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S Grinstein

Publications and source records attributed to S Grinstein.

At least 199 records · Page 11Linked to original sources
PubMed1991

Tyrosine phosphorylation and oxygen consumption induced by G proteins in neutrophils.

In neutrophils, receptor-mediated activation of the respiratory burst requires ATP, possibly for phosphotransferase reactions. The oxidative response is only partially inhibited by blockers of protein kinase C, suggesting the involvement of other kinases. Recent evidence has demonstrated activation of tyrosine phosphorylation in chemoattractant-stimulated cells. This effect is likely mediated by G proteins because it is obliterated by pretreatment with pertussis toxin. In this report we have attempted to correlate the respiratory burst and phosphotyrosine accumulation induced by activation of G proteins, accomplished by treatment of electroporated cells with nonhydrolyzable analogues of GTP. In cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) both responses displayed similar time course and concentration dependence. The guanine nucleotide selectivity sequence and the divalent cation requirements were also similar for both responses. These similarities suggest a relationship between tyrosine phosphorylation and the activation of the NADPH oxidase. GTP gamma S-induced phosphotyrosine accumulation was found to be inhibited by pretreatment of the cells with phorbol esters, underlining the existence of regulatory interactions between different signal transduction pathways in neutrophils.

Adenine Nucleotides↗
PubMed1991

Cytoplasmic pH regulation in monocytes and macrophages: mechanisms and functional implications.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is critical to optimal cell function. Monocytes and macrophages (Møs) actively regulate their pHi through three distinct plasma membrane ion transport systems: (1) Na+/H+ exchange; (2) Na(+)-dependent anion exchange; and (3) vacuolar-type H+ ATPases. Alterations in the functional state of monocytes and Møs have been linked to changes in pHi and/or its regulation by these ion transport systems. Differentiation, proliferation, and activation of Møs in response to a variety of agents are associated with increased Na+/H+ exchange. The resultant cytoplasmic alkalinization typically observed in HCO3(-)-free media likely plays a permissive, rather than a triggering, role in mediating Mø response to most of these agents. Prevention of cytoplasmic acidification is essential during Mø activation, when production of metabolic acid increases. This is of particular importance within the in vivo microenvironment of an abscess or tumour, where pHi is further threatened by the low extracellular pH (pHo) which typically prevails. At low pHo, H+ ATPase-mediated H+ extrusion plays a critical role in maintenance of pHi, preserving the ability of Møs to generate a respiratory burst. The requirement for maintenance of pHi within a range conducive to efficient Mø function may explain why Møs have acquired a variety of parallel systems for pHi regulation.

Acid-Base Equilibrium↗
PubMed1990

Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages.

The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.

Animals↗
PubMed1990

ATP and guanine nucleotide dependence of neutrophil activation. Evidence for the involvement of two distinct GTP-binding proteins.

In phagocytes, activation of the respiratory burst by chemoattractants requires ATP and involves a pertussis toxin-sensitive G protein. ATP is also required for the response elicited in permeabilized neutrophils by nonhydrolyzable GTP analogs, indicating that at least one of the ATP-dependent steps lies downstream of the receptor-coupled G protein(s). A respiratory burst can also be produced in a reconstituted cell-free system by addition of arachidonic acid. Most investigators find this response to be independent of ATP, yet stimulated by GTP analogs, implying that the ATP-dependent steps observed in the unbroken cells must precede the guanine nucleotide-requiring event. To resolve this apparent discrepancy, we studied the ATP and guanine nucleotide dependence of the oxidative response elicited by arachidonic acid in electrically permeabilized human neutrophils. Two components of the response were apparent: one was ATP-dependent, the other ATP-independent. The ATP-dependent component was partially inhibited by staurosporine, suggesting involvement of protein kinase C. This kinase signals activation of the NADPH oxidase without intervening G proteins, since stimulation by phorbol ester was unaffected by guanosine 5'-(beta-thio)diphosphate (GDP beta S). Although nonhydrolyzable GTP analogs failed to stimulate the oxidase in the absence of ATP, the ATP-independent response stimulated by arachidonic acid was found to require GTP or one of its analogs and to be inhibited by GDP beta S. The relative potency of the guanine nucleotides to support the arachidonic acid response in the absence of ATP (5'-guanylyl imidodiphosphate (GMP-PNP) greater than or equal to guanosine 5'-(gamma-thio)triphosphate GTP gamma S) greater than or equal to (GTP) differed from their efficacy to stimulate the burst in the presence of ATP (GTP gamma S greater than GMP-PNP much greater than GTP). These observations suggest the involvement of two distinct GTP-binding proteins in oxidase activation: a receptor-coupled, heterotrimeric, pertussis toxin-sensitive G protein, and a second GTP-binding protein(s) located downstream of the ATP-requiring steps, which may lie in close proximity to the NADPH oxidase. This secondary GTP-binding protein could be part of the pathway activated by chemoattractants, but does not mediate stimulation via protein kinase C. Therefore multiple parallel routes may exist for activation of the NADPH oxidase.

