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-m1 cells). Stimulation of CHO-m1 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 1 μM phorbol 12,13-dibutyrate and were not attenuated by pertussis toxin (100 ng/ml, 20 h). Agonist-stimulated cAMP accumulation was also observed in CHO-m1 cell membranes incubated in a buffer containing 100 nM free Ca2+. Guanosine 5´-[γ-thio]triphosphate (10 μM) potentiated agonist-stimulated cAMP accumulation in CHO-m1 cell membranes, implicating a G-protein involvement in this response. Co-incubation of carbachol with forskolin (10 μM) produced a greater than additive accumulation of cAMP in CHO-m1 cells. Furthermore, a C-terminal-directed anti-Gsα serum attenuated both carbachol-stimulated (in CHO-m1 cell membranes) and isoprenaline-stimulated (in CHO-β2 cell membranes) cAMP accumulation with a similar dose-dependency. These results suggest that muscarinic agonist-stimulated cAMP accumulation in CHO-m1 cells occurs via activation of Gsα and not as a consequence of phosphoinositidase C activation.
- The Biochemical Society, London © 1996