Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells.

TRH stimulation of prolactin secretion is thought to be mediated by an elevation of free cytoplasmic Ca2+. However, whether TRH-induced influx of extracellular Ca2+ is required to elevate cytoplasmic Ca2+ remains controversial. We measured cytoplasmic free Ca2+ concentration in GH3 cells with an intracellularly trapped fluorescent indicator, Quin 2. In unstimulated cells incubated in medium containing 1.5 mM Ca2+, cytoplasmic free Ca2+ concentration was 118 +/- 18 nM (mean +/- SD). TRH (1 microM) caused a rapid transient elevation of free cytoplasmic Ca2+ to a level estimated to be at least 500 nM. High extracellular K+, which induces extracellular Ca2+ influx, caused an elevation of free cytoplasmic Ca2+ which was greater and longer in duration that that caused by TRH. When cells were incubated in medium containing 3 mM EGTA, the K+ depolarization-induced increase in free cytoplasmic Ca2+ was abolished. By contrast, the TRH-induced increase was not affected by incubating cells in medium with 3 mM EGTA, or high K+, or both; incubation of cells in medium with EGTA and high K+ abolishes the electrochemical driving force for Ca2+ influx. These data demonstrate that Ca2+ influx is not required for TRH-induced elevation of free cytoplasmic Ca2+ in GH3 cells. We conclude that in GH3 cells TRH induces an elevation of free cytoplasmic Ca2+ leading to stimulated prolactin secretion by mobilizing cellular Ca2+.