CARBACHOL STIMULATES ADENYLATE-CYCLASE AND PHOSPHOLIPASE-C AND MUSCLE CONTRACTION-RELAXATION IN A RECIPROCAL MANNER IN DOG IRIS SPHINCTER SMOOTH-MUSCLE

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100-mu-M). In the dog sphincter, lower concentrations (< 5-mu-M) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (> 5-mu-M) enhanced cAMP formation, inhibited IP3 production and induced relaxation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25-mu-M) increased both basal and isoproterenol- and forskolin-stimulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC50 of 9 nM. Intracellular Ca2+, derived from IP3-induced Ca2+ release and/or from muscarinic receptor-operated Ca2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (i) At short time intervals (< 1 min) carbachol (25-mu-M) increased IP3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated IP3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbol ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca2+-calmodulin stimulated adenylate cyclase was demonstrated in membranes from dog iris sphincter but not in that from rabbit and bovine. (4) Trifluoperazine (0.1-mu-M), a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (5) The Ca2+ ionophore A23187 and the phorbol ester increased cAMP production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carbachol or by the phorbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol ester or staurosporine. (7) Nifedipine (0.01-0.5-mu-M), a Ca2+ channel antagonist, inhibited carbachol stimulation of cAMP production, suggesting the presence of a muscarinic receptor-operated Ca2+ influx pathway in this tissue. The above data are interesting in three ways: (a) They demonstrate an important species difference in the second messenger systems coupled to the activation of muscarinic receptors, (b) they demonstrate a reciprocal relationship (cross-talk) between cAMP and IP3 production, and (c) they suggest that both intracellular and extracellular Ca2+ mobilization may be involved in muscarinic stimulation of cAMP production. We suggest that in dog iris sphincter a rise in intracellular cAMP levels by cholinergic stimulation is needed to regulate muscarinic receptor-linked Ca2+ mobilization and muscle contraction-relaxation. Cyclic AMP production through muscarinic activation could also be required for the functioning of Ca2+ channels in this excitable tissue.