title: Delay of acute intracellular pH recovery after acidosis decreases endothelial cell activation.
abstract: Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion.
authors.affiliation: Institute of Physiology II-Nanolab, University of Muenster, Muenster, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Huck
authors.forename: André
authors.initials: A
authors.lastname: Niemeyer
authors.forename: Tobias
authors.initials: T
authors.lastname: Goerge
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Schnaeker
authors.forename: Rainer
authors.initials: R
authors.lastname: Ossig
authors.forename: Phillip
authors.initials: P
authors.lastname: Rogge
authors.forename: Matthias F
authors.initials: MF
authors.lastname: Schneider
authors.forename: Hans
authors.initials: H
authors.lastname: Oberleithner
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Schneider
mesh_terms: D000138:Acidosis / Q000097:blood / Q000378:metabolism* / Q000503:physiopathology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters / Q000037:antagonists & inhibitors / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D005089:Exocytosis; D006146:Guanidines / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016209:Interleukin-8 / Q000378:metabolism; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D016014:Linear Models; D008689:Methacrylates / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism; D010973:Platelet Adhesiveness; D015427:Reperfusion Injury / Q000097:blood / Q000378:metabolism* / Q000503:physiopathology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: C118956:3-(2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methylphenyl)-N-isopropylidene-2-methyl-acrylamide dihydrochloride; D027963:Chloride-Bicarbonate Antiporters; D006146:Guanidines; D016209:Interleukin-8; D008689:Methacrylates; D019007:P-Selectin; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C069139:growth factor-activatable Na-H exchanger NHE-1; D014841:von Willebrand Factor; D000584:Amiloride; C093373:cariporide; D006632:Histamine; D000242:Cyclic AMP; D002118:Calcium; C039614:ethylisopropylamiloride