Adenosine Triphosphate↗
PubMed1990

Activation of permeabilized HL60 cells by vanadate. Evidence for divergent signalling pathways.

The possible role of tyrosine phosphorylation in the activation of granulocytic HL60 cells was examined using vanadate, a phosphotyrosine phosphatase inhibitor. Treatment of permeabilized cells with micromolar concentrations of vanadate resulted in a substantial accumulation of tyrosine-phosphorylated proteins, detected by immunoblotting. At comparable concentrations, vanadate was also found to elicit an NADPH-dependent burst of oxygen utilization. Actin assembly, studied using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, was similarly stimulated by vanadate, though considerably higher concentrations were required to observe this effect. In contrast with these responses, the secretion of lysozyme was not stimulated by vanadate, nor did vanadate affect calcium-induced secretion. Therefore, accumulation of tyrosine-phosphorylated proteins is associated with stimulation of some, but not all, of the responses characteristic of granulocytic cell activation. This indicates that the effects of vanadate are selective and suggests divergence of the signalling pathways leading to the individual effectors.

Actins↗
PubMed1990

A vacuolar type H(+)-ATPase regulates cytoplasmic pH in murine macrophages.

An Na(+)- and HCO3(-)-independent mechanism of cytoplasmic pH (pHi) recovery was previously demonstrated in acid-loaded macrophages (Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1988) J. Biol. Chem. 263, 19558-19563). Acid extrusion was found to be ATP-dependent and sensitive to N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide, suggesting involvement of an H(+)-pumping ATPase. In this report, the properties and mode of activation of this putative pump were studied in detail. In acid-loaded cells, pHi recovery, measured using a fluorescent probe, was found to be insensitive to azide or oligomycin, which are inhibitors of F0F1 (mitochondrial) H(+)-ATPases, and to vanadate, an inhibitor of E1E2-type ATPases. Instead, the recovery was sensitive to the vacuolar type H(+)-ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, p-chloromercuribenzenesulfonic acid, and bafilomycin A1. Using the fluorescent probes bisoxonol and 3,3'-dipropylthiodicarbocyanide iodide to measure the membrane potential of intact cells, acid loading of macrophages was shown to result in an N,N'-dicyclohexylcarbodiimide-sensitive hyperpolarization of approximately 15 mV. This hyperpolarization was not inhibited by charybdotoxin, suggesting that it was not due to efflux of K+ through Ca2(+)-activated K+ channels, but may instead be due to electrogenic pumping of protons across the plasma membrane. This was consistent with the partial dependence of the Na(+)- and HCO3(-)-independent pHi recovery on the presence of intracellular Cl-. As in vacuolar membranes, Cl- appears to act as a counterion to H+, preserving electroneutrality and thus facilitating pHi recovery. In acid-loaded urinary epithelial cells, activation of H+ pumping occurs by exocytic insertion of intracellular (vacuolar) H(+)-ATPases into the plasma membrane. In this system, exocytosis is triggered by an associated increase in the cytoplasmic free Ca2+ concentration and is microtubule-dependent. We determined whether an analogous process exists in macrophages. Acid loading of macrophages induced an approximately 120 nM increase in cytoplasmic free Ca2+ concentration due to mobilization of Ca2+ from an intracellular source. However, preventing this increase by preloading macrophages with bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid did not inhibit the Na+ and HCO3(-)-independent pHi recovery, neither was the recovery inhibited by microtubular disruption using 0.1 mM colchicine. Furthermore, cytoplasmic acid loading did not cause a detectable release of secretory granular, endosomal, or lysosomal contents, suggesting that activation of H+ pumping at the cell surface is not mediated by exocytic fusion of these compartments with the plasma membrane. Taken together, these data suggest that H(+)-ATPases are constitutively present in the macrophage plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages.

