ROLE OF CYTOCHROME-P-450 EPOXYGENASE METABOLITES IN EGF SIGNALING IN RENAL PROXIMAL TUBULE

Epidermal growth factor (EGF) is a potent epithelial cell mitogen and induces eicosanoid production in many cell types. The present study examined signaling mechanisms for EGF in the renal proximal tubule, where high concentrations of cytochrome P-450 epoxygenase have been reported. In primary cultures of rabbit proximal tubule cells, EGF (30 nM) increased endogenous epoxyeicosatrienoic acid (EET) levels 5.3 +/- 1.4-fold within 10 min (n = 6). In these cells EGF-stimulated [H-3]thymidine incorporation was significantly inhibited by the cytochrome P-450 inhibitors ketoconazole or clotrimazole but not by the cyclooxygenase inhibitor indomethacin. In fura 2-loaded proximal tubule cells, EGF caused a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+](i)), due to Ca2+ influx, which was inhibited by either ketoconazole or SKF-525A but not by indomethacin. Addition of 5,6-EET (0.5 mu M) also induced Ca2+ influx in proximal tubule cells, whereas 8,9-,11,12-, or 14,15-EET did not. In cells treated with bis(2-amino-5-methylphenoxy)ethane N,N,N ', N'-tetraacetic acid tetraacetoxy-methyl ester to chelate [Ca2+](i), EGF-stimulated [H-3]thymidine incorporation was significantly inhibited. Pretreatment of cells with 5.6-EET also augmented EGF-stimulated [H-3]thymidine incorporation. These results indicate that EGF increases EET levels in proximal tubule and suggest that 5,6-EET or its metabolites may be a modulator of EGF-induced[Ca2+](i) increases and involved in mitogenesis.