Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on intracellular cAMP and Ca 2+ levels in common carp (Cyprinus carpio) pituitary cells in vitro

Abstract

In order to study signal transduction in PACAP-stimulated GH release, effects of ovine PACAPs (oPACAPs) on cAMP synthesis and intracellular free Ca 2+ ([Ca 2+ ] i) levels in primarily cultured common carp (Cyprinus carpio) pituitary cells were investigated using radioimmunoassay (RIA) for cAMP and [Ca 2+ ] i Indo1/AM loading techniques. The main results obtained could be briefly summarized as follows: parallel with their GH-releasing effects, oPACAP-38 (0.01 nmol/L to 1 μmol/L) and oPACAP-27 (0.01 nmol/L to 1 μmol/L) were also effective in increasing cAMP release, cellular cAMP content, and total cAMP production in a dose-dependent manner in common carp pituitary cells. In addition to their cAMP synthesis, 0.5 μmol/L oPACAP-38 and 0.5 μmol/L oPACAP-27 were also effective in elevating [Ca 2+ ] i levels in common carp pituitary cells preloaded with the Ca 2+ -sensitive dye Indo-1; the stimulatory actions were dependent on the presence of extracellular Ca 2+ ([Ca 2+ ] e), as removal of [Ca 2+ ] e by adding the Ca 2+ chelator 4 mmol/L EGTA blocked the rise of [Ca 2+ ] i induced by oPACAP-38 and oPACAP-27, respectively. Besides, the rise in [Ca 2+ ] i induced by 0.5 μmol/L oPACAP-38 was blocked by treatment with 10 μmol/L nifedipine, an inhibitor of "L-type" voltage-sensitive Ca 2+ channels (VSCC); but 0.5 μmol/L oPACAP-38 was not effective in elevating [Ca 2+ ] i levels after the cells treated with Ca 2+ -sensitive dye Indo-1 were challenged with 10 μmol/L nifedipine. These results, taken as a whole, strongly suggest the involvement of the cAMP- and Ca 2+ -dependent pathways in PACAP-stimulated GH release in common carp.