Dynamic video imaging of cystolic Ca(2+) in the alphaT3-1, gonadotrope-derived cell line

Studies with pituitary cell cultures have revealed Ca(2+) as a mediator of gonadotropin-releasing hormone (GnRH) action. Here we have used dynamic video imaging to characterize Ca(2+) metabolism in the gonadotrope-derived, alphaT3-1 cell line focusing on stimuli that influence Ca(2+) metabolism of gonadotropes in primary culture. At least 90% of the cells imaged responded to 100 nMGnRH with an increase in cytosolic Ca(2+) (typically 10- to 20-fold). GnRH (10-1000 nM) caused a biphasic "spikeplateau" increase, whereas the spike phase was not seen with 0.01-1 nM GnRH. The plateau response was blocked by Ca(2+)-free medium and inhibited by 10 muM nifedipine (a dihydropyridine Ca(2+) channel antagonist) but not by blockade of voltage-sensitive Na(+) channels with 10 muM tetrodotoxin. In Ca(2+)-free medium GnRH caused only a transient Ca(2+) increase. Endothelin-1 (400 nM) also increased Ca(2+) although only 30-50% of the cells responded and the average response was less than that with 100 nM GnRH. Depolarization with 30 mM KCl increased Ca(2+) in >90% of the cells with an average effect comparable to the 100 nM GnRH effect. Protein kinase C activation approximately doubled the Ca(2+) concentration and induced Ca(2+) oscillations in some cells. The close parallels between responses of these cells and those observed with primary cultures suggests that alphaT3-1 cells will provide a valuable model for further studies of the involvement of Ca(2+) in GnRH action.