Platelet activating factorstimulates intracellular calcium transients in human neutrophils: involvement of endogenous 5-lipoxygenase products (original) (raw)
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European Journal of Pharmacology, 1990
The inhibitory action of calcium channel blockers on platelet-activating factor (PAF)-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]PAF to neutrophils was studied. Verapamil and diltiazem inhibited PAF (1O-s-1O-s M)-induced degranulation and superoxide production in a dose-dependent manner, with PA, values of 5.6 and 6.1 for verapamil and 5.9 and 6.2 for diltiazem, respectively. Both channel blockers inhibited the specific binding of ['H]PAF to PMNL in dose-dependent fashion, with Ki values of 3.9 f 0.6 x lo-' RI and 3.2 f 0.4 x lo-' M for verapamil and diltiazem, respectively. Scatchard analysis of the binding data revealed that both calcium channel blockers decreased the receptor binding affinity and slightly increased the number of high-affinity PAF receptors. whereas they did not affect the binding affinity and number of low-affinity receptors. These results indicate that calcium channel blockers can inhibit neutrophil responses elicited by PAP by a mechanism other than inhibition of calcium influx and suggest that the PAF receptor may be closely associated with calcium channels. PAF (platelet activating factor, PAF-acether); Ca 2+ channel blockers; PAF receptors; Degranuiation; Superoxide production; Neutrophil granulocytes (human) An increase in intracellular calcium levels is a critical, early event in the activation of human polymorphonuclear leukocytes (PMNL) challenged with platelet-activating factor (1-Q-alkyl-2-0-acetyl-sn-glycero-3-phosphorylcholine) (O'Flaherty et al., 1981). Calcium influx may account for the bulk of the increase in intracellular calcium concentrations; thus PMNL activation can not be observed in the absence of extracellular calcium (O'Flaherty et al., 1981; Pennington et al., 1986
Biochemical Journal, 1995
The relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB4 synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca(2+)-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB4. The [Ca2+]i values for threshold and maximal arachidonic acid release were found to be 150 nM and 350 nM respectively, suggesting the involvement of cytosolic phosphol...
FEBS Letters, 1984
Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.
Biphasic increase in intracellular calcium induced by platelet-activating factor in macrophages
FEBS Letters, 1989
In single mouse macrophages stimulated by platelet-activating factor (PAF), the intracellular calcium concentration (Cai) monitored with fura-2 at room temperature presents a biphasic increase, including a transient and a more sustained component. After pulse administration of PAF, the first phase lasts for a few seconds and reaches a peak value of 0.5-1 /~M Ca z+ at high PAF concentration. The amplitude of this peak is independent of extracellular Ca z÷ concentration, suggesting that the initial Ca 2÷ transient is due to the release of Ca 2÷ from intracellular stores. The second phase of the response lasts for several minutes; its maximum amplitude is reached 1-2 min after the brief initial PAF stimulation. This phase, suppressed in zero external Ca 2÷ and increased in 10 mM Ca z+, is probably due to influx of Ca 2÷ through the plasma membrane. This secondary Ca 2÷ increase is blocked by 10-50/tM lanthanum. At low PAF concentration, the initial Ca z+ transient is not followed by a second phase, showing that the initial rises of Ca 2÷ and of its activator (presumably inositol trisphosphate) are not sufficient to trigger the second phase of Ca 2÷ increase.
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
Journal of Biological Chemistry, 2002
The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl--cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn 2؉ induced by fMet-Leu-Phe or thapsigargin. Methyl--cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10 ؊10 M platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10 ؊7 M platelet-activating factor. On the other hand, methyl--cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of Fc␥RIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl--cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.
Biochemical Journal, 1984
The role of Ca2+ in the activation of the enzyme lyso-(platelet-activating factor): acetyl-CoA acetyltransferase was studied in rat peritoneal macrophages in response to complement-coated zymosan particles and ionophore A23187. By using Ca2+-containing buffers, a threshold concentration of extracellular Ca2+ above 1 microM was found to be necessary to observe the activation of the enzyme in response to zymosan. By contrast, a significant role of intracellular Ca2+ in this process could be ruled out, since the putative intracellular calcium-transport antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] did not inhibit the activation of the acetyltransferase induced by zymosan in the presence of extracellular Ca+. The link between acetyltransferase activation and extracellular Ca2+ transport was studied by measuring Ca2+ uptake in response to the stimuli. Zymosan particles induced a rapid increment in cell-associated Ca2+ which correlated well with the extent of acetyl...
Cell Calcium, 1984
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, PAF) is a potent inducer of shape-change, aggregation and secretion in platelets. PAF causes a rapid increase in intracellular calcium, but has no calcium gating effect in intact lipid bilayers. Human red cells (RBC) did not metabolize either PAF or PAF-phosphatidate (PAF-PA). While PAF (10 PM) was devoid of calcium ionophoretic activity, PAF-PA (l-5 uM) stimulated calcium influx into intact human RBC. In addition, PAF-PA (l-10 PM), but not PAF (10 PM), elicited a series of satellite effects related to the rise of intracellular calcium: 1) increased efflux of intracellular potassium (G&-dos effect); 2) alkalinization of unbuffered RBC suspensions; 3) stimulation of ATP consumption and production, and enhancement of glycolytic flux with crossover at the glyceraldehyde 3-phosphate dehydrogenase step. These effects exactly duplicate those brought about by the calcium ionophore A23187. The ionophoretic potency of PAF-PA was about half that of A23187. Approximately the same concentrations of PAF-PA as those that stimulate calcium influx into RBC elicit full aggregatory response in human platelets. It is possible that transformation of PAF into PAF-PA by the combined action of phospholipase C and diacylglycerol kinase contributes to the increase of calcium influx in platelets.