CYTOSOLIC ACIDIFICATION STIMULATES A CALCIUM INFLUX THAT ACTIVATES NA+-H+ EXCHANGE IN LLC-PK1

We have previously shown that cytosolic acidification-stimulated Na+-H+ exchange in LLC-PK1 cells is inhibited by calmodulin antagonists. To investigate further the role of Ca2+-calmodulin-dependent processes in intracellular pH (pH(i)) regulation in these cells, we studied the effects of pH(i) changes on cytosolic Ca2+ concentration ([Ca2+]i). In fura-2/acetoxymethylester (fura-2/AM)-loaded cells maintained in isotonic Na+-free buffer containing 1.8 mM CaCl2, [Ca2+]i was 168 +/- 59 nM (n = 5). After NH4Cl-induced alkalinization, [Ca2+]i decreased to 83 +/- 28 nM and partially recovered to 126 +/- 42 nM. Cytosolic acidification, after NH4Cl washout, caused an increase in [Ca2+]i to 481 +/- 166 nM (P < 0.05; n = 5) that was dependent on extracellular Ca2+. An increase in [Ca2+]i was also observed in cells acidified with KCl-nigericin, with a return of [Ca2+]i to baseline with cell alkalinization. No increase in Ca-45(2+) flux occurred in association with initial NH4Cl-induced [Ca2+]i decrease, suggesting Ca2+ flux into an intracellular store during alkalinization. Membrane depolarization did not alter [Ca2+]i. The acidification-induced [Ca2+]i increase was inhibited by preincubation with verapamil or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Na+-dependent pH(i) recovery in 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded LLC-PK1 cells and cytosolic acidification-stimulated basolateral Na+-H+ exchange activity in LLC-PK1/CL4 cells were both attenuated in the absence of extracellular Ca2+. The results indicate that cytosolic acidification activates an influx of extracellular Ca2+ in LLC-PK1 cells. Furthermore, in the absence of Ca2+ influx, Na+-H+ exchange is inhibited. We postulate that a pH(i)-sensitive Ca2+ influx pathway exists in these cells.