The cyclic AMP-protein kinase A pathway restrains islet phospholipase A(2) activation

Although phospholipase A(2) (PLA(2)) is of importance for insulin secretion, it is not established how it relates to other signalling mechanisms. This study examined the crosstalk between PLA(2) and the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in isolated rat islets. Forskolin, IBMX, and dbcAMP reduced [(3)H]arachidonic acid ([(3)H]AA) efflux from prelabelled islets during PLA(2) activation by mellitin or cholecystokinin (CCK-8), while efflux induced by carbachol was unaffected. The PKA inhibitor myrPKI(14-22) prevented this reduction of CCK-8-induced efflux. Glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and vasoactive intestinal polypeptide (VIP) diminished CCK-8-induced efflux. Also in the absence of Ca(2+), forskolin/IBMX and dbcAMP reduced CCK-8-induced efflux. In parallel with effects on [(3)H]AA, the expected additive insulin secretion induced by mellitin or CCK-8 in combination with forskolin or GLP-1, respectively, was reduced. In conclusion, the cAMP-PKA pathway restrains both Ca(2+)-dependent and Ca(2+)-independent PLA(2) activation, indicating a regulating crosstalk between these two pathways.     

Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D

Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.     

Arachidonic acid modulates the spatiotemporal characteristics of agonist-evoked Ca2+ waves in mouse pancreatic acinar cells

In pancreatic acinar cells analysis of the propagation speed of secretagogue-evoked Ca2+ waves can be used to examine coupling of hormone receptors to intracellular signal cascades that cause activation of protein kinase C or production of arachidonic acid (AA). In the present study we have investigated the role of cytosolic phospholipase A2 (cPLA2) and AA in acetylcholine (ACh)- and bombesin-induced Ca2+ signaling. Inhibition of cPLA2 caused acceleration of ACh-induced Ca2+ waves, whereas bombesin-evoked Ca2+ waves were unaffected. When enzymatic metabolization of AA was prevented with the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid, ACh-induced Ca2+ waves were slowed down. Agonist-induced activation of cPLA2 involves mitogen-activated protein kinase (MAPK) activation. An increase in phosphorylation of p38(MAPK) and p42/44(MAPK) within 10 s after stimulation could be demonstrated for ACh but was absent for bombesin. Rapid phosphorylation of p38(MAPK) and p42/44(MAPK) could also be observed in the presence of cholecystokinin (CCK), which also causes activation of cPLA2. ACh-and CCK-induced Ca2+ waves were slowed down when p38(MAPK) was inhibited with SB 203580, whereas inhibition of p42/44(MAPK) with PD 98059 caused acceleration of ACh- and CCK-induced Ca2+ waves. The spreading of bombesin-evoked Ca2+ waves was affected neither by PD 98059 nor by SB 203580. Our data indicate that in mouse pancreatic acinar cells both ACh and CCK receptors couple to the cPLA2 pathway. cPLA2 activation occurs within 1-2 s after hormone application and is promoted by p42/44(MAPK) and inhibited by p38(MAPK). Furthermore, the data demonstrate that secondary (Ca2+-induced) Ca2+ release, which supports Ca2+ wave spreading, is inhibited by AA itself and not by a metabolite of AA.     

Independent functioning of cytosolic phospholipase A2 and phospholipase D1 in Trp-Lys-Tyr-Met-Val-D-Met-induced superoxide generation in human monocytes

Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes. The peptide stimulated phospholipase A2 (PLA2) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA2 (cPLA2)-specific inhibitors. The involvement of cPLA2 in the peptide-induced AA release was further supported by translocation of cPLA2 to the nuclear membrane of monocytes incubated with WKYMVm. WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D1 (PLD1), but not PLD2. GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes. Furthermore, the interrelationship between the two phospholipases, cPLA2 and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA2 did not blunt peptide-stimulated PLD1 activation or vice versa. Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA2. The WKYMVm-dependent stimulation of cPLA2 activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD1. Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+.     

Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.     

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.     

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.     

Characterization of CCK(A) receptor affinity states and Ca(2+) signal transduction in vagal nodose ganglia

CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.     

Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.     

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.     

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.     

Hydrogen peroxide activation of Ca(2+)-independent phospholipase A(2) in uterine stromal cells

In rat uterine stromal cells (U(III) cells), an oxidative stress induced by H(2)O(2) caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca(2+) concentration and was not inhibited by Ca(2+)-dependent phospholipase A(2) (cPLA(2)) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H(2)O(2) treatment did not impair AA esterification but significantly increased Ca(2+)-independent PLA(2) (iPLA(2)) activity. Since iPLA(2) specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H(2)O(2), we conclude that iPLA(2) activity represents the major mechanism by which H(2)O(2) increases the availability of non-esterified AA in U(III) cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H(2)O(2), suggesting a regulatory mechanism of iPLA(2) by PKC that remains to be clarified.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Direct relationship between intracellular calcium mobilization and phospholipase D activation in prostaglandin E-stimulated human erythroleukemia cells.

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.     

Ca2+-Independent, Ca2+-Dependent, and Carbachol- Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C]arachidonic acid [( 14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly-PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C (PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 microM CaCl2 enhance AA liberation by approximately 25%, whereas 2 mM CaCl2 resulted in a 50% increase in AA release relative to EGTA. The source for Ca(2+)-dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca(2+)-dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10-15%. This agonist-mediated AA release occurs specifically from phosphoinositides (PI + poly-PI). Quinacrine almost completely suppresses calcium-and carbachol-mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca(2+)-independent AA release. Ca(2+)-dependent and carbachol-mediated AA liberation occurs mainly as the result of PLA2 action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.