Amiloride inhibits phorbol ester-stimulated Na⁺/H⁺ exchange and protein kinase C. An amiloride analog selectively inhibits Na⁺/H⁺ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na⁺-dependent H⁺ efflux. In efforts to determine whether stimulation of Na⁺/H⁺ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na⁺/H⁺ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na⁺/H⁺ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na⁺/H⁺ exchange than is amiloride, while being no more potent that amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na⁺/H⁺ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na⁺/H⁺ exchange-coupled events in phorbol ester-stimulated cells.