PROTEIN-KINASE REGULATION OF MUSCARINIC RECEPTOR SIGNALING IN COLONIC SMOOTH-MUSCLE

1 We have previously demonstrated that M2 and M3 muscarinic receptors coexist in the circular smooth muscle of canine promixal colon. Activation of receptors of the M2 subtype leads to inhibition of adenylyl cyclase activity through the GTP-binding protein, G(i), while M3 receptors are coupled to a pertussis toxin-insensitive GTP-binding protein and mediate phosphoinositide hydrolysis. 2 In the present study, the interactions between these second messenger systems were examined. Activation of either protein kinase C or adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase attenuated carbachol-stimulated phosphoinositide hydrolysis without affecting basal activity. Activation of both protein kinases produced greater attenuation of inositol 1,4,5-trisphosphate formation than activation of either kinase alone. 3 In contrast to its inhibitory effect on phosphoinositide hydrolysis, activation of protein kinase C had no effect on adenylyl cyclase activity. 4 Activation of protein kinase C by phorbol ester treatment resulted in the sequestration Of M3 muscarinic receptors from the cell surface without effecting the M, muscarinic receptor population. Sequestered M3 muscarinic receptors were not rapidly degraded. 5 In contrast, elevation of cellular cyclic AMP decreased the affinity of cell surface muscarinic receptors for an antagonist radioligland without affecting their density. 6 Muscarinic agonist binding was not affected by either activation of protein kinase C or elevation of cellular cyclic AMP. 7 These data support the notion of negative feedback by protein kinase C and cyclic AMP-dependent protein kinase on phosphoinositide hydrolysis. In canine colonic circular smooth muscle this negative feedback regulation of inositol phosphate generation by muscarinic receptor stimulation does not appear to involve the guanine nucleotide binding protein:receptor interaction.