Abstract
The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.
Highlights
The m l muscarinic acetylcholine receptor gene was systems are activated upon receptor occupation. a-l-adrenertransfected into and stably expressed in A 9 L cells. gic stimulation of both phospholipase AZ and phospholipase
Carbachol stimulated arachidonic acid and inositol phosphartelease with similar potencies, while cAMP generation required a higherconcentration.Studieswereperformed todetermine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m l muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messenpholipase Az and phospholipase C was shown in Swiss 3T3 cells and MDCK cells (3, 4).Dopamine-1-stimulated adenylate cyclase and phospholipase C activity has been described in renal membrane preparations ( 5 )
Gene Generates Multiple Second Messengers-In A9 L fibroblasts transfected with and stably expressing the m l muscarinic receptor, carbachol stimulated inositol phosphate release (EC50IP = 6 g ~IP,2= g ~IP, = PM)and arachidonic acid release (EC50 = 7 p ~ w)ithsimilar potencies when assayed under identical assay conditions (Fig. 1)
Summary
Vol 264, No 34, Issue of December 5, pp. 20356-20362,1989 Printed in U.S.A. A Transfected m l Muscarinic Acetylcholine Receptor Stimulates Adenylate Cyclasevia Phosphatidylinositol Hydrolysis*. PMA in- coupling of a single receptor subtypeto second messenger hibited inositol phosphate release in response to car- systems without the presence of multiple receptor subtypes bachol, suggesting that activationof phospholipase C from the same receptor family (8-10). From these model might be involveidn cAMP accumulation. TMB-8, an inhibitor of intracellular calcium release, and W7,a calmodulin antagonist These observations suggest that carbachol-stimulatedcAMP accumulation does not occur through direct m l muscarinic receptor coupling or through the releaosfearachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. A number of more complex systems have been described in which multiple transduction
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