title: Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and 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 CO(2)/HCO(3)(-), removal of bath Na(+) produced endothelial acidification (DeltapHi = -0.71 +/- 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pHi (DeltapHi=-0.28 +/- 0.08). Insulin and H2O2 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon Na+ removal by approximately 65%. Cariporide abolished the effect of insulin and H2O2. In vascular smooth muscle cells, H2O2 produced intracellular acidification (DeltapHi = -0.48 +/- 0.06) as did high concentrations of insulin (DeltapHi = -0.03 +/- 0.01). NHE activity after an NH4+ prepulse was approximately 80% attenuated by H2O2 and approximately 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 pHi 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 pHi 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.
authors.affiliation: Institute of Physiology and Biophysics, The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark. eb@fi.au.dk
authors.forename: Ebbe
authors.initials: E
authors.lastname: Boedtkjer
authors.forename: Christian
authors.initials: C
authors.lastname: Aalkjaer
mesh_terms: D000818:Animals; D002374:Catalase / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D008297:Male; D008638:Mesenteric Arteries / Q000201:enzymology; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology; D013997:Time Factors
chemical_list: D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D006146:Guanidines; D007328:Insulin; C512715:NSC-87877; D011804:Quinolines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C490997:Slc9a1 protein, mouse; D027982:Sodium-Bicarbonate Symporters; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C070295:catalase-polyethylene glycol; D011092:Polyethylene Glycols; C093373:cariporide; D006861:Hydrogen Peroxide; D002374:Catalase; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516725:Ptpn11 protein, mouse