title: Gonadotropin-releasing hormone-induced calcium signaling in clonal pituitary gonadotrophs.
abstract: In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.
authors.affiliation: Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: F
authors.initials: F
authors.lastname: Merelli
authors.forename: S S
authors.initials: SS
authors.lastname: Stojilković
authors.forename: T
authors.initials: T
authors.lastname: Iida
authors.forename: L Z
authors.initials: LZ
authors.lastname: Krsmanovic
authors.forename: L
authors.initials: L
authors.lastname: Zheng
authors.forename: P L
authors.initials: PL
authors.lastname: Mellon
authors.forename: K J
authors.initials: KJ
authors.lastname: Catt
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D004095:Dihydropyridines / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D013729:Terpenes; D007987:Gonadotropin-Releasing Hormone; D015759:Ionomycin; D019284:Thapsigargin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000252:Calcium-Transporting ATPases; D009543:Nifedipine; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium