Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) caused complex transient perturbations of amiloride-sensitive short- circuit Na⁺ currents (I(Na)) in A6 epithelia and frog skins that were tissue and concentration dependent. A noninvasive channel blocker pulse method of noise analysis (18) was used to investigate how PMA caused time-dependent changes of apical membrane epithelial Na⁺ channel (ENaC) single-channel currents, channel open probabilities (P(o)), and channel densities (N(T)). In A6 epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependent sustained decreases of P(o) (~55% below control, 50 nM) and rapid compensatory transient increases of N(T) within 7 min (~220% above control, 50 nM), resulting in either small transient increases of I(Na) at 5 nM PMA or small biphasic decreases of I(Na) at 50 nM PMA. In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin caused after a delay of at least 10 min transient increases of N(T) to ~60-70% above control at 30-60 min. Unlike A6 epithelia, P(o) was increased ~15% above control within 7 min and remained within ±10-15% of control for the duration of the 2-h experiments. Despite differences in the time courses of secondary inhibition of transport in A6 epithelia and frog skin, the delayed downregulation of transport was due to time-dependent decreases of N(T) from their preelevated levels in both tissues. Whereas P(o) is decreased within minutes in A6 epithelia as measured by noise analysis or by patch clamp (8), the discrepancy in regulation of N(T) in A6 epithelia as measured by noise analysis and patch clamp is most likely explained by the inability of on-cell patches formed before treatment of tissues with PMA to respond to regulation of their channel densities.