Muscarinic m1 receptor-stimulated adenylate cyclase activity in Chinese hamster ovary cells is mediated by Gs alpha and is not a consequence of phosphoinositidase C activation.
The mechanism underlying muscarinic m1 receptor-mediated increases in adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in Chinese hamster ovary (CHO) cells expressing human recombinant m1 muscarinic receptors (CHO-ml cells). Stimulation of CHO-ml cells with carbachol resulted in marked accumulation of Ins(1,4,5)P3 and cAMP, in an atropine-sensitive manner, with EC50 values (log M) of -5.16 +/- 0.06 and -3.93 +/- 0.07 respectively. Basal and agonist-stimulated cAMP accumulation were unaffected by a 5 min pretreatment with l microM phorbol 12,13-dibutyrate and were not attenuated by pertussis toxin (100 ng/ml, 20h). Agonist-stimulated cAMP accumulation was also observed in CHO-ml cell membranes incubated in a buffer containing 100 nM free Ca2+. Guanosine 5'- [gamma-thio]triphosphate (10 microM) potentiated agonist-stimulated cAMP accumulation in CHO-ml cell membranes, implicating a G-protein involvement in this response. Co-incubation of carbachol with forskolin (10 microM) produced a greater than additive accumulation of cAMP in CHO-ml cells. Furthermore, a C-terminal-directed anti-Gs alpha serum attenuated both carbachol-stimulated (in CHO-ml cell membranes) and isoprenaline-stimulated (in CHO-beta 2 cell membranes) cAMP accumulation with a similar dose-dependency. These results suggest that muscarinic agonist-stimulated cAMP accumulation in CHO-ml cells occurs via activation of Gs alpha and not as a consequence of phosphoinositidase C activation.
Burford
N T
NT
Department of Cell Physiology and Pharmacology, University of Leicester, U.K.
Nahorski
S R
SR
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol