bafilomycin-a1 and Acidosis

Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites.. Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages.. Acidic pH represents a novel danger signal alerting the innate immunity.. Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Topics: Acidosis; Animals; Carrier Proteins; Caspase 1; Cell Hypoxia; Cells, Cultured; Culture Media; Cytokines; Enzyme Activation; Extracellular Fluid; Humans; Hydrogen-Ion Concentration; Immunity, Innate; Inflammasomes; Inflammation Mediators; Lipopolysaccharides; Macrolides; Macrophages; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Potassium; Proton Pump Inhibitors; Transcriptional Activation

Localized acidification of the osteoclast-bone interface is driven by a vacuolar-type H+-ATPase (V-ATPase) in the plasma membrane in a process thought to be associated with bone resorption. The present study investigated the mechanism underlying the roles of V-ATPase-induced acidosis in osteoclastogenesis. Active proton pumping due to increased V-ATPase activity during RANKL-induced osteoclastogenesis induced intracellular and extracellular acidification of osteoclast precursors. Subsequent analysis revealed blockage of extracellular acidification and induction of intracellular acidification by bafilomycin A1, a specific inhibitor of V-ATPase, indicating that extracellular acidification is mostly induced by V-ATPase-mediated proton pumping into extracellular space. Low-pH media controlled by HEPES-buffered conditions to mimic metabolic acidosis led to synergistic activation of RANKL-stimulated signals, including mitogen-activated protein kinases and transcription factor NF-kappaB, resulting in enhanced osteoclastogenesis. Low-pH media also upregulated the expression of osteopontin secreted into extracellular space, which is required for cell migration by binding to cell surface integrin alphavbeta3. Osteoclast precursor migration was significantly inhibited by treatment of antibodies to integrin alphavbeta3, resulting in the retardation of osteoclastogenesis. Taken together, these findings indicate that V-ATPase-driven acidosis modulates osteoclastogenesis.

Topics: Acidosis; Animals; Cell Differentiation; Cell Movement; Female; Gene Expression Regulation; Hydrogen-Ion Concentration; Integrin alphaVbeta3; Macrolides; Mice; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteopontin; RANK Ligand; RNA, Messenger; Signal Transduction; Stem Cells; Vacuolar Proton-Translocating ATPases

Net HCO3- transport in the rabbit kidney cortical collecting duct (CCD) is mediated by simultaneous H+ secretion and HCO3- secretion, most likely occurring in a alpha- and beta-intercalated cells (ICs), respectively. The polarity of net HCO3- transport is shifted from secretion to absorption after metabolic acidosis or acid incubation of the CCD. We investigated this adaptation by measuring net HCO3- flux before and after incubating CCDs 1 h at pH 6.8 followed by 2 h at pH 7.4. Acid incubation always reversed HCO3- flux from net secretion to absorption, whereas incubation for 3 h at pH 7.4 did not. Inhibition of alpha-IC function (bath CL- removal or DIDS, luminal bafilomycin) stimulated net HCO3- secretion by approximately 2 pmol/min per mm before acid incubation, whereas after incubation these agents inhibited net HCO3- absorption by approximately 5 pmol/min per mm. Inhibition of beta-IC function (luminal Cl- removal) inhibited HCO3- secretion by approximately 9 pmol/min per mm before incubation, whereas after incubation HCO3- absorption by only 3 pmol/min per mm. After acid incubation, luminal SCH28080 inhibited HCO3- absorption by only 5-15% vs the circa 90% inhibitory effect of bafilomycin. In outer CCDs, which contain fewer alpha-ICs than midcortical segments, the reversal in polarity of HCO3- flux was blunted after acid incubation. We conclude that the CCD adapts to low pH in vitro by downregulation HCO3- secretion in beta-ICs via decreased apical CL-/base exchang activity and upregulating HCO3- absorption in alpha-ICs via increased apical H+ -ATPase and basolateral CL-/base exchange activities. Whether or not there is a reversal of IC polarity or recruitment of gamma-ICs in this adaptation remains to be established.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Animals; Anti-Bacterial Agents; Bicarbonates; Biological Transport; Chlorides; Culture Techniques; Enzyme Inhibitors; Female; Imidazoles; Kidney Cortex; Kidney Tubules, Collecting; Macrolides; Membrane Potentials; Proton Pump Inhibitors; Proton-Translocating ATPases; Rabbits

We carried out in vivo microperfusion experiments in acid-loaded rats to characterize the adaptive response of the unidirectional components secretory flux (Jsec) and reabsorptive flux (Jreab)] of distal tubule bicarbonate reabsorption and to test the hypothesis that Jreab is dependent on bafilomycin A1-sensitive H(+)-adenosinetriphosphatase activity. During 18 h of severe acidosis there was a significant decrease in Jsec (-15 +/- 3 vs. -38 +/- 5 pmol.min-1.mm-1, P < 0.05) and a significant increase in Jreab (37 +/- 6 vs. 0 +/- 5 pmol.min-1.mm-1, P < 0.05), which was insensitive to 10(-5) M bafilomycin A1, 10(-5) M Sch-28080, and 3 mM amiloride. After 3 days of acid loading, these same inhibitors reduced Jreab by approximately 60%. However, when water flux was completely inhibited by isosmotic perfusion, a significant Jreab (15 +/- 2 pmol.min-1.mm-1) resistant to 10(-5) M bafilomycin A1 persisted, as in severe acidosis. In reabsorbing distal tubules of overnight-fasted rats, Sch-28080 elicited no inhibition, whereas bafilomycin A1 and amiloride had significant effects (28 +/- 5, 24 +/- 4, respectively, vs. 50 +/- 4 pmol.min-1.mm-1 for fasted rats, P < 0.05). Thus, although Jsec is reduced in the transition from mild to severe metabolic acidosis of 18-h duration, the predominant effect is a stimulation of bafilomycin A1-resistant Jreab.

Topics: Acclimatization; Acidosis; Acute Disease; Amiloride; Ammonium Chloride; Animals; Anti-Bacterial Agents; Anti-Ulcer Agents; Antifungal Agents; Bicarbonates; Chronic Disease; Diet; Fasting; Hydrochloric Acid; Imidazoles; In Vitro Techniques; Infusions, Intravenous; Kidney Tubules, Distal; Macrolides; Male; Perfusion; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Reference Values