REGULATION OF THE RENAL NA-BCO3 COTRANSPORTER .6. MECHANISM OF THE STIMULATORY EFFECT OF PROTEIN-KINASE-C

We have previously shown that the activity of the Na-HCO3 cotransporte r is stimulated by protein kinase C (PKC) activation, but the mechanis m responsible for this effect is not clear. We have shown that culture d proximal tubule cells of the rabbit have DIDS-sensitive Na-HCO3 cotr ansporter activity as assessed by HCO3-dependent Na-22 uptake or by me asurement of intracellular pH. In cells loaded with BCECF and treated with the amiloride analogue, ethylisopropyl amiloride, removal of extr acellular Na was associated with a rapid decrease in pH which returned to normal with re-addition of Na. This pH recovery was inhibited by D IDS and was used to quantify the activity of the Na-HCO3 cotransporter . In the present study, we utilized primary cultures of the proximal t ubule of the rabbit to examine the effect of PKC activation on the act ivity 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 a ctivity as compared to controls. Incubation for two hours also caused a significant stimulation of the Na-HCO3 cotransporter activity. The i nactive analogue of PMA, 4-alpha phorbol, failed to alter the cotransp orter. Similar results were observed when we examined the effect of PM A on HCO3-dependent Na-22 uptake. The affect of PMA to stimulate the c otransporter was mediated by PKC activation since it could be prevente d 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 stimu late the Na-HCO3 cotransporter activity was prevented by the protein s ynthesis inhibitors, actinomycin D or cycloheximide. The early effect of PKC to stimulate the cotransporter appeared to be associated with i ncreased phosphorylation of a 56 kD protein band, while the late effec t 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 cotransporte r by two distinct mechanisms: a long term effect which is protein synt hesis-dependent and a short term effect which is protein synthesis-ind ependent and is likely mediated by phosphorylation.