PKC activation is required by EGF-stimulated Na(+)-H+ exchanger in human pleural mesothelial cells

Epidermal growth factor (EGF) stimulates the Na(+)-H+ exchanger, leading to enhanced cell proliferation. In human pleural mesothelial cells (PMCs), the intracellular signaling mechanism mediating the EGF-induced stimulation of the Na(+)-H+ exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, to measure changes in intracellular pH (pHi), we found that 1) EGF and 12-O-tetradecanoylphorbol 13-acetate (TPA; a phorbol ester) both stimulate the ethylisopropyl amiloride-sensitive Na(+)-H+ exchanger; 2) TPA-induced alkalosis can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and staurosporine) or by PKC down-regulation, indicating that PKC activation is involved in the stimulation of the Na(+)-H+ exchanger. However, TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; and 3) the stimulatory effect of EGF on the Na(+)-H+ exchanger acts via stimulation of tyrosine kinase-receptor activity because it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A, and herbimycin A). It also involves PKC activation because EGF-induced alkalosis was blocked by PKC inhibitors. These results suggest that PKC activation is one of the downstream signals for EGF-induced activation of the Na(+)-H+ exchanger in primary cultures of human pleural mesothelial cells.