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.

Amiloride↗
PubMed1990

Hepoxilin A3 induces changes in cytosolic calcium, intracellular pH and membrane potential in human neutrophils.

The effects of hepoxilin A3 (HxA3), a 12-lipoxygenase metabolite of arachidonic acid, on cytosolic calcium ([Ca2+]i), intracellular pH (pHi), transmembrane potential and right-angle light scattering in human neutrophils were investigated. A rapid, transient elevation of [Ca2+]i was observed with HxA3 which was dependent on the concentration used. The effect of HxA3 on [Ca2+]i was blocked by pertussis toxin, suggesting involvement of receptors coupled to GTP-binding proteins. Experiments in Ca2(+)-free medium and using intracellular Ca2+ chelators indicated that HxA3 mobilized Ca2+ from intracellular stores. At similar concentrations, HxA3 altered pHi, producing an initial acidification followed by an alkalinization. The initial acidification was decreased in cells loaded with a Ca2+ chelator. In the presence of N-ethyl-N-(1-methylethyl)amino amiloride, an inhibitor of the Na+/H+ antiport, HxA3 induced a greater acidification but failed to elicit the recovery phase, suggesting that the latter is due to activation of the antiport. HxA3 also depolarized the membrane potential, although this effect was small. A decrease in right-angle light scattering, qualitatively similar to that observed with chemotactic peptides, was seen with HxA3, indicating that the 12-lipoxygenase metabolite can induce shape changes in neutrophils. At the concentrations used for the above effects, HxA3 was unable to generate a respiratory burst. These findings suggest that hepoxilins, which are formed by stimulated neutrophils, may have a role as messengers in neutrophil activation.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Vanadate stimulates oxygen consumption and tyrosine phosphorylation in electropermeabilized human neutrophils.

To determine the role of protein phosphorylation in neutrophil activation, electropermeabilized cells were treated with vanadate, a phosphatase inhibitor. Micromolar concentrations of vanadate elicited a NADPH-dependent burst of oxygen utilization in permeabilized, but not in intact cells, indicating an intracellular site of action. Stimulation of oxygen consumption by vanadate was reversible, concentration dependent and required the presence of ATP and Mg2+. Generation of a respiratory burst by vanadate was associated with accumulation of phosphorylated proteins. Such accumulation was due, at least in part, to inhibition of phosphoprotein phosphatase activity, as indicated by pulse-chase experiments. No evidence for stimulation of protein kinases by vanadate was found. Phosphoamino acid analysis revealed that a large fraction of the vanadate-induced phosphorylation occurred on tyrosine residues. The pronounced accumulation of tyrosine-phosphorylated proteins was confirmed by immunoblotting with anti-phosphotyrosine antibodies. The data suggest that neutrophils possess one or more constitutively active tyrosine kinases and that phosphoprotein accumulation is normally prevented by vigorous concomitant phosphatase activity. Inhibition of the latter by vanadate leads to phosphoprotein accumulation and is accompanied by stimulation of oxygen consumption.

Adenosine Triphosphate↗
PubMed1990

Serotyping of human rotaviruses in Argentina by ELISA with monoclonal antibodies.

