Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes.
Mechanisms of intracellular pH (pHi) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi. Under nominally HCO3(-)-free conditions, resting pHi of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97). In the presence of HCO3-/CO2, pHi values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively. Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pHi. Inhibitors of a vacuolar type H(+)-ATPase [bafilomycin A1, N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)] reduced pHi by at least 0.2 pH units. Inhibitors of other classes of H(+)-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect. Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pHi was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pHi recovered exponentially to normal within 2 min. The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine. Inhibitors of the vacuolar H(+)-ATPase also inhibited recovery. NEM and NBD nonspecifically inhibited all recovery. Bafilomycin A1 and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pHi was above 7.0. We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pHi. Conversely, a vacuolar H(+)-ATPase regulates the resting pHi of J774 cells but contributes little to recovery from acidification.
Department of Physiology, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603.
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McKinney
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Moran
D000143:Acids / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012146:Rest; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors
D000143:Acids; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D006180:Proton-Translocating ATPases