Alkalinizing the intralysosomal pH inhibits degranulation of human neutrophils.

Klempner, Mark S.; Styrt, Barbara

doi:10.1172/jci111139

Cited by 74 publications

(51 citation statements)

References 28 publications

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“…The decrease of cell recovery rates suggests that numbers of the cells might have been destroyed due to cytotoxic or cytolytic effects of ammonia, such as disturbance of acid-base balance or interference with essential metabolic cycles. Ammonia has also been found to inhibit neutrophil function including chemotaxis, phagocytosis, degranulation and bactericidal activity by alkalizing the intralysosomal pH [1,6]. The enhancement of chemiluminescence per cell at lower ammonia concentrations in this study, however, seems contradictory to those findings.…”

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confidence: 91%

Effects of In vitro Atmospheric Ammonia Exposure on Recovery Rate and Luminol-dependent Chemiluminescence of Bovine Neutrophils and Bronchoalveolar Macrophages.

Murata

Horino

1999

J. Vet. Med. Sci.

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ABSTRACT. The effects of atmospheric ammonia, a major pollutant in animal confinement facilities, on bovine neutrophils and bronchoalveolar macrophages were evaluated in vitro. Ammonia exposure at concentrations 50, 100 and 200 ppm for one hour impaired recovery rates of neutrophils dose-dependently but enhanced their chemiluminescence activity per cell at lower concentrations (50 and 100 ppm). Macrophages were resistant to the exposure. Their recovery rates and chemiluminescence remained unaffected even at 200 ppm exposure. The present results suggest that ammonia exposure is unfavorable for bovine neutrophils in vitro, and probably in vivo also, in light of causing cell damage and triggering wider inflammatory responses.-KEY WORDS: ammonia exposure, bovine bronchoalveolar macrophage, bovine neutrophil.J. Vet. Med. Sci. 61(3): 279-281, 1999 monitored with a gas detector (Gastec model 801, Japan) before and after the cell exposure. Preliminary experiments have confirmed that ammonia concentration can be kept at each designed level at least for 2 hr at 37°C. Two ml of cell suspension were placed into a siliconcoated polypropylene V-bottom tube with a 25 mm diameter and a 50 mm depth in the chamber and exposed to each ammonia concentration for one hour at 37°C. After the exposure, pH values of the cell suspensions were measured with a pH meter (pHBOY-P2, Shin-Dengen, Japan). Ammonia concentrations dissolved in the suspension were estimated with a pH-ammonia concentration standard curve obtained preliminarily. Cell counting was made on each suspension and cell recovery rate at each concentration was calculated as a percentage of cell count ratio (Exposure/ Control). Recovered cells were centrifuged and resuspended in HBSS (pH 7.4) at a concentration of 1 × 10 6 /ml.The chemiluminescence assay of the recovered cells was performed as previously described [7]. Briefly, photon emission levels of the cells (2 × 10 5 cells) in response to phorbol myristate acetate (PMA: Sigma, 1.6 × 10 6 M at final concentration) were evaluated in the presence of luminol (Sigma, 0.5 mg/ml at final concentration) with a photon-counting luminescence analyzer (Berthold LUMAT LB9505C, Germany). The peak chemiluminescence (cpm) per cell at each ammonia exposure was calculated.Data were analyzed using analysis of variance and Student's t-test. A level of p<0.05 was regarded as significant. Table 1 shows recovery rates of neutrophils and macrophages exposed to ammonia at concentrations of 50, 100 and 200 ppm for one hour. Neutrophils were sensitive to ammonia exposure and their recovery rates decreased dose-dependently. In contrast, macrophages remained resistant to the exposure. The pH values and estimated ammonia concentrations (parentheses) in the cell suspensions were at pH8.4-8.6 (1-2 mg/dl), pH8.7-8.9 (2-3 mg/dl) and pH9.1-9.2 (5-6 mg/dl) at 50, 100 and 200

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confidence: 91%

Effects of In vitro Atmospheric Ammonia Exposure on Recovery Rate and Luminol-dependent Chemiluminescence of Bovine Neutrophils and Bronchoalveolar Macrophages.

Murata

Horino

1999

J. Vet. Med. Sci.

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“…We then applied external ammonium chloride to supply NH 3 , a membrane-permeant base that neutralizes the acidic pH of organelles (31,32). Fig.…”

Section: Most Granules Do Not Collapse Into the Plasma Membrane Aftermentioning

confidence: 99%

Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells

Taraska

Perrais

Ohara‐Imaizumi

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

340

404

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Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH 4Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.PC-12 cells ͉ evanescent-field microscopy ͉ endocytosis ͉ kiss and run

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“…To determine the rate of cholesteryl ester hydrolysis, 2 g/ml of Sandoz 58-035 (an ACAT inhibitor) was added during lipoprotein or liposome incubation to prevent reesterification of liberated [ 14 C]oleate and free cholesterol. To examine whether the rate of cholesteryl ester hydrolysis involved the lysosomal CEH, the cells were cultured in the presence of 50 g/ml of either native HDL or SAA-HDL together with CQ (100 M), an agent that neutralizes the lysosomal proton gradient (34,35). At various time points, cellular lipids were extracted and analyzed for cholesteryl ester radioactivity as described above.…”

Section: Rates Of Hydrolysis Of Cholesteryl Ester In J774 Cellsmentioning

confidence: 99%