ACTIVATION OF NO-CGMP PATHWAY BY ACETYLCHOLINE IN BOVINE CHROMAFFIN CELLS - POSSIBLE ROLE OF CA2+ IN THE DOWN-REGULATION OF CGMP SIGNALING

The production of cyclic GMP (cGMP) induced by acetylcholine and other stimuli was studied in bovine chromaffin cells. Acetylcholine increased intracellular cGMP in a transitory (peak at 2 min) and concentration-dependent manner (estimated half maximal increase, EC(50) = 61 +/- 5 mu M). N-G-nitro-L-arginine methyl ester (NAME) inhibited such a rise in cGMP with a half maximal inhibitory concentration (IC50) of 231 +/- 55 mu M. The acetylcholine-induced increase in cGMP was also inhibited by a calmodulin antagonist (calmidazolium, 30 mu M) and by the absence of extracellular calcium. Other agents that strongly increased cytosolic calcium concentration ([Ca2+](i)) as acetylcholine did, such as the nicotinic-agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), high-KCl (50 mM), and ionomycin, also caused a rise in cGMP in cultured bovine chromaffin cells. Veratridine, an activator of sodium channels, produced a slowly developing calcium increase and no significant cGMP production. The muscarinic-agonist, muscarine, failed to increase cytosolic calcium, and was the weakest stimulator of cGMP production. cGMP formation, induced by sodium nitroprusside (SNP, 100 mu M) and by C-type natriuretic peptide (CNP, 100 nM), was inhibited by 30-40% by increasing [Ca2+](i) with ionomycin. This inhibition was abolished by calmidazolium (30 mu M) and by the absence of calcium in the extracellular medium. In conclusion, bovine chromaffin cells synthesize nitric oxide (NO) to activate guanylate cyclase in response to several stimuli, which increase [Ca2+](i). Moreover, the increase in [Ca2+](i) also stimulates a Ca2+/calmodulin phosphodiesterase, which could down-regulate the levels of cGMP in these cells.