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 agent that stimulates polyphosphoinositide hydrolysis, was studied in thyroid cells. Incubation of cultured dog thyroid cells with 10 microM carbachol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stimulation of adenylyl cyclase activity of membrane preparations by approximately 40%. This inhibition was reversed by atropine, occurred even in a Ca(2+)-free medium containing ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ ionophore 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. Pretreatment of cells with pertussis toxin to inactivate the Gi inhibitory protein also failed to affect the carbachol inhibition. Although carbachol 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-stimulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on 125I-TSH binding to membranes but reduced the cyclase stimulation by not only TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated AD[32P]-ribosylation of Gs in membranes. These data indicate that carbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that Gs is a possible site of its action. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, can reduce the adenylyl cyclase activity by a process that does not involve Ca2+ or activation of phosphodiesterase.
Pasquali
D
D
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Rani
C S
CS
Deery
W J
WJ
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003864:Depression, Chemical; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D007457:Iodine Radioisotopes; D008566:Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D037342:Pertussis Toxin; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011989:Receptors, Thyrotropin / Q000187:drug effects / Q000378:metabolism; D013268:Stimulation, Chemical; D013961:Thyroid Gland / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D013972:Thyrotropin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D007457:Iodine Radioisotopes; D010726:Phosphodiesterase Inhibitors; D011989:Receptors, Thyrotropin; D010566:Virulence Factors, Bordetella; D002217:Carbachol; D013972:Thyrotropin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D000262:Adenylyl Cyclases; D002118:Calcium