CHARACTERIZATION OF THE GONADOTROPIN-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.