Human rotaviruses are the major, recognized cause of infantile diarrhea worldwide. Characterization of naturally occurring human isolates indicates that there are six human rotavirus serotypes, four of which (serotype 1 to 4) are widespread. We utilized monoclonal antibodies specific for the VP of serotypes 1, 2, 3, and 4 as capture antibody in a sandwich enzyme-linked immunosorbent to serotype rotaviruses directly in stool samples. The stool samples were collected from 1983 through 1986, from two epidemiologic studies in the area of Buenos Aires, Argentina. All four serotypes assayed were found Serotype 2 and 3 viruses, which were detected most frequently in 1983 and 1984, were virtually undetected in 1985 and 1986 (chi 2 = 23, P = less than 0.001 for this difference). No significant difference was noted among the three collection sites for serotype prevalence. These results indicate that the changing predominance of rotavirus serotype in a given region can involve multiple serotypes at the same time. Analysis of an outbreak of diarrhea in two neighboring families which occurred during a prospective study of community diarrhea documented inter- and intra-family spread of one serotype of virus.

Antibodies, Monoclonal↗
PubMed1990

Effect of cytoplasmic acidification on the membrane potential of T-lymphocytes: role of trace metals.

The effect of lowering intracellular pH on the membrane potential (Em) of rat thymic lymphocytes was studied using the potential-sensitive dye bis-oxonol. Cells were acid loaded by addition of the electroneutral K+/H+ exchanging ionophore nigericin. Acidification to pH 6.3 in Na(+)-free solution resulted in a biphasic change in Em: an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca2+ ([Ca2+]i). The hyperpolarization was eliminated when the change in [Ca2+]i was prevented using BAPTA, an intracellular Ca2+ chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca2+]i at physiological pHi using ionomycin, suggesting involvement of Ca2(+)-activated K+ channels. In contrast, the depolarization phase could not be mimicked by raising [Ca2+]i with ionomycin. However, intracellular BAPTA effectively inhibited the acidification-induced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca2+ concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes in Em. Accordingly, similar changes in Em can be induced without acidification by the addition of small amounts of Cu2+ to the medium. The ionic basis of the Em changes induced by acidification and the significance of these observations are discussed.

Animals↗
PubMed1990

Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera.

Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.

Acute Disease↗
PubMed1990

Actin assembly in electropermeabilized neutrophils: role of intracellular calcium.

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.

Actins↗
PubMed1990

Calcium-independent cell volume regulation in human lymphocytes. Inhibition by charybdotoxin.

The properties of the K+ pathway underlying regulatory volume decrease (RVD) in human blood lymphocytes were investigated. Evidence is presented for the existence of three types of K+ conductance in these cells. Ionomycin, a Ca2+ ionophore, induced a K(+)-dependent hyperpolarization, indicating the presence of Ca2(+)-activated K+ channels, which were blocked by charybdotoxin (CTX). CTX also induced a depolarization of the resting membrane potential, even at subphysiological cytosolic [Ca2+]([Ca2+]i), which suggests the existence of a second CTX-sensitive, but Ca2(+)-independent conductance. A CTX-resistant K+ conductance was also detected. RVD in blood lymphocytes was partially (approximately 75%) blocked by CTX. However, volume regulation was not accompanied by detectable changes in [Ca2+]i, nor was it prevented by removal of extracellular Ca2+ and depletion or buffering of intracellular Ca2+. These observations suggest that K+ loss during RVD is mediated by Ca2(+)-independent, CTX-sensitive channels or that Ca2(+)-dependent channels can be activated by cell swelling at normal or subnormal [Ca2+]i. The former interpretation is supported by findings in rat thymic lymphocytes. These cells also displayed a CTX-sensitive Ca2(+)-dependent hyperpolarization. However, CTX did not significantly alter the resting potential, suggesting the absence of functional Ca2(+)-independent, toxin-sensitive channels. Volume regulation in thymic lymphocytes was less efficient than in human blood cells. In contrast to blood lymphocytes, RVD in thymocytes was not affected by CTX. These observations indicate that, though present in lymphocytes, Ca2(+)-activated K+ channels do not play an important role in volume regulation. Instead, RVD seems to be mediated by Ca2(+)-independent K+ channels. We propose that two types of channels, one CTX sensitive and the other CTX insensitive, mediate RVD in human blood lymphocytes, whereas only the latter type is involved in rat thymocytes.

