fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.     

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.     

Role of Ca2+-independent phospholipase A2 on arachidonic acid release induced by reactive oxygen species

Previous studies have shown that reactive oxygen species (ROS) enhance arachidonic acid (AA) release and the subsequent AA metabolism in macrophages. The purpose of this study was determined the implication of phospholipases A2 (PLA2s) in these events. Our results show that oxidative stress induced by exogenous adding of hydrogen peroxide or superoxide anion in macrophage RAW 264.7 and mouse peritoneal macrophage cultures caused a marked enhancement of calcium-independent PLA2 (iPLA2) activity,whereas the increment of secreted PLA2 (sPLA2) and calcium-dependent cytosolic PLA2 (cPLA2) activities were slight. This increase of iPLA2 activity by ROS was rapid and dose-dependent. ROS also induced a significant [3H] arachidonic acid (AA) release. The iPLA2 selective inhibitor, bromoenol lactone, almost completely suppressed the mobilization of [3H]AA induced by ROS whereas antisense oligonucleotide against cPLA2 did not have any appreciable effect. Thus, our data show that iPLA2 activity is involved in the mechanism by which ROS increases the availability of free AA in macrophages RAW 264.7. Moreover, the protein kinase C (PKC) inhibitor, calphostin C, and calcium chelators had no effect on the [3H]AA release induced by ROS, suggesting this is a regulatory role of iPLA2.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Phospolipase A2 and apoptosis

Phospolipase A(2) (PLA(2)) is the esterase activity that cleaves the sn-2 ester bond in glycerophospholipids, releasing free fatty acids and lysophospholipids. The PLA(2) activity is found in a variety of enzymes which can be divided in several types based on their Ca(2+) dependence for their activity; Ca(2+)-dependent secretory phosholipases (sPLA(2)s) and cytosolic phospholipases (cPLA(2)s), and Ca(2+)-independent phospholipase A(2)s (iPLA(2)s). These enzymes also show diverse size and substrate specificity (i.e., in the fatty acid chain length and extent of saturation). Among the fatty acids released by PLA(2), arachidonic acid (AA) is of particular biological importance, because it is subsequently converted to prostanoids and leukotrienes by cyclooxygenases (COX) and lipoxygenases (LOX), respectively. Free AA may also stimulate apoptosis through activation of sphingomyelinase. Alternatively, it is suggested that oxidized metabolites generated from AA by LOX induce apoptosis. Although the precise mechanisms remain to be elucidated, changes are observed in glycerolipid metabolism during apoptotic processes. In some cells induced to undergo apoptosis, AA is released concomitant with loss of cell viability, caspase activation and DNA fragmentation. Such AA releases appear to be mediated by activation of cPLA(2) and/or iPLA(2). For example, tumor necrosis factor-alpha (TNF-alpha)-induced cell death is mediated by cPLA(2), whereas Fas-induced apoptosis appears to be mediated by iPLA(2). Some discrepancies among early experimental results were probably caused by differences in the experimental conditions such as the serum concentration, inhibitors used that are not necessarily specific to a single-type enzyme, or differential expression of each PLA(2) in cells employed in the experiments. Recent studies eliminated such problems, by carefully defining the experimental conditions, and using multiple inhibitors that show different specificities. Accordingly, more convincing data are available that demonstrate involvement of some PLA(2)s in the apoptotic processes. In addition to cPLA(2) and iPLA(2), sPLA(2)s were recently found to play roles in apoptosis. Moreover, new proteins that appear to control PLA(2)s are being discovered. Here, the roles of PLA(2)s in apoptosis are discussed by reviewing recent reports.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells

Previous studies have demonstrated that U937 cells are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in response to receptor-directed and soluble stimuli by a mechanism that involves the activation of Group IV cytosolic phospholipase A(2)alpha. In this paper we show that these cells also mobilize AA in response to an oxidative stress induced by H(2)O(2) through a mechanism that appears not to be mediated by cytosolic phospholipase A(2)alpha but by the calcium-independent Group VI phospholipase A(2) (iPLA(2)). This is supported by the following lines of evidence: (i) the response is essentially calcium-independent, (ii) it is inhibited by bromoenol lactone, and (iii) it is inhibited by an iPLA(2) antisense oligonucleotide. Enzyme assays conducted under a variety of conditions reveal that the specific activity of the iPLA(2) does not change as a result of H(2)O(2) exposure, which argues against the activation of a specific signaling cascade ending in the iPLA(2). Rather, the oxidant acts to perturb membrane homeostasis in a way that the enzyme susceptibility/accessibility to its substrate increases, and this results in altered fatty acid release. In support of this view, not only AA, but also other fatty acids, were found to be liberated in an iPLA(2)-dependent manner in the H(2)O(2)-treated cells. Collectively, these studies underscore the importance of the iPLA(2) in modulating homeostatic fatty acid deacylation reactions and document a potentially important route under pathophysiological conditions for increasing free fatty acid levels during oxidative stress.     

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.     

