H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis. Patients with ATP6B1 mutations also have sensorineural hearing loss; consistent with this finding, we demonstrate expression of ATP6B1 in cochlea and endolymphatic sac. Our data, together with the known requirement for active proton secretion to maintain proper endolymph pH, implicate ATP6B1 in endolymph pH homeostasis and in normal auditory function. ATP6B1 is the first member of the H+-ATPase gene family in which mutations are shown to cause human disease.
The kinetics of phagocytosis of Candida albicans by human polymorphonuclear leukocytes was studied. The basis for these studies was a phagocytic assay with use of C. albicans radiolabeled with [3H]adenine. After incubation of leukocytes with C. albicans, extracellular C. albicans was separated from phagocytes by centrifugation through Ficoll-Hypaque suspensions (specific density, 1.175 g/cm3). Recovery of leukocytes by this technique was greater than or equal to 85%. The initial rate of phagocytosis was more rapid than that previously reported for bacteria. Ethylenediaminetetraacetate, vinblastine, ethylmaleimide, NaF, and ice bath temperature completely inhibited phagocytosis. Colchicine had no effect, and NaN3 was partially inhibitory. Pooled sera possessed low titers (greater than or equal to 1:40) of heat-stable opsonins. The opsonic activity of pooled sera was shown to depend primarily upon complement activated through both the alternative and classical pathways. Decomplemented hyperimmune sera were opsonic at high dilutions (greater than or equal to 1:160), and complement amplified the initial rate of ingestion seen with hyperimmune sera.
Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl- phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.
We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10- negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.
The antiinflammatory drug, phenylbutazone (PBZ), has been studied in terms of its influence on chemotactic deactivation of human neutrophils. When PBZ was present during the time of preincubation of cells with N-formyl-methionyl-phenylalanine (F-Met-Phe), loss of subsequent spontaneous mobility and chemotactic responsivity to F-Met- Phe did not occur. The action of PBZ to protect neutrophils from peptide-mediated chemotactic deactivation was found to involve in part its inhibitory influence on hexose monophosphate shunt activity and in part its antagonistic effect on interaction of peptide receptors with N- formyl peptide. Phenylbutazone interfered with binding of N-formyl- methionyl-leucyl-[3H]phenylalanine but not [125I]C5a to the neutrophil, displaced labeled tripeptide bound in the absence of PBZ, increased the dissociation constant (KD) for labeled tripeptide, and limited down regulation of peptide receptor function. These results provide an example of drug-mediated modulation of the interaction of neutrophils with N-formyl peptide and strongly support the possibility that PBZ interacts directly and specifically with the human neutrophil peptide receptor as a competitive antagonist. They also provide an additional example of a compound outside of th N-formyl peptide series that interacts with the peptide receptor.
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