Phorbol-ester is bound specifically to renal luminal membranes and stimulates the Na-H antiporter

We characterized binding of [³H]-phorbol-12,13-dibutyrate (an activator of protein kinase C) to highly purified rabbit cortical renal luminal membranes and measured its effect on the kinetics of the Na-H antiporter. There was 95% specific binding to luminal membranes and this binding was time, temperature and pH dependent with optimal binding at 4°C and pH 7.4. Scatchard analysis of the binding revealed Kd of 0.8 μM and Bmax of 19.4 pmoles/mg protein. Phorbol-12,13-dibutyrate stimulated the Vmax of the Na-H antiporter by 16.5% ± 4.7 at 10^-6 M and by 19.9% ± 5.4 at 10^-5 M. The protein kinase C inhibitor H7¹ prevented the observed stimulation. Thus, there is a correlation between phorbol ester binding and phorbol ester stimulation of the Na-H antiporter. These data demonstrate that protein kinase C plays a role in the stimulation of the Na-H antiporter in renal luminal membranes.