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Mechanisms of activation of Na+/H+ exchange in human osteoblast-like SaOS-2 cells.

ABSTRACT: Because of the importance of pH homeostasis in bone and the current uncertainty about the mechanisms by which intracellular pH (pHi) 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 pHi were measured using Fura-2 and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) respectively. The basal pHi in HCO3(-)-free buffer was 7.36 +/- 0.04 units (mean +/- S.D.). Addition of ionomycin in Ca(2+)-containing buffer did not cause a rise in basal pHi; however, addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) did cause a slowly developing rise in resting pHi of 0.14 +/- 0.02 unit over 4-5 min. Nigericin, a K+/H+ ionophore, caused an abrupt fall in pHi to 6.70 +/- 0.07 units. In nigericin-pretreated cells, PMA caused a rapid rise in pHi without changing the [Ca2+]i. In acidified cells, ionomycin increased [Ca2+]i and pHi 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 pHi was inhibited. Staurosporine, a protein kinase C inhibitor, blocked the action of PMA on pHi, but it had no effect on the ionomycin-induced increase in pHi. 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 non-protein kinase C pathway.

SUBMITTER: Graham CS

PROVIDER: S-EPMC1132090 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Mechanisms of activation of Na+/H+ exchange in human osteoblast-like SaOS-2 cells.

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