Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils.
Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.
Kaba
Nubia K
NK
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
Schultz
Joanne
J
Law
Foon-Yee
FY
Lefort
Craig T
CT
Martel-Gallegos
Guadalupe
G
Kim
Minsoo
M
Waugh
Richard E
RE
Arreola
Jorge
J
Knauf
Philip A
PA
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000643:Ammonium Chloride / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019041:L-Selectin / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015427:Reperfusion Injury / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D039481:CD11b Antigen; D018821:CD18 Antigens; D006146:Guanidines; C509295:ITGAM protein, human; D016177:Macrophage-1 Antigen; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; D000643:Ammonium Chloride; D019041:L-Selectin; C057360:5-(N,N-hexamethylene)amiloride; D019344:Lactic Acid; C039465:5-dimethylamiloride; D000584:Amiloride; C093373:cariporide; D048051:p38 Mitogen-Activated Protein Kinases; D045726:Metalloproteases