CARBACHOL-INDUCED DECREASE IN THYROID-CELL ADENYLYL CYCLASE ACTIVITY IS INDEPENDENT OF CALCIUM AND PHOSPHODIESTERASE ACTIVATION

The mechanism of adenylyl cyclase desensitization by carbachol, an age nt that stimulates polyphosphoinositide hydrolysis, was studied in thy roid cells. Incubation of cultured dog thyroid cells with 10-mu-M carb achol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stim ulation of adenylyl cyclase activity of membrane preparations by appro ximately 40%. This inhibition was reversed by atropine, occurred even in a Ca2+-free medium containing ethylene glycol bis(beta-aminoethylet her)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ io nophore A23187. The carbachol effect was not prevented by simultaneous incubation of cells with either isobutylmethylxanthine, an inhibitor of phosphodiesterase, or H-7, an inhibitor of protein kinase. Pre-trea tment of cells with pertussis toxin to inactivate the G(i) inhibitory protein also failed to affect the carbachol inhibition. Although carba chol did not reduce the basal or the TSH-stimulated cyclase activities when added to membranes directly during the assay, exposure of cells to carbachol for 2-4 hr resulted in long lasting inhibition of TSH-sti mulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on I-125-T SH binding to membranes but reduced the cyclase stimulation by not onl y TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated A D[P-32]-ribosylation of G(s) in membranes. These data indicate that ca rbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that G(s) is a possible site of its actio n. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, ca n reduce the adenylyl cyclase activity by a process that does not invo lve Ca2+ or activation of phosphodiesterase.