Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes

L. C. McKinney, A. Moran

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14 Scopus citations


Mechanisms of intracellular pH (pH(i)) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein to measure pH(i). Under nominally HCO3/--free conditions, resting pH(i) 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, pH(i) 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 pH(i). 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 pH(i) 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 pH(i) was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pH(i) 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 pH(i) 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 pH(i). Conversely, a vacuolar H+- ATpase regulates the resting pH(i) of J774 cells but contributes little to recovery from C acidification.

Original languageEnglish
Pages (from-to)C210-C217
JournalAmerican Journal of Physiology - Cell Physiology
Issue number1 37-1
StatePublished - 1 Jan 1995
Externally publishedYes

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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