title: 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.
abstract: 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 alphaT3-1 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.
authors.affiliation: Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: K
authors.initials: K
authors.lastname: Rao
authors.forename: W Y
authors.initials: WY
authors.lastname: Paik
authors.forename: L
authors.initials: L
authors.lastname: Zheng
authors.forename: R M
authors.initials: RM
authors.lastname: Jobin
authors.forename: M
authors.initials: M
authors.lastname: Tomić
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.forename: S
authors.initials: S
authors.lastname: Nakanishi
authors.forename: S S
authors.initials: SS
authors.lastname: Stojilkovic
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects; D006065:Gonadotropins, Pituitary / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011966:Receptors, LHRH / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002120:Calcium Channel Agonists; D004791:Enzyme Inhibitors; D006065:Gonadotropins, Pituitary; D007295:Inositol Phosphates; D010703:Phorbol Esters; D011966:Receptors, LHRH; D015759:Ionomycin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D007986:Luteinizing Hormone; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; D000077191:Wortmannin