Calcium↗
PubMed1990

Determinants of the transmembrane distribution of chloride in rat lymphocytes: role of Cl(-)-HCO3- exchange.

The purpose of the present experiments was to establish the factors that determine the intracellular Cl- concentration ([Cl-]i) of lymphocytes. Coulometric and isotope equilibration determinations indicated that [Cl-]i was in the range of 70-85 mmol/l cells. Since the membrane potential (Em) of these cells approximates -55 mV, [Cl-]i is severalfold higher than the level expected at electrochemical equilibrium (approximately 16 mM). This suggests that conductive pathways contribute marginally to the distribution of Cl-. Accordingly, altering the force driving conductive Cl- fluxes by manipulating Em had little effect on [Cl-]i. The possible role of electroneutral cation-Cl- cotransport in the accumulation of intracellular Cl- was also assessed. 36Cl- uptake was largely unaffected by omission of extracellular Na+ and K+ or by addition of bumetanide, a potent cotransport inhibitor. Moreover, [Cl-]i remained unaltered for at least 1 h in cells incubated without Na+ or K+ or in the presence of loop diuretics. Thus it appears unlikely that Cl(-)-anion cotransport plays a major role in maintaining [Cl-]i. A vigorous stilbene disulfonate-sensitive anion exchanger was detected in thymocytes. This system constitutes a large fraction of the Cl- flux pathways and is possibly a major contributor to the establishment of [Cl-]i. Accordingly, modifying the force driving Cl- through the exchanger, by altering pH at constant PCO2, resulted in changes in cellular Cl- content and associated changes in cell volume. These effects were markedly reduced in the nominal absence of HCO3- or in the presence of disulfonic stilbene derivatives, suggesting that they are mediated by Cl(-)-HCO3- exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Modulation of the macrophage respiratory burst by an acidic environment: the critical role of cytoplasmic pH regulation by proton extrusion pumps.

Within the acidic milieu of an abscess or tumor, macrophages must be able to maintain their cytoplasmic pH (pHi) close to the physiologic range to ensure optimal cell function. Our recent studies have demonstrated that a proton-extrusion mechanism with the characteristics of an H+ adenosine triphosphatase mediates pHi recovery in acid-loaded macrophages. These studies were designed to examine the role of these H+ pumps in maintaining cell function in an acidic extracellular environment. Peritoneal macrophages were tested for superoxide production in response to phorbol myristate acetate at either physiologic or acidic extracellular pH (pHo; 7.35 or 6.70, respectively). pHi was measured with the fluorescent dye 2',7'-biscarboxy-ethyl-5(6)-carboxy-fluorescein. Bafilomycin A1, a specific H+ adenosine triphosphatase inhibitor, was used to examine the contribution of the H+ pump to pHi regulation and cell function. At pHo 7.35, bafilomycin A1 had no effect on pHi or phorbol myristate acetate-stimulated superoxide production. However, at pHo 6.70, bafilomycin A1 reduced pHi to 6.61 +/- 0.01 versus 6.79 +/- 0.01 in control cells (p less than 0.001) and caused a concomitant reduction in superoxide production to 4.8 +/- 1.2 versus 13.0 +/- 1.2 nmol/10(6) cells/40 min in control cells (p less than 0.001). To determine whether the observed reduction in superoxide formation was the result of the pHi reduction, superoxide production was measured in cells whose pHi was pharmacologically clamped at various levels according to the K+/nigericin method. Lowering pHi from 6.80 to 6.60 caused a significant reduction in superoxide production from 13.1 +/- 1.8 to 7.5 +/- 0.9 nmol/10(6) cells/40 min (p less than 0.01). Thus H+ extrusion pumps are important to maintenance of macrophage pHi at low pHo, permitting continued superoxide production under these conditions. By keeping pHi close to the physiologic range, these pumps serve to optimize cell function in an acidic extracellular environment.

Acids↗