Regulation of the renal Na-HCO3 cotransporter: VI. Mechanism of the stimulatory effect of protein kinase C

Abstract

We have previously shown that the activity of the Na-HCO3 cotransporter is stimulated by protein kinase C (PKC) activation, but the mechanism responsible for this effect is not clear. We have shown that cultured proximal tubule cells of the rabbit have DIDS-sensitive Na-HCO3 cotransporter activity as assessed by HCO3-dependent 22Na uptake or by measurement of intracellular pH. In cells loaded with BCECF and treated with the amiloride analogue, ethylisopropyl amiloride, removal of extracellular Na was associated with a rapid decrease in pH which returned to normal with re-addition of Na. This pH recovery was inhibited by DIDS and was used to quantify the activity of the Na-HCO3 cotransporter. In the present study, we utilized primary cultures of the proximal tubule of the rabbit to examine the effect of PKC activation on the activity of the Na-HCO3 cotransporter. Short term incubation (5 min) with the active phorbol ester, phorbol 12-myristate, 13-acetate (PMA), 10(-7) M, caused a significant stimulation of the Na-HCO3 cotransporter activity as compared to controls. Incubation for two hours also caused a significant stimulation of the Na-HCO3 cotransporter activity. The inactive analogue of PMA, 4-alpha phorbol, failed to alter the cotransporter. Similar results were observed when we examined the effect of PMA on HCO3-dependent 22Na uptake. The effect of PMA to stimulate the cotransporter was mediated by PKC activation since it could be prevented by the PKC inhibitors, calphostin C or sphingosine, or by prior PKC depletion. The long term but not the short term effect of PMA to stimulate the Na-HCO3 cotransporter activity was prevented by the protein synthesis inhibitors, actinomycin D or cycloheximide. The early effect of PKC to stimulate the cotransporter appeared to be associated with increased phosphorylation of a 56 kD protein band, while the late effect appeared to be associated with an increase in immunoreactive content of a 56 kD protein which is thought to be an active component of the cotransporter. Thus PKC stimulation activates the Na-HCO3 cotransporter by two distinct mechanisms: a long term effect which is protein synthesis-dependent and a short term effect which is protein synthesis-independent and is likely mediated by phosphorylation.