Insulin inhibits [Na.sup.+]/[H.sup.+] exchange in vascular smooth muscle and endothelial cells in situ: involvement of [H.sub.2][O.sub.2] and tyrosine phosphatase SHP-2

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates [Na.sup.+]/[H.sup.+] exchange (NHE) activity in vascular cells through [H.sub.2][O.sub.2]-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH ([pH.sub.i]) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of C[O.sub.2]/HC[O.sub.3], removal of bath [Na.sup.+] produced endothelial acidification ([DELTA][pH.sub.i] = -0.71 [+ or -] 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state [pH.sub.i] ([DELTA][pH.sub.i] = -0.28 [+ or -] 0.08). Insulin and H202 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon [Na.sup.+] removal by ~65%. Cariporide abolished the effect of insulin and [H.sub.2][O.sub.2]. In vascular smooth muscle cells, [H.sub.2][O.sub.2] produced intracellular acidification ([DELTA][pH.sub.i] = -0.48 [+ or -] 0.06) as did high concentrations of insulin ([DELTA][pH.sub.i] = -0.03 [+ or -] 0.01). NHE activity after an N[H.sup.+.sub.4] prepulse was ~80% attenuated by [H.sub.2][O.sub.2] and ~40% by high insulin concentrations. [H.sub.2][O.sub.2] had no effect on [Na.sup.+]-HC[O.sup.-.sub.3] cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on [pH.sub.i]. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)chloromethyl-2',7'-dichlorodihydrofluorescein. The SHP-2 selective inhibitor NSC-87877 and protein tyrosine phosphatase (PTP) inhibitor IV reduced steady-state [pH.sub.i] up to 0.3 pH units and inhibited NHE activity 60-80%; when applied in combination with insulin or [H.sub.2][O.sub.2], no further effect was obtained. We conclude that NHE contributes to phi regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and [H.sub.2][O.sub.2]. We propose that insulin signaling involves [H.sub.2][O.sub.2] and inhibition of PTP SHP-2. sodium/hydrogen exchanger 1; protein tyrosine phosphatase; hydrogen peroxide; intracellular pH; mesenteric artery