Background:Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. Results: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1␤ secretion in human macrophages. Conclusion: Acidic pH represents a novel danger signal alerting the innate immunity. Significance: Local acidosis may promote inflammation at ischemic and inflammatory sites.

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Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H ؉ pump activity, estimated as a bafilomycin A 1 -sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzymelinked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H ؉ -ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H ؉ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na ؉ /H ؉ antiport activity. In contrast, chronic acidosis activated vacuolar H ؉ pumps in the osteoclasts. Although only ϳ5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6.5 for 4 -6 h were able to recover from the acute acid load by means of bafilomycin A 1 -sensitive proton extrusion. Conversely, the H ؉ -selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H ؉ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.Bone resorption is a multistep process involving migration of osteoclasts and/or osteoclast precursors to the bone surface, attachment to the bone matrix, and subsequent degradation of the underlying bone mineral by local acidification of the osteoclast-bone interface. When resorbing bone, osteoclasts display a specialized attachment zone, called the clear zone, which delimits a sealed compartment characterized by the presence of an extensive ruffled cell membrane (1). Demineralization of the bone matrix requires acidification of this extracellular compartment. Two lines of evidence suggest that the primary cellular mechanism responsible for this acidification is a vacuolar type H ϩ -ATPase (V-ATPase) 1 localized to the ruffled border of these cells. First, immunohistochemical studies demonstrated a marked accumulation of V-ATPases on the ruffled membrane of osteoclasts adherent to bone (2, 3). Second, the bone-resorbing capacity of osteoclasts is effectively inhibited by the specific V-ATPase inhibitor bafilomycin A 1 (4, 5). Considered together, these observations indicate a central role for the plasmalemmal V-ATPase in osteoclastic bone resorptio...