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This Article Full Text Full Text (PDF) Supplemental Figures All Versions of this Article: 296/2/H247    most recent 00725.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Boedtkjer, E. Articles by Aalkjaer, C. Search for Related Content PubMed PubMed Citation Articles by Boedtkjer, E. Articles by Aalkjaer, C.

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

Institute of Physiology and Biophysics, The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark

Submitted 14 July 2008 ; accepted in final form 24 November 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 (pH_i = –0.71 ± 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pH_i (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 65%. Cariporide abolished the effect of insulin and H₂O₂. In vascular smooth muscle cells, H₂O₂ produced intracellular acidification (pH_i = –0.48 ± 0.06) as did high concentrations of insulin (pH_i = –0.03 ± 0.01). NHE activity after an NH₄⁺ prepulse was 80% attenuated by H₂O₂ and 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 pH_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_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.

sodium/hydrogen exchanger 1; protein tyrosine phosphatase; hydrogen peroxide; intracellular pH; mesenteric artery