Insulin inhibits Na+/H+exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2and tyrosine phosphatase SHP-2

Abstract

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates Na+/H+exchange (NHE) activity in vascular cells through H2O2-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH (pHi) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of CO2/HCO3−, removal of bath Na+produced endothelial acidification (ΔpHi= −0.71 ± 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pHi(ΔpHi=−0.28 ± 0.08). Insulin and H2O2acidified endothelial cells 0.2–0.3 pH units and reduced the acidification upon Na+removal by ∼65%. Cariporide abolished the effect of insulin and H2O2. In vascular smooth muscle cells, H2O2produced intracellular acidification (ΔpHi= −0.48 ± 0.06) as did high concentrations of insulin (ΔpHi= −0.03 ± 0.01). NHE activity after an NH4+prepulse was ∼80% attenuated by H2O2and ∼40% by high insulin concentrations. H2O2had no effect on Na+-HCO3−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 pHi. 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 pHiup to 0.3 pH units and inhibited NHE activity 60–80%; when applied in combination with insulin or H2O2, no further effect was obtained. We conclude that NHE contributes to pHiregulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and H2O2. We propose that insulin signaling involves H2O2and inhibition of PTP SHP-2.