Possible Involvement of Mitogen-Activated Protein Kinase in Phospholipase D Activation Induced by H2O2, but Not by Carbachol, in Rat Pheochromocytoma PC12 Cells

Abstract: We have previously reported that hydrogen peroxide (H₂O₂) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H₂O₂-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H₂O₂, but not by carbachol. In the present study, H₂O₂ also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H₂O₂-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A₂ (PLA₂) inhibitor, almost completely prevented the release of [³H]OA by H₂O₂ treatment. From the preferential release of OA and sensitivity to other PLA₂ inhibitors, the involvement of a Ca²⁺-independent cytosolic PLA₂-type enzyme was suggested. In contrast, to OA release, MAFP did not inhibit PLD activation by H₂O₂. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H₂O₂-induced PLD activation may be mediated by MAP kinase and also that H₂O₂-mediated OA release, which would be catalyzed by a Ca²⁺-independent cytosolic PLA₂-like enzyme, is not linked to the PLD activation in PC12 cells.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with ³H-arachidonic acid, ionomycin and Ca²⁺-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC₅₀ = 1.6 ± 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca²⁺-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 μM), a blocker of PLA₂, completely inhibited bradykinin-induced arachidonic acid release. The B₂ bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B₁-selective agonist DesArg⁹-bradykinin was inactive and the B₁-selective antagonist [Leu⁸]DesArg⁹-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 μM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 μM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA₂ by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca²⁺-calmodulin appears to be the prominent activator of PLA₂ in this system. © 1996 Wiley-Liss, Inc.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Thapsigargin-induced calcium entry in FRTL-5 cells: Possible dependence on phospholipase A2 activation

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A₂ (PLA₂) is activated. Since PLA₂ is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA₂ activity and the entry of calcium. Stimulating ³H-arachidonic acid (³H-AA)-labelled cells with thapsigargin resulted in a substantial release of ³H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA₂ activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA₂ in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA₂ activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca²⁺ in FRTL-5 cells. © 1994 Wiley-Liss, Inc.     

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A₂ (PLA₂) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H₂O₂). Exposure of the smooth muscle cells to H₂O₂ dose-dependently stimulates [¹⁴C] AA release, and enhances the cell membrane associated PLA₂ activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H₇ and sphingosine prevent the cell membrane associated PLA₂ activity, and AA release caused by H₂O₂. Treatment of the smooth muscle cells with H₂O₂ stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H₂O₂ caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA₂ and PKC activities correlate linearly. These results suggest that H₂O₂ caused stimulation of the smooth muscle cell membrane associated PLA₂ activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)Abbreviations AA Arachidonic Acid - PLA₂ Phospholipase A₂ - PKC Protein Kinase C - PBS Phosphate Buffered Saline - HBPS Hank's Buffered Physiological Saline - HEPES 4-(2-Hydroxyethyl)-1-Piperazine N-2-Ethanesulfonate - FCS Fetal Calf Serum - ATP Adenosine Triphosphate - H₇ 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine - DMEM Dulbecco's Modified Eagles Medium - TCA Trichloroacetic Acid     

PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A₂ (PLA₂) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA₂-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca²⁺-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA₂ activity and the release of free arachidonic acid (AA) from 1-acyl 2-¹⁴C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA₂ hyperactivity is Ca²⁺-dependent. There was no considerable alteration in the PLA₂ activity by stimulation of the activity by tyrosine kinase (with vanadate, H₂0₂), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF₄ thus in Tetrahymena PLA₂ activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA₂ activity is in connection with the phospholipid metabolism of Tetrahymena.     

Role of MAP kinase in neurons

Extracellular stimuli such as neurotransmitters, neurotrophins, and growth factors in the brain regulate critical cellular events, including synaptic transmission, neuronal plasticity, morphological differentiation and survival. Although many such stimuli trigger Ser/Thr-kinase and tyrosine-kinase cascades, the extracellular signal-regulated kinases, ERK1 and ERK2, prototypic members of the mitogen-activated protein (MAP) kinase family, are most attractive candidates among protein kinases that mediate morphological differentiation and promote survival in neurons. ERK1 and ERK2 are abundant in the central nervous system (CNS) and are activated during various physiological and pathological events such as brain ischemia and epilepsy. In cultured hippocampal neurons, simulation of glutamate receptors can activate ERK signaling, for which elevation of intracellular Ca²⁺ is required. In addition, brain-derived neurotrophic factor and growth factors also induce the ERK signaling and here, receptor-coupled tyrosine kinase activation has an association. We describe herein intracellular cascades of ERK signaling through neurotransmitters and neurotrophic factors. Putative functional implications of ERK and other MAP-kinase family members in the central nervous system are give attention.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Mitochondrial lipid pore in the mechanism of glutamate-induced calcium deregulation of brain neurons

