD > PRZ > AF-DX 116. in addition, CCh increased inositol-1,4,5-trisphosphate level in a time- and concentration-dependent fashion (EC(50) of 1.5 +/- 0.5 mu M). CCh inhibited both isoproterenol- and forskolin-induced cyclic AMP accumulation in isolated smooth muscle cells. Moreover, CCh inhibited forskolin-stimulated adenylate cyclase activity in smooth muscle homo genates (EC(50) of 10.0 +/- 22.1 mu M); the CCh-induced inhibition of forskolin-stimulated adenylate cyclase activity was reversed significa ntly by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffec tive. In contraction experiments, CCh reduced the mean cell length (EC ,, of 3.94 +/- 1.23 pM). The order of potency of antagonists for the i nhibition of the CCh-induced contraction was atropine > p-F-HHSiD > PR Z > AF-DX 116. In conclusion, our data suggest that smooth muscle cell s isolated from the circular layer of the rabbit cecum contain M(2) an d M(3) muscarinic receptor subtypes coupled to different transduction pathways (adenylate cyclase inhibition and phospholipase C activation, respectively). Moreover, the direct myogenic effect of CCh on cell co ntraction is mediated through interaction of the agonist with muscarin ic M(3)-subtype receptors.