Functional uncoupling between intracellular calcium dynamics and secretion in the alphaT3-1 gonadotropic cell line.
Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.
Trueta
C
C
Neurobiology Center, National University of Mexico, Querétaro.
Díaz
M
M
Vaca
L A
LA
Clapp
C
C
Martinez de la Escalera
G
G
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005682:Gadolinium / Q000494:pharmacology; D015292:Glycoprotein Hormones, alpha Subunit / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007211:Indoles / Q000493:pharmacokinetics; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D010902:Pituitary Gland / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D011966:Receptors, LHRH / Q000502:physiology*; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000378:metabolism; D013462:Sulfur Radioisotopes; D019284:Thapsigargin / Q000494:pharmacology; D014316:Tritium
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D015292:Glycoprotein Hormones, alpha Subunit; D006023:Glycoproteins; D007211:Indoles; D007476:Ionophores; D011966:Receptors, LHRH; D019837:Ryanodine Receptor Calcium Release Channel; D013462:Sulfur Radioisotopes; D014316:Tritium; D012433:Ryanodine; D007987:Gonadotropin-Releasing Hormone; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D005682:Gadolinium; D007770:L-Lactate Dehydrogenase; D009543:Nifedipine; C048960:indo-1; D002118:Calcium