Submitted on November 10, 2007
Accepted on September 22, 2008
Inhibition of Na+/H+ Exchanger Enhances Low pH Induced L-selectin Shedding and 
2-integrin Surface Expression in Human Neutrophils
Nubia K. Kaba1, Joanne Schultz2, Foon-Yee Law2, Craig T. Lefort3, Guadalupe Martel-Gallegos4, Minsoo Kim3, Richard E. Waugh3, Jorge Arreola5*, and Philip A. Knauf6
1 Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York, United States
2 Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, United States
3 Department of Biomedical Engineering, University of Rochester Medical Center, Rochester, New York, United States
4 Physiology and Pharmacology, Universidad Autonoma de San Luis Potosi, School of Medicine, San Luis Potosi, San Luis Potosi, Mexico
5 Physics Institute, Universidad Autonoma de San Luis Potosi, San Luis Potosi, SLP, Mexico
6 Biochemistry & Biophysics, University of Rochester Medical Center, Rochester, New York, United States
* To whom correspondence should be addressed. E-mail: Arreola{at}dec1.ifisica.uaslp.mx.
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 lactoacidosis and consequent reductions in intracellular pH (pHi) have on surface expression of adhesion molecules on neutrophils. When human neutrophils where 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 HMA. Similar effects were observed when pHi was reduced while maintaining normal extracellular pH, by using an NH4Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors (HMA, cariporide or DMA). The amount of L-selectin shedding induced by different [NH4Cl] in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, 
2-integrin (CD11b and CD18) was only slightly up-regulated in the low pHi 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 (SB203580 and SB239063), implying a transmembrane effect of pHi. 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.