Role of iPLA(2) in the regulation of Src trafficking and microglia chemotaxis

Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca(2+) -independent phospholipase A(2) (iPLA(2)) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA(2) by 4-bromoenol lactone (BEL) or iPLA(2) knockdown exerted a significant inhibition on phosphatidylinositol-3-kinase (PI3K) activation and chemotaxis. Further examination revealed that iPLA(2) knockdown abrogated Src activation, which is required for PI3K activation and chemotaxis. Colocalization studies showed that cSrc-GFP was retained in the endosomal recycling compartment (ERC) in iPLA(2) knockdown cells, but the addition of arachidonic acid (AA) could restore cSrc trafficking to the plasma membrane by allowing the formation/release of recycling endosomes associated with cSrc-GFP. Using BODIPY-AA, we showed that AA is selectively enriched in recycling endosomes. These results suggest that AA is required for the cSrc trafficking to the plasma membrane by controlling the formation/release of recycling endosomes from the ERC.     

Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation

Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated, but the mechanism is unclear. U937 cells express group IV phospholipase A(2) (cPLA(2)), but neither its level nor its phosphorylation state increases upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phospholipids in some cells and facilitates arachidonate incorporation into glycerophosphocholine (GPC) lipids in others, but it is not known whether U937 cells express iPLA(2). We confirm that ionophore A23187 induces substantial [(3)H]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containing GPC species than control cells. U937 cells express iPLA(2) mRNA and activity, but iPLA(2) inhibition impairs neither [(3)H]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohydrolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS analyses of PLD-PAPH products indicate that differentiated cells gain the ability to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely independent of protein kinase C, and requires cPLA(2) activity. This may reflect DAG effects on cPLA(2) substrate state.     

H(2)O(2): a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca(2+) stores. We therefore measured cytosolic Ca(2+) in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H(2)O(2) to the reduction in Ca(2+) signal. In normal cells, the ACh-induced Ca(2+) increase was the same in normal-Ca(2+) and Ca(2+)-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca(2+) release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca(2+) increase in normal-Ca(2+) medium was significantly lower in esophagitis than in normal cells and was further reduced ( approximately 70%) when the cells were incubated in Ca(2+)-free medium. This reduction was partially reversed by the H(2)O(2) scavenger catalase. H(2)O(2) measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H(2)O(2), the ACh-induced Ca(2+) increase was significantly reduced in normal-Ca(2+) and Ca(2+)-free medium and was similar to that observed in animals with esophagitis. The initial ACh-induced contraction was also reduced in normal cells incubated with H(2)O(2). H(2)O(2), when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca(2+) signal in normal cells. H(2)O(2)-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H(2)O(2) reduced TG-induced contraction, suggesting that TG and H(2)O(2) may operate through similar mechanisms. Ca(2+)-ATPase activity measurement indicates that H(2)O(2) and TG reduced Ca(2+)-ATPase activity, confirming similarity of mechanism of action. We conclude that H(2)O(2) may be at least partly responsible for impairment of Ca(2+) release in acute experimental esophagitis by inhibiting Ca(2+) uptake and refilling Ca(2+) stores.     

Role of protein kinase C in 1,25(OH)(2)-vitamin D(3) modulation of intracellular calcium during development of skeletal muscle cells in culture

Regulation of muscle cell Ca(2+) metabolism by 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] is mediated by the classic nuclear mechanism and a fast, nongenomic mode of action that activates signal transduction pathways. The role of individual protein kinase C (PKC) isoforms in the regulation of intracellular Ca(2+) levels ([Ca(2+)](i)) by the hormone was investigated in cultured proliferating (myoblasts) and differentiated (myotubes) chick skeletal muscle cells. 1,25(OH)(2)D(3) (10(-9) M) induced a rapid (30- to 60-s) and sustained (>5-min) increase in [Ca(2+)](i) which was markedly higher in myotubes than in myoblasts. The effect was suppressed by the PKC inhibitor calphostin C. In differentiated cells, PKC activity increased in the particulate fraction and decreased in cytosol to a greater extent than in proliferating cells after 5-min treatment with 1,25(OH)(2)D(3). By Western blot analysis, these changes were correlated to translocation of the PKC alpha isoform from cytosol to the particulate fraction, which was more pronounced in myotubes than in myoblasts. Specific inhibition of PKC alpha activity using antibodies against this isoform decreased the 1, 25(OH)(2)D(3)-induced [Ca(2+)](i) sustained response associated with Ca(2+) influx through voltage-dependent calcium channels. Neomycin, a phospholipase C (PLC) inhibitor, blocked its effects on [Ca(2+)](i), PKC activity, and translocation of PKC alpha. Exposure of myotubes to 1,2-dioleyl-rac-glycerol (1,2-diolein), also increased [Ca(2+)](i), PKC activity, and the amount of PKC alpha associated with the particulate fraction. Changes in [Ca(2+)](i) induced by diolein were inhibited by calphostin C and nifedipine. The results indicate that PKC alpha activation via PLC-catalyzed phosphoinositide hydrolysis is part of the mechanism by which 1, 25(OH)(2)D(3) regulates muscle intracellular Ca(2+) through modulation of the Ca(2+) influx pathway of the Ca(2+) response to the sterol.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

Role of ca(2+) in the control of h(2)o(2)-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(2)O(2)-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(2)O(2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(2+) ([Ca(2+)](i)), and document that activity of the L-type Ca(2+) channel is necessary for the H(2)O(2)-promoted increase in sarcomere shortening and of [Ca(2+)](i). Using the chemical NO sensor Cu(2)(FL2E), we discovered that the H(2)O(2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(2+) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(2)O(2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(2+)](i) as well as the activation of protein kinase C (PKC). We also found that H(2)O(2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(2+) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(2)O(2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(2)O(2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(2+) channel, but is independent of PKC. These studies establish key roles for Ca(2+)- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(2)O(2).     

Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.