Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

Abstract

The inward L-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulinrelated signaling pathways involved in the increase of L-dopa transport into isolated rat PTCs. Insulin (50?500 U/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 U/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 M). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2?100 M) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 M), but not maximal (5 M), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 M LY-294002) and protein kinase C (PKC; 1 M RO-318220). Insulin-induced activation of PKC- was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC- , and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.