MECHANISMS OF ACTIVATION OF NA+/H+ EXCHANGE IN HUMAN OSTEOBLAST-LIKE SAOS-2 CELLS

Because of the importance of pH homeostasis in bone and the current uncertainty about the mechanisms by which intracellular pH (pH(i)) is regulated in this tissue, we have investigated the roles of cytosolic free Ca2+ concentrations ([Ca2+]i) and protein kinase C on the activation of Na+/H+ exchange in human osteoblast-like SaOS-2 cells. [Ca2+]i and pH(i) were measured using Fura-2 and 2'7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) respectively. The basal pH(i) in HCO3--free buffer was 7.36 +/- 0.04 units (mean +/- S.D.). Addition of ionomycin in Ca2+-containing buffer did not cause a rise in basal pH(i); however, addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) did cause a slowly developing rise in resting pH(i) of 0.14 +/- 0.02 unit over 4-5 min. Nigericin, a K+/H+ ionophore, caused an abrupt fall in pH(i) to 6.70 +/- 0.07 units. In nigericin-pretreated cells, PMA caused a rapid rise in pH(i) without changing the [Ca2+]i. In acidified cells, ionomycin increased [Ca2+]i and pH(i) in a parallel concentration-dependent (30-500 nM) manner. This action of ionomycin occurred in both the presence and the nominal absence of extracellular Ca2+. Ionomycin-induced alkalinization depended on extracellular Na+ and was inhibited in cells incubated with hexamethylene amiloride. When the incremental increase in [Ca2+]i induced by ionomycin was blocked by preincubation with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA)/AM, the effect on pH(i) was inhibited. Staurosporine, a protein kinase C inhibitor, blocked the action of PMA on pH(i), but it had no effect on the ionomycin-induced increase in pH(i). The action of ionomycin was not due to osmotic shock. We conclude that SaOS-2 cells have a protein kinase C-activatable Na+/H+ antiporter that is also stimulated, in acidified cells, in a concentration-dependent fashion by transients in [Ca2+]i which act via a nonprotein kinase C pathway.