The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca²⁺ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria with Sr²⁺ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation of Ca²⁺/Sr²⁺-dependent phospholipase A₂ (PLA₂), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca²⁺ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated earlier on both artificial and mitochondrial membranes. The inhibitors of PLA₂ have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the PLA₂ inhibitors prolonged the lag of the delayed Sr²⁺ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that the initial stages of glutamate-induced Ca²⁺ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria.     

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10^?4 M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂‐induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂‐induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX‐2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley‐Liss, Inc.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E₁ (10⁻⁸ − 10⁻⁷M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE₁, tetracaine and PMSF. The inhibitory effect of PGE₁ is strongly potentiated by theophylline (1.0 mM).Addition of the Ca²⁺ ionophore A23187 to platelet suspensions overcomes PGE₁ and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O₂ consumption. Tetracaine and dibucaine, however, block the responses to A23187.Formation of MDA and RCS (a mixture of PG endoperoxides and TXA₂) due to stimulation by thrombin and collagen depends upon activation of Ca²⁺-dependent phospholipase A₂ (PLA₂) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE₁, antagonizes the mobilization of the Ca²⁺ which is normally required for PLA₂ activity. Thrombin-stimulated platelets exhibit enhanced ⁴⁵Ca uptake which probably reflects exchange of extracellular Ca²⁺ with an increased available pool of exchangeable intracellular Ca²⁺. PGE₁ strongly suppresses this ⁴⁵Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca²⁺ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca²⁺ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O₂ uptake are nearly normal indicating that activation of PLA₂ can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca²⁺ to activate PLA₂. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA₂ activity.     

Release of arachidonic acid via Ca2+ increase stimulated by pyrophosphonucleotides and bradykinin in mammary tumour cells

The relationship between the increase of intracellular Ca²⁺ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca²⁺ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca²⁺ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A₂ and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca²⁺-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A₂.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

Protein tyrosine phosphorylation and calcium signaling in thyroid FRTL-5 cells

We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca²⁺ entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72-kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca²⁺ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca²⁺, but the capacitative Ca²⁺ or Ba²⁺ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca²⁺ ([Ca²⁺]_i), whereas the Ba²⁺ entry was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP-evoked phosphorylation of MAP kinase and attenuated both the ATP- and the PMA-evoked DNA synthesis. However, genistein did not inhibit the ATP-evoked expression of c-fos. Furthermore, genistein enhanced the ATP-evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca²⁺-dependent mechanism. A genistein-sensitive mechanism participates, in part, in the ATP-evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca²⁺ entry in FRTL-5 cells. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc.     

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H₂O₂ generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.

     

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The presentstudy was performed to characterize thrombin-stimulated phospholipaseA₂(PLA₂) activity and theresultant release of lysophospholipids from endothelial cells. Themajority of PLA₂ activity inendothelial cells was membrane associated,Ca²⁺ independent, and arachidonateselective. Incubation with thrombin increased membrane-associatedPLA₂ activity using bothplasmenylcholine and alkylacyl glycerophosphocholine substrates in theabsence of Ca²⁺, with no increasein activity observed with phosphatidylcholine substrate. The increasedPLA₂ activity was accompanied byarachidonic acid and lysoplasmenylcholine (LPlasC) release fromendothelial cells into the surrounding medium. Thrombin-induced changeswere duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with theCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked thrombin-stimulated increases inPLA₂ activity, arachidonic acid,and LPlasC release. Stimulation of protein kinase C (PKC) increasedbasal PLA₂ activity and LPlasCproduction. Thrombin-stimulatedPLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cellsactivates a PKC-activated, membrane-associated,Ca²⁺-independentPLA₂ that selectively hydrolyzesarachidonylated, ether-linked phospholipid substrates, resulting inLPlasC and arachidonic acid release.