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.

Intracellular pH on Translocation of Protein Kinase C Isozymes in Rat Pinealocytes

“…Our results add to the understanding of transduction mechanisms that mediate the NE‐mediated increase in Na + /H + exchange activity in rat pinealocytes. In support of the established link between elevation of [Ca 2+ ] i and activation of the Na + /H + exchange (Berk et al, 1987; Graham and Tashjian, 1992), we now confirm the importance of elevation of [Ca 2+ ] i in the NE‐mediated elevation of pH i . This is based on the observations that pretreatment with BAPTA‐AM is effective in blocking the NE‐mediated elevation of pH i .…”

Dual regulation of the Na+/H+‐exchange in rat peritoneal mast cells: role of protein kinase C and calcium on pHi regulation and histamine release

“…The additive effects of ionomycin and PMA further support the independent pathways for Na+/H+ exchange activation by the two drugs. In a human U937 cell line, a similar additive effect of calcium and PKC on Na+/H+ exchange has been described (Alvarez et al, 1989), whereas others have shown partial additivity between these two pathways in acidified cells only (T6rnquist & Tashjian, 1992; Graham & Tashjian, 1992).…”

A New Action of Parathyroid Hormone

“…To ensure that the Na ϩ -free BSS effectively inhibited Na ϩ /H ϩ exchange, SaOS-2 cells were loaded with the pH-sensitive fluorescent probe BCECF/AM and then acidified with 1 M nigericin (a K ϩ /H ϩ ionophore). In nigericin-acidified SaOS-2 cells, PMA induces alkalinization via a Na ϩ /H ϩ exchanger-dependent mechanism (42). This PMA-induced alkalinization was completely blocked in Na ϩ -free buffer (data not shown).…”