Irish thoracic society

Abstract

Regulation of cytosolic pH is vital for normal cellular function. Pathological microenvironments are associated with acid loading and cell metabolic processes generate acid equivalents. Cells have therefore evolved several mechanisms for proton extrusion, including plasmalemmal H+ATPase (active. when cells are acid loaded) and the Na/H+ antiporter (which in HPBMs is activated during formylmethionyl-leucyl phenylalanine induces acidosis. We have shown that inhibition of H+ATPase is associated with a mild intracellular acidosis (approx. 00.2-0.3 pH unit below physiological pH) in HAMs and that this is associated with reduced Fc mediated phagocytosis. The aim of these experiments was to assess the role of H+ATPase in HPBM pH regulation and phagocytosis and assess its role in the generation of intracellular reactive oxygen species (a vital cytotoxic mechanism) in HAMs and HPBM. PH changes were monitored flow cytometrically using pH sensitive intracellular probes. Respiratory burst was assessed by the reduction of dihydrorhodamine. Fc mediated phagocytosis was assessed flow cytometrically following exposure to Fc opsonised FITC labelled E.Coli HPBMs failed to recover physiological pH following acid loading in the presence of a specific H+ATPase inhibitor bafilomycin A 1 (0.002 pH units/min(baf) vs 0.21 pH units/min, p<0.05), but recovery was insensitive to the antiporter inhibitor amiloride. Bafilomycin reduced Fc mediated phagocytosis (mean channel fluorescence (mcf) vs 465, p<0.005. Respiratory burst was reduced in HAMs (mcf 379 vs 480, p<0.05) and in HPBMs (224 vs 302, p<0.05). These results suggest a critical role for H+ATPases in regulation of cytosolic pH and effector function of these cells.