Effect of protein kinase C activation on Na +–H + exchange in erythrocytes of frog Rana temporaria

Abstract

The treatment of frog erythrocytes incubated in standard nitrate medium with 100 nM phorbol ester (PMA) induced a sharp increase in the 22Na uptake by the cells and intracellular Na + concentration. The PMA-induced enhancement in 22Na uptake was stimulated by the addition of 0.1 mM ouabain to the incubation medium and completely blocked by 1 mM amiloride. The time course of 22Na uptake by frog red cells in the presence of PMA showed a lag phase (∼5 min), after which was linear within 5–15 min. The calculated Na + influx in erythrocytes treated with PMA was 49.4±3.7 mmol l −1 cells h −1 as compared with 1.2±0.25 mmol l −1 h −1 for control cells. 5-( N-ethyl- N-isopropyl)-amiloride, selective blocker of NHE1, caused a dose-dependent inhibition of the PMA-induced Na + influx with IC 50 of 0.27 μM. The PMA-induced Na + influx was almost completely inhibited by 0.1 μM staurosporine, protein kinase C blocker. Pretreatment of frog red blood cells for 5, 10 or 15 min with 10 mM NaF, non-selective inhibitor of protein phosphatase, led to a progressive stimulation of the PMA effect on Na + influx. Both amiloride and NaF did not affect the basal Na + influx in frog erythrocytes. The data indicate that the Na +–H + exchanger in the frog erythrocytes is quiescent under basal conditions and can be markedly stimulated by PMA.