Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and tyrosine phosphatase SHP-2

Boedtkjer E, Aalkjaer C. Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and tyrosine phosphatase SHP-2. Am J Physiol Heart Circ Physiol 296: H247-H255, 2009. First published November 26, 2008; doi:10.1152/ajpheart.00725.2008.-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 (pH(i)) 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 (Delta pH(i) = -0.71 +/- 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pH(i) (Delta pH(i)=-0.28 +/- 0.08). Insulin and H2O2 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon Na+ removal by similar to 65%. Cariporide abolished the effect of insulin and H2O2. In vascular smooth muscle cells, H2O2 produced intracellular acidification (Delta pHi = -0.48 +/- 0.06) as did high concentrations of insulin (Delta pH(i) = -0.03 +/- 0.01). NHE activity after an NH4+ prepulse was similar to 80% attenuated by H2O2 and similar to 40% by high insulin concentrations. H2O2 had 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 pH(i) up 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 pH(i) regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and H2O2. We propose that insulin signaling involves H2O2 and inhibition of PTP SHP-2.