Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils (original) (raw)
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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...
Journal of Leukocyte Biology, 2003
A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca 2؉ -dependent signaling. The lyso-PC mix (0.45-14.5 M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8-to 15.7-fold and 1.7-to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca 2؉ . Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca 2؉ functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca 2؉ chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca 2؉ -dependent manner that involves the activation of a pertussis toxin-sensitive G-protein. J. Leukoc. Biol. 73: 511-524; 2003.
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.
Extracellular Ca2+ regulates the respiratory burst of human neutrophils
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998
The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca 2-ATPase inhibitor, thapsigargin, was used to activate higher Ca 2 influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca 2 entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca 2 influx under normal physiological conditions. The higher Ca 2 influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra-and extracellular Ca 2 are required for full activation of the respiratory burst of human neutrophils, and the Ca 2 influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.
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].
Ca2+ influx shutdown in neutrophils induced by Fas (CD95) cross-linking
Immunology, 2004
In neutrophils, as in most other cell types, Ca 2+ + signalling is important for a number of cellular activities. Although inositol(1,4,5)trisphosphate-mediated release of Ca 2+ + from intracellular stores is a necessary prelude, it is the Ca 2+ + influx that is responsible for many of the neutrophil responses. We report here that although elevations of cytosolic Ca 2+ + do not accompany Fasmediated apoptosis in neutrophils, the Ca 2+ + influx component of the response to N-formylmethionyl-leucyl-phenylalanine (FMLP) becomes selectively inactived as the neutrophils progress towards accelerated apoptosis induced by Fas (CD95) cross-linking. After 4 hr incubation at 37°, untreated neutrophils display an exaggerated Ca 2+ + influx phase in response to FMLP. This was absent in neutrophils that had been Fas-activated at the same time. No Ca 2+ + influx component was demonstrable by the removal of extracellular Ca 2+ + or by Ca 2+ + channel blockade with Ni 2+ + and no Mn 2+ + influx was detectable. The defect could not be attributed to a decrease in receptor sensitivity, receptor coupling or receptor number because the release of stored Ca 2+ + remained constant during incubation and was unaffected by Fas activation. Ca 2+ + influx became uncoupled from store release before detectable gross morphological changes or phosphatidyl serine externalization and was also insensitive to caspase 3 and 8 inhibitors. These results suggest a mechanism other than caspase-mediated proteolytic damage to components important for Ca 2+ + influx.
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.
Calcium Pathways in Human Neutrophils—The Extended Effects of Thapsigargin and ML-9
Cells, 2018
In neutrophils, intracellular Ca2+ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca2+-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca2+ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca2+ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due...