Ca2+ influx shutdown in neutrophils induced by Fas (CD95) cross-linking (original) (raw)
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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].
Near membrane Ca2+ changes resulting from store release in neutrophils: detection by FFP-18
Cell Calcium, 1996
FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca2+ changes resulting from the release of Ca2+ from intracellular stores. Near membrane and cytosolic Ca2+ changes were measured under conditions in which store release and Ca2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca2+ changes near the plasma membrane and bulk cytosolic Ca2+ concentration, which were consistent with a Ca2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca2+ storage locations in neutrophils. tNTRODUCTtON
PAF-mediated Ca2+ influx in human neutrophils occurs via store-operated mechanisms
Journal of Leukocyte Biology
Many inflammatory mediators activate neutrophils (PMN) partly by increasing cytosolic calcium concentration ([Ca2+]i). Modulation of PMN [Ca2+]i might therefore be useful in regulating inflammation after shock or sepsis. The hemodynamic effects of traditional Ca2+ channel blockade, however, could endanger unstable patients. Store-operated calcium influx (SOCI) is known now to contribute to Ca2+ flux in “nonexcitable” cells. Therefore, we studied the role of SOCI in human PMN responses to the proinflammatory ligand PAF. PMN [Ca2+]i was studied by spectrofluorometry with and without external calcium. We studied the effects of PAF on Mn2+ entry into and on Ca2+ efflux from thapsigargin (Tg)-treated cells. Influx was assessed in the presence and absence of the blockers SKF-96365 (SKF), TMB-8, and 2-APB. Half of PAF [Ca2+]i mobilization occurs via calcium influx. The kinetics of calcium entry were typical of SOCI rather than receptor-mediated calcium entry (RMCE). SKF had multiple nonspe...
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.
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...
Two distinct Ca2+ storage and release sites in human neutrophils
Journal of leukocyte biology, 1998
It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor cross-linking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores. The release sites also correlated with organelles that stained with DiOC6(3...
The Journal of Immunology, 2002
Human polymorphonuclear neutrophil (PMN) responses to G protein-coupled chemoattractants are highly dependent upon storeoperated Ca 2؉ entry (SOCE). Recent research suggests that SOCE currents can be mediated by a variety of related channel proteins of the transient receptor potential superfamily. SOCE has been regarded as a specific response to depletion of cell calcium stores. We hypothesized that net SOCE might reflect the contributions of more than one calcium entry pathway. SOCE was studied in normal human PMN using Ca 2؉ and Sr 2؉ ions. We found that PMN SOCE depends on at least two divalent cation influx pathways. One of these was nonspecific and Sr 2؉ permeable; the other was Ca 2؉ specific. The two pathways show different degrees of dependence on store depletion by thapsigargin and ionomycin, and differential sensitivity to inhibition by 2-aminoethyoxydiphenyl borane and gadolinium. The inflammatory G protein-coupled chemoattractants fMLP, platelet-activating factor, and IL-8 elicit unique patterns of Sr 2؉ and Ca 2؉ influx channel activation, and SOCE responses to these agonists displayed differing degrees of linkage to prior Ca 2؉ store depletion. The mechanisms of PMN SOCE responses to G protein-coupled chemoattractants are physiologically diverse. They appear to reflect Ca 2؉ transport through a variety of channels that are independently regulated to varying degrees by store depletion and by G protein-coupled receptor activation.
Science Signaling, 2015
cytosolic Ca 2+ is regulated by multiple proteins, including the plasma membrane Ca 2+ -ATPases (PMCAs), which use ATP to transport Ca 2+ out of cells. PMCA isoforms exhibit different kinetic and regulatory properties, thus the presence and relative abundance of individual isoforms may help shape Ca 2+ transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, PMCA2b) on Ca 2+ transients elicited by conditions that trigger storeoperated Ca 2+ entry (SOCE) and that blocked Ca 2+ uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced longlasting Ca 2+ oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundancesensitive clearance of SOCE-mediated Ca 2+ transients; whereas PMCA4a reduced cytosolic Ca 2+ resulting in the establishment of a higher than baseline cytosolic Ca 2+ concentration.
Biochemical Pharmacology, 2005
The current study was designed to probe Ca 2+ shuttling between intracellular stores and the cytosol as a potential mechanism contributing to the prolongation of elevated Ca 2+ transients in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Cytosolic Ca 2+ concentrations and transmembrane fluxes of the cation were measured using spectrofluorimetric and radiometric procedures, respectively, while inositol 1,4,5-triphosphate (IP 3 ) was measured using a radioreceptor assay. The Ca 2+chelating agent, ethylene glycol-bis (b-aminoethyl ether) N,N,N 0 N 0 -tetraacetic acid (EGTA; 10 mM), was used to exclude store-operated influx of Ca 2+ into neutrophils, while the IP 3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 mM), added to the cells 10 s after FMLP (0.01 and 1 mM), at which time the increases in IP 3 and cytosolic Ca 2+ were maximal, was used to eliminate both sustained release from stores and influx of Ca 2+ . Addition of FMLP at 0.01 or 1 mM resulted in equivalent peak increases in cytosolic Ca 2+ , while the increase in IP 3 was greater and the rate of clearance of Ca 2+ from the cytosol slower, in cells activated with 1 mM FMLP. Treatment of the cells with either EGTA or 2-APB following addition of 1 mM FMLP, completely (EGTA) or almost completely (2-APB) abolished the influx of Ca 2+ and accelerated the rate of clearance of the cation from the cytosol. Post-peak cytosolic Ca 2+ concentrations were lower, and the Ca 2+ content of the stores higher, in cells treated with 2-APB. The involvement of IP 3 was confirmed by similar findings in cells treated with U-73122 (1 mM), a selective inhibitor of phospholipase C. Taken together, these observations are compatible with IP 3 -mediated Ca 2+ shuttling in neutrophils activated with FMLP. #