Agonist-stimulated Cl− efflux from human neutrophils

Shimizu, Yoshiyuki; Daniels, Robert; Elmore, Moira A.; Finnen, Michael J.; Hill, Maxine E.; Lackie, J. M.

doi:10.1016/0006-2952(93)90429-z

Cited by 24 publications

(3 citation statements)

References 32 publications

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“…Neutrophils have a particularly high resting cytosolic concentration of Cl - ; approximately 80 meq/L or 80 mM ( Simchowitz and De Weer, 1986 ). After stimulation by soluble agonists ( Yasuaki et al, 1993 ) or phagocytosis ( Busetto et al, 2007 ), they release large quantities of Cl - into the extracellular medium ( Menegazzi et al, 1999 ), which is thought to adjust for phagocytosis-induced cellular swelling ( Stoddard et al, 1993 ).…”

Section: Discussionmentioning

confidence: 99%

An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

et al. 2017

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Neutrophils phagocytosing bacteria and fungi exhibit a burst of non-mitochondrial respiration that is required to kill and digest the engulfed microbes. This respiration is accomplished by the movement of electrons across the wall of the phagocytic vacuole by the neutrophil NADPH oxidase, NOX2. In this study, we have attempted to identify the non-proton ion channels or transporters involved in charge compensation by examining the effect of inhibitors on vacuolar pH and cross-sectional area, and on oxygen consumption. The chloride channel inhibitors 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) and flufenamic acid (FFA) were the most effective inhibitors of alkalinisation in human neutrophil vacuoles, suggesting an efflux of chloride from the vacuole. The proton channel inhibitor, zinc (Zn2+), combined with DCPIB caused more vacuolar swelling than either compound alone, suggesting the conductance of osmotically active cations into the vacuole. Support for cation influx was provided by the broad-spectrum cation transport inhibitors anandamide and quinidine which inhibited vacuolar alkalinisation and swelling when applied with zinc. Oxygen consumption was generally unaffected by these anion or cation inhibitors alone, but when combined with Zn2+ it was dramatically reduced, suggesting that multiple channels in combination can compensate the charge. In an attempt to identify specific channels, we tested neutrophils from knock-out mouse models including CLIC1, ClC3, ClC4, ClC7, KCC3, KCNQ1, KCNE3, KCNJ15, TRPC1/3/5/6, TRPA1/TRPV1, TRPM2, and TRPV2, and double knockouts of CLIC1, ClC3, KCC3, TRPM2, and KCNQ1 with HVCN1, and humans with channelopathies involving BEST1, ClC7, CFTR, and MCOLN1. No gross abnormalities in vacuolar pH or area were found in any of these cells suggesting that we had not tested the correct channel, or that there is redundancy in the system. The respiratory burst was suppressed in the KCC3-/- and enhanced in the CLIC1-/- cells, but was normal in all others, including ClC3-/-. These results suggest charge compensation by a chloride conductance out of the vacuole and by cation/s into it. The identity of these channels remains to be established.

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Section: Discussionmentioning

confidence: 99%

An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

et al. 2017

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“…Recently a Na + /HCO 3 − cotransporter has been identified (22, 23). Efflux of chloride is commonly accompanying activation of neutrophils (24). Substitution of chloride with glucuronate leads to an outward flux of chloride (25).…”

Section: Discussionmentioning

confidence: 99%

Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs

et al. 2016

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In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation.

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“…These include anion exchangers (Simchowitz and De Weer, 1986), Na + /K + /2Cl - co-transporters (Bialasiewicz et al, 2004), and calcium- and swell-activated channels (Krause and Welsh, 1990; Schumann et al, 1993; Stoddard et al, 1993). Amongst the diffusive pathways available to Cl - in neutrophils; the evidence of a large Cl - efflux (Shimizu et al, 1993; Busetto et al, 2007), the swelling of the phagocytic vacuole (Reeves et al, 2002; Levine et al, 2015), and the recent molecular identification of LRRC8A as an indispensable component of the volume-regulated anion channel (VRAC) or volume-sensitive outwardly rectifying (VSOR) current (Qiu et al, 2014; Voss et al, 2014) prompted us to consider this channel as a possible participant in compensation of the current generated by the oxidase.…”

Section: Introductionmentioning

confidence: 99%

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

et al. 2017

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The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl-) current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A); the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

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Agonist-stimulated Cl− efflux from human neutrophils

Cited by 24 publications

References 32 publications

An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

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