Characterization of the gonadotrophin-releasing hormone calcium response in single alpha T3-1 pituitary gonadotroph cells.
Intracellular calcium ([Ca2+]i) was measured in single immortalized gonadotroph alpha T3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. Gonadotrophin-releasing hormone (GnRH, 10(-8) M) produced a biphasic rise in [Ca2+]i which could be abolished by a GnRH antagonist. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. The calcium spike was reduced by nifedipine (10(-6) M), a calcium channel blocker, and thapsigargin (10(-6) M) which inhibits inositol 1,4,5-trisphosphate (IP3) mediated release of [Ca2+]i but abolished by the intracellular calcium antagonist TMB-8 (10(-6) M). The secondary phase was reduced following pretreatment with either nifedipine or the protein kinase C (PKC) antagonist, H-7 (10(-6) M). The PKC agonist PMA (phorbol 12-myristate 13-acetate, 10(-6) M) produced a small rise in basal [Ca2+]i and abolished the GnRH calcium response. The initial calcium response to GnRH therefore involves both an IP3-mediated rise in cytosolic calcium due to the release from intracellular stores and an influx of extracellular calcium through second messenger-operated calcium channels. In contrast the secondary calcium response mainly involves the influx of extracellular calcium through PKC-activated calcium channels.
Anderson
L
L
MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK.
Hoyland
J
J
Mason
W T
WT
Eidne
K A
KA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000502:physiology*; D007546:Isoquinolines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002121:Calcium Channel Blockers; D007546:Isoquinolines; D010879:Piperazines; D007987:Gonadotropin-Releasing Hormone; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium