N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils (original) (raw)
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Altered Ca2+ signalling in human neutrophils from inflammatory sites
Annals of the Rheumatic Diseases, 1994
Objectives-To determine whether the intracellular store release of Ca2" in neutrophils from patients with rheumatoid arthritis, other joint disease and active leg ulceration was different from normal neutrophils. Methods-The release into the cytosol of Ca2" from stores within individual neutrophils was determined using ratiometric imaging of fura2. The size of the elevated Ca2" 'cloud' and its concentration were quantified in neutrophils from the circulation of patients with rheumatoid arthritis, other joint diseases, and leg ulcers and from the joints of those with joint disease. Results-In neutrophils isolated from both the synovial fluid of patients with rheumatoid arthritis and other joint conditions, and also arising from leg ulcers, the amount of the cell cytosol occupied by elevated Ca21 was significantly increased compared with neutrophils from healthy subjects; for neutrophils from rheumatoid, non-rheumatoid joints and leg ulcers p values were 0-0006, <0-0001, 0-016 respectively (Student's t test). There was also a significant increase in Ca2' release from circulating neutrophils from patients with rheumatoid arthritis (p = 0.09), but not in circulating neutrophils from patients with leg ulcers or nonrheumatoid joint conditions. Conclusions-It is proposed that the increased release of free Ca2" into the
Biochimica et Biophysica Acta (BBA) - General Subjects, 1981
The relative importance of intracellular and extracellular calcium in the stimulation of human neutrophils was studied by use of chelators and calcium antagonists. In the first series of experiments, EGTA was used to see if extracellular calcium was an absolute requirement for neutrophil activation. Stimulated neutrophils secreted lysosomal enzymes and generated superoxide anion radicals in the presence or absence of 5 mM EGTA or Mg-EGTA. Seven different stimuli were employed: the chemotactic peptide N-formylmethionylleucylphenylalanine (10 -7 M), calcium ionophore A23187 (10 -s M), concanavaiin A (30/.tg/ml), serum-treated zymosan (2 mg/ml), zymo. san-treated serum (10%), an immune complex (150/.tg/ml) and phorbol myristate acetate (1 gg/ml). Release of the lysosomal enzymes/~-glucuronidase and lysozyme and generation of superoxide anions in response to these stimuli were readily demonstrable in the presence of EGTA. Each response of neutrophils was variably, but not completely, reduced in the presence of the chelator; EGTA did not completely block the responses elicited by any of the stimuli. Consequently, it is unlikely that an influx of divalent cations from the extracellular medium is obligatory for stimulus-response coupling in human neutrophils. The role of intracellular calcium was then studied using calcium antagonists. Inhibition of neutrophil responses was readily obtained using TMB-8, an antagonist of the mobilization of intracellular calcium, and trifluoperazine and W-7, inhibitors of calmodulin. These results suggest that intracellular, but not extracellular, calcium plays an obligatory role in the responses of neutrophils to a variety of soluble and insoluble stimuli. 0304-4165[81[0000-0000[$02.50
Biochemical Society Transactions, 1994
Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca2+-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B released different amounts of calcium from the stores and induced Ca2`(Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca2+-free medium, but returned to basal levels in cells incubated in Ca2+-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2`(Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].
Proceedings of the National Academy of Sciences, 1984
The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximat...
Biochimica Et Biophysica Acta-molecular Cell Research, 2009
Extracellular agonists increase the cytosolic free Ca 2+ concentration ([Ca 2+ ] c ) by Ca 2+ influx and by stimulating Ca 2+ release from intracellular stores, mainly the endoplasmic reticulum and to a lesser extent also later compartments of the secretory pathway, particularly the Golgi. The Golgi takes up Ca 2+ via Sarco/Endoplasmic Reticulum Ca 2+ ATPases (SERCAs) and the Secretory-Pathway Ca 2+ ATPases (SPCAs). The endogenous expression of SERCAs and SPCAs neutrophils was demonstrated by Western blotting and immunocytochemistry. Up till now, all cytosolic Ca 2+ transients due to intracellular Ca 2+ release have been found to originate from SERCA-dependent stores. We found that human neutrophils also present Ca 2+ release from a SERCA-independent store. Changes in [Ca 2+ ] c of neutrophils were investigated during chemokinesis induced by chemotactic factors in Ca 2+ -free solution with and without the SERCA-specific inhibitor thapsigargin. Using N-formyl-methionyl-leucyl-phenylalanine or interleukin-8 as agonists, Ca 2+ release from intracellular stores was observed in respectively about 40% and 20% of the neutrophils pre-treated with Ca 2+free solution and thapsigargin. In the latter condition, 20-30% of the cells preserved migratory behaviour. These results indicate that both SERCA-dependent and SERCA-independent (presumably SPCA-dependent) intracellular Ca 2+ stores contribute to Ca 2+ signaling during chemokinesis of human neutrophil granulocytes.
Na+/Ca2+ exchange-mediated calcium entry in human lymphocytes
Journal of Clinical Investigation, 1994
Regulation of cytosolic Ca2+ and cytosolic Na+ is critical for lymphocyte cation homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura-2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a potent inhibitor of store-dependent calcium influx pathways. These results show lymphocytes demonstrate Na+/Ca2+ exchange activity and suggest the Na+/Ca2+ exchanger modulates cytosolic Ca2+ following intracellular Ca2+ store depletion.
Free Radical Biology and Medicine, 2007
Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2 O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane o op pe en nU UP P ((J Ju un ne e 2 20 00 07 7)) potential and influx of Ca 2+. Treatment of the cells with H 2 O 2 , and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.
Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production
The FASEB Journal
Recently, it was demonstrated that liver injury and TNF-␣ production as a result of endotoxin (lipopolysaccharide, LPS) were attenuated by feeding animals a diet enriched with glycine. This phenomenon was shown to be a result of, at least in part, activation of a chloride channel in Kupffer cells by glycine, which hyperpolarizes the cell membrane and blunts increases in intracellular calcium concentrations ([Ca 2؉ ] i) similar to its action in the neuron. It is well known that hepatotoxicity due to LPS has a neutrophil-mediated component and that activation of neutrophils is dependent on increases in [Ca 2؉ ] i. Therefore, the purpose of this study was to determine if glycine affected agonistinduced increases in [Ca 2؉ ] i in rat neutrophils. The effect of glycine on increases in [Ca 2؉ ] i elicited either by the bacterial-derived peptide formyl-methionine-leucine-phenylalanine (FMLP) or LPS was studied in individual neutrophils using Fura-2 and fluorescence microscopy. Both FMLP and LPS caused dose-dependent increases in [Ca 2؉ ] i , which were maximal at 1 M FMLP and 100 g/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in [Ca 2؉ ] i in a dosedependent manner with an IC 50 of ϳ0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist-induced increases in [Ca 2؉ ] i in chloride-free buffer. Moreover, strychnine (1 M), an antagonist of the glycine-gated chloride channel in the central nervous system, reversed the effects of glycine (1 mM) on FMLP-or LPS-stimulated increases in [Ca 2؉ ] i. To provide hard evidence for a glycine-gated chloride channel in the neutrophil, the effect of glycine on radioactive chloride uptake was determined. Glycine caused a dosedependent increase in chloride uptake into neutrophils with an ED 50 of ϳ0.4 mM, an effect also prevented by 1 M strychnine. Glycine also significantly reduced the production of superoxide anion from FMLP-stimulated neutrophils. Taken together, these data provide clear evidence that neutrophils contain a glycine-gated chloride channel that can attenuate increases in [Ca 2؉ ] i and diminish oxidant production by this important leukocyte.
A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes
Biochimica Et Biophysica Acta-biomembranes, 1979
An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.