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Wortmannin-Sensitive and -Insensitive Steps in Calcium-Controlled Exocytosis in Pituitary Gonadotrophs: Evidence That Myosin Light Chain Kinase Mediates Calcium-Dependent and Wortmannin-Sensitive Gonadotropin Secretion

Kang Rao, Won-Young Paik, Lixin Zheng, Richard M. Jobin, Melanija Tomi, He Jiang, Satoshi Nakanishi and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. (S.N.), Tokyo, Japan

Address all correspondence and requests for reprints to: Dr. Stanko S. Stojilkovic, National Institute of Child Health and Human Development, Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, 49 Covent Drive, MSC 4510, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca²⁺]_i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca²⁺]_i by ionomycin and activation of voltage-sensitive Ca²⁺ channels by Bay K 8644 or high K⁺, as well as Ca²⁺-induced LH release in permeabilized pituitary cells. The [Ca²⁺]_i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca²⁺-dependent step in exocytosis without affecting Ca²⁺ signaling. In perifused pituitary cells, the GnRH-induced early spike phase 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 production were observed in wortmannin-treated pituitary cells during the sustained 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 kinase 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 action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and T3–1 gonadotrophs treated with GnRH, K⁺, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca²⁺ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

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