WORTMANNIN-SENSITIVE AND WORTMANNIN-INSENSITIVE STEPS IN CALCIUM-CONTROLLED EXOCYTOSIS IN PITUITARY GONADOTROPHS - EVIDENCE THAT MYOSIN LIGHT-CHAIN KINASE MEDIATES CALCIUM-DEPENDENT AND WORTMANNIN-SENSITIVE GONADOTROPIN-SECRETION

In cultured rat pituitary cells, increases in the cytosolic calcium co ncentration ([Ca2+](i)) and LW release are induced by activation of Gn RH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, w ortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LW responses to nonreceptor-mediated elevation of [Ca2+](i) by io nomycin and activation of voltage-sensitive Ca2+ channels by Bay K 864 4 or high K+, as well as Ca2+-induced LH release in permeabilized pitu itary cells. The [Ca2+](i) responses to these stimuli were unaltered i n wortmannin-treated pituitary cells, indicating that this compound in hibits a Ca2+-dependent step in exocytosis without affecting Ca2+ sign aling. In perifused pituitary cells, the GnRH-induced early spike phas e of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol productio n were observed in wortmannin-treated pituitary cells during the susta ined phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13- acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein ki nase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an ac tion supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alpha T3-1 gonadotrophs tre ated with GnRH, K+, and Bay K 8644. This protein was coprecipitated fr om pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PA GE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensit ive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB ligh t chain by MLCK.