Intracellular pH regulation in U937 human monocytes: roles of V-ATPase and Na+/H+ exchange

The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal V-ATPase and Na+/H+ exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pHi was approximately 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0-7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5-7.4; pHi declined approximately 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5-7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na+/H+ exchange (EIPA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values > or = 7.0, the V-ATPase (bafilomycin-sensitive component) and NHE contributed almost equally to H+ extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H+-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes.