Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction

The present study investigated the signal-transduction pathway responsible for the epidermal growth factor (EGF) stimulation of phosphate transport (JPhos) in the rabbit proximal convoluted tubule (PCT). Genistein, 10(-4) M, bath and lumen, an inhibitor of EGF receptor tyrosine kinase activity, blocked the EGF effect on JPhos, consistent with a role for tyrosine kinase in the signal-transduction pathway. Both staurosporine (5 x 10(-8) M) and calphostin C (10(-8) M), inhibitors of protein kinase C, blocked the EGF stimulation of JPhos, indicating that protein kinase C is involved in EGF signaling. Intracellular calcium (Ca2+i) concentrations were measured in perfused tubules using fura PE3 to determine whether changes in Ca2+i were also part of the signaling pathway. After addition of 3 nM EGF, there was no change in Ca2+i, suggesting that stimulation of protein kinase C is not from phosphatidylinositol hydrolysis by phospholipase C-gamma. To determine whether phospholipase A2 (PLA2) is involved, the inhibitor mepacrine was used. Mepacrine (5 x 10(-5) M) had no direct effect on PCT transport but blocked the stimulatory effect of EGF on JPhos. PLA2 activity, assessed as free arachidonic acid release from proximal tubules in suspension, increased by 18.8% with 3 nM EGF. Thus the stimulation of JPhos by EGF is mediated via a signal-transduction pathway involving tyrosine kinase, protein kinase C, and PLA2.