kn-62 and cobaltous-chloride

The possible involvement of extracellular Ca2+ ([Ca2+]o) in mediating the acute gonadotropin (GtH) response to salmon gonadotropin-releasing hormone (sGnRH) and chicken gonadotropin-releasing hormone-II (cGnRH-II) in goldfish was examined using dispersed pituitary cells in perifusion. Perifusion with Ca(2+)-deficient medium reduced the GtH responses to 5-min pulses of either GnRH, indicating the participation of [Ca2+]o in acute GnRH action. Using a 10-min GnRH pulse application protocol, the dependence of the acute GtH responses to the two GnRHs on [Ca2+]o entry through voltage-sensitive Ca2+ channels (VSCC) was examined using the dihydropyridine VSCC blocker nifedipine and the cation Co2+. Treatment with nifedipine consistently reduced the acute GtH response to either sGnRH or cGnRH-II. Similarly, perifusion with CoCl2 reduced the sGnRH-induced GtH release. In contrast to its effects on sGnRH, CoCl2 abolished the cGnRH-II-induced GtH release. These results indicate that [Ca2+]o entry through VSCC participates in the acute GtH response to both native GnRHs; however, the cGnRH-II-stimulated acute release is relatively more dependent on [Ca2+]o and VSCC functions than sGnRH-induced secretion. The involvement of calmodulin (CaM) in mediating GnRH action was also examined. Treatment with a CaM antagonist, calmidazolium, or with a Ca2+/CaM-dependent protein kinase II inhibitor, KN62, reduced GtH responses to sGnRH and cGnRH-II in 2-hr static incubation, but not in perifusion studies with dispersed goldfish pituitary cells using 5- or 10-min GnRH pulses. These results suggest that CaM-dependent mechanisms participate in mediating the long-term, but not the acute, GtH response to GnRH. Compared to sGnRH, cGnRH-II-induced GtH release was more sensitive to inhibition by KN62, indicating a higher degree of dependence of cGnRH-II action on CaM. These results extend our understanding of the differential involvement of [Ca2+]o and CaM in mediating the short-term and long-term actions of the two native GnRH peptides on GtH release in goldfish.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Cobalt; Culture Media; Enzyme Inhibitors; Female; Goldfish; Gonadotropin-Releasing Hormone; Gonadotropins; Imidazoles; Isoquinolines; Male; Nifedipine; Piperazines; Pituitary Gland