Thyrotropin-Releasing Hormone (TRH) Stimulates Biphasic Elevation of Cytoplasmic Free Calcium in GH3Cells. Further Evidence that TRH Mobilizes Cellular and Extracellular Ca2+*

Abstract

TRH stimulation appears to be coupled to PRL [prolactin] secretion, at least in part, by elevation of the concentration of Ca2+ free in the cytoplasm ([Ca2+]i). An intracellularly trapped fluorescent probe of Ca2+, Quin 2, was employed to measure [Ca2+]i in GH3 cells, cloned rat pituitary tumor cells. Basal [Ca2+]i in GH3 cells incubated in medium containing 1.5 mM Ca2+ was 148 .+-. 8.6 nM (mean .+-. SE). TRH caused a biphasic elevation of [Ca2+]i to 517 .+-. 29 nM at less than 10 s after TRH addition, followed by a decline towards the resting level over 1.5 min (1st phase) and then a sustained elevation to 261 .+-. 14 mM (2nd phase). Whether mobilization of cellular Ca or enhanced influx of extracellular Ca2+, or both, were involved in the elevation of [Ca2+]i during each of the 2 phases was determined. In all experiments, the elevation of [Ca2+]i stimulated by TRH was compared with that induced by depolarization of the plasma membrane with high extracellular K+, which enhances Ca2+ influx. In medium with 1.5 mM Ca2+, K+-depolarization caused an elevation of [Ca2+]i to 780 .+-. 12 nM. When the concentration of Ca2+ in the medium was lowered to 0.1 mM and 0.01 mM, basal [Ca2+]i was lowered to 114 .+-. 3.4 and 110 .+-. 11 nM, respectively. In medium with 0.1 and 0.01 mM Ca2+, peak K+ depolarization-induced elevation of [Ca2+]i was lowered to 30 .+-. 3.9% and 7.3 .+-. 2.0% of control, respectively. The peak 2nd phase increase caused by TRH was reduced to 33 .+-. 2.8% and 16 .+-. 5.6% of control, respectively; the peak 1st phase elevation of [Ca2+]i was lowered only to 79 .+-. 5.5% and 52 .+-. 10% of control in medium with 0.1 mM and 0.01 mM Ca2+, respectively. When cells were incubated in medium with 1.5 mM Ca2+ containing the Ca2+-channel blocking agents, nifedipine and verapamil, basal [Ca2+]i was not affected. Nifedipine plus verapamil, each at a maximally effective dose, lowered K+ depolarization induced elevation of [Ca2+]i to 6.5 .+-. 1.0% of control, the peak 2nd phase increase caused by TRH to 28 .+-. 4.3% of control, but the peak 1st phase elevation only to 64 .+-. 3.7% of control. The decrease in the 1st phase response to TRH caused by the channel blockers appeared to be secondary to partial depletion of an intracellular, nonmitochondrial calcium pool. Apparently, the 2nd phase elevation of [Ca2+]i caused by TRH is inhibited to a greater extent by lowering extracellular Ca2+ or by blocking Ca2+ influx than is the 1st phase elevation and are consistent with the notion that the 2nd phase increase is due largely to enhanced Ca2+ influx; the 1st phase elevation of [Ca2+]i induced by TRH is caused mainly, if not solely, by mobilization of cellular Ca2+.