PH HOMEOSTASIS IN PITUITARY GH4C1 CELLS - BASAL INTRACELLULAR PH IS REGULATED BY CYTOSOLIC FREE CA-2+ CONCENTRATION

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pH(i)). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pH(i) from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P < 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pH(i) by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pH(i). To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pH(i) to 6.75 +/- 0.03 (P < 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pH(i); however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pH(i) markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pH(i) through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pH(i) in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.