Phospholipase C and phosphatidylinositol 3-kinase signaling are involved in the exogenous arachidonic acid-stimulated respiratory burst in human neutrophils (original) (raw)
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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.
FEBS Letters, 1985
The relationships between the changes of cellular Ca 2+ the activation of phosphoinositide turnover and , the functional responses induced by the stimulus-receptor interactions in neutrophils are matter of controversy. By measuring the concentration dependency of different formyl-leucyl-methionyl-phenylalanine (FMLP)-induced changes, the following values of ED,, were found: 1.6 and 0.8 nM for the rise in [Ca2+], monitored with Quin-2, in the presence and absence of exogenous Ca2+, respectively; 20 nM for the activation of phosphoinositide metabolism, monitored as change in the 32P, of phosphatidate; 14 nM for membrane-bound Ca2+ mobilization, monitored with chlorotetracycline (CTC); 34 nM for %a2+ influx and 32 nM for the respiratory burst. Furthermore, low dose of FMLP causes an increase in [Ca"], in absence of activation of breakdown of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phospha-tidylinositol4,5-biphosphate monitored as changes in [3H]glycerol radioactivity. The results clearly demonstrate that the increase in [Ca2+],, due to the release from intracellular stores, is not caused by the breakdown of phosphatidylinositides.
Journal of Allergy and Clinical Immunology, 1998
Human basophils secrete leukotriene C4 (LTC4) in response to various stimuli, and a short treatment with IL-3 enhances LTC4 release, although IL-3 alone does not induce LTC4 release. However, the mechanism of this priming effect of IL-3 for LTC4 generation remains unknown in human basophils. This study was designed to explore the mechanisms by which short treatments with IL-3 enhance stimulated secretion of LTC4, with a focus on the activation of cytosolic phospholipase A2 (cPLA2). The phosphorylation state of cPLA2 in human basophils was examined by its shift in electrophoretic mobility as detected by Western blotting. Free arachidonic acid (AA) and LTC4 were measured by gas chromatography-negative ion chemical ionization mass spectrometry and LTC4-specific RIA, respectively. Human basophils expressed cPLA2. IL-3, as well as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, caused a shift in the electrophoretic mobility of cPLA2, which indicated phosphorylation of cPLA2 and therefore its activation. Ionomycin at a concentration of 0.1 microg/mL was used to induce a modest elevation of cytosolic calcium response ([Ca2+]I), no apparent cPLA2 phosphorylation, and little free AA and LTC4 generation. Pretreatment with IL-3 (1 to 10 ng/mL) markedly enhanced ionomycin (0.1 microg/mL)-mediated AA and LTC4 generation. The concentration dependence of cPLA2 phosphorylation by IL-3 and its effects on free AA and LTC4 generation were similar. The selective PKC inhibitors, bis-indolylmaleimide II and Ro-31-8220 inhibited the phorbol 12-myristate 13-acetate-mediated cPLA2 electrophoretic mobility shift, but not the IL-3-mediated shift, suggesting that the IL-3 effect is PKC independent. Both the anaphylatoxin split product of the C component C5 (C5a) and f-Met-Leu-Phe induced PKC-independent cPLA2 phosphorylation with a similar time course most notable for the absence of observable changes in cPLA2 phosphorylation before 30 seconds. These results suggested an explanation for the absence of free AA generation by C5a. When [Ca2+]I was elevated in response to C5a, there was no phosphorylation of cPLA2, and by the time cPLA2 became phosphorylated, [Ca2+]I had returned to resting levels. Treatment with IL-3 preconditioned the cPLA2 by causing its phosphorylation so that the transient [Ca2+]I response, which followed stimulation by C5a, could induce the generation of free AA and LTC4. Taken together, these results suggest that the effect of IL-3 for free AA generation and LTC4 release might be due to induction of cPLA2 phosphorylation. The studies demonstrated a need for synchronous cPLA2 phosphorylation and elevations in [Ca2+]I.
Cellular Signalling, 1993
In this paper it has been shown that increase in intracellular cAMP by epinephrine or its analogue dibutyryl cAMP (Bt2cAMP) abolishes in a dose-dependent manner the protein kinase C (PKC)-mediated respiratory burst in polymorphonuclear leukocytes. The mechanism of inhibition has been shown to involve induction of cytosolic phosphoprotein phosphatase activity specific to cells receiving dual signals (PKC, PKA), as minimum respiratory burst was associated with cells with maximum phosphatase activity. Inclusion of specific PKA inhibitor completely restricted the development of'dual signal-induced phosphatase activity in vitro, demonstrating the requirement of multisite phosphorylation of the phosphatase for the development of its activity. Purified phosphatase had a molecular weight of 78,000 and could exert its inhibitory effect on PKC-triggered respiratory burst in permeabilized cells, clearly showing that down-regulation of oxidase activity involved dephosphorylation by the phosphatase. Interaction of the purified phosphatase with eightfold purified NADPH oxidase as revealed by fluorescence studies further confirmed the role of the phosphatase in the respiratory burst event. Taken together, we have been able to establish that cross-talk between protein kinase C and protein kinase A is essential to 'turn olV generation of reactive oxygen species.
Roles of phospholipase D in phorbol myristate acetate-stimulated neutrophil respiratory burst
Journal of Cellular and Molecular Medicine, 2010
The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10-fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA-stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA-stimulated respiratory burst. Using 1-butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA-stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA-stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA-stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA-stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2-dioctanoyl-sn-glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA-stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA-stimulated respiratory burst.