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 (pH(i)) 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 pH(i) and the kinetics of pH(i) recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A(1) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pH(i) was similar to7.13 at an extracellular pH (pH(o)) of 7.4 and fell progressively at lower pH(o) values. EIPA had no effect on baseline pH(i) at pH(o) 7.4, but caused a sustained decrement in pH(i) at pH(o) 6.0-7.0. Bafilomycin A(1) had biphasic effects on baseline pH(i) at pH(o) 6.5-7.4: pH(i) declined similar to 0.1 units over the course of several minutes and then recovered. At pH(o) 6.0, bafilomycin A(1) caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pH(o) 6.5-7.4 was prevented by EIPA. Similarly, pH(i) recovery from NH4Cl prepulse acid-loads (pH(o) 7.4) was sensitive to both EIPA and bafilomycin A(1) During this recovery process, Na+/H+ exchange (EITA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pH(i) values < 7.0. At acid-loaded pH(i) values > 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 pH(i) regulation in U937 cells. The H+-pump and NHE interacted to set baseline pH(i) and for pH(i) recovery following cytosolic acid-loading of the monocytes.