Polyunsaturated fatty acids potentiate interleukin-1-stimulated arachidonic acid release by cells overexpressing type IIA secretory phospholipase A2.

By analyzing human embryonic kidney 293 cell transfectants stably overexpressing various types of phospholipase A2 (PLA2), we have shown that polyunsaturated fatty acids (PUFAs) preferentially activate type IIA secretory PLA2 (sPLA2-IIA)-mediated arachidonic acid (AA) release from interleukin-1 (IL-1)-stimulated cells. When 293 cells prelabeled with 13H]AA were incubated with exogenous PUFAs in the presence of IL-1 and serum, there was a significant increase in [3H]AA release (in the order AA > linoleic acid > oleic acid), which was augmented markedly by sPLA2-IIA and modestly by type IV cytosolic PLA2 (cPLA2), but only minimally by type VI Ca2(+)-independent PLA2, overexpression. Transfection of cPLA2 into sPLA2-IIA-expressing cells produced a synergistic increase in IL-1-dependent [3H]AA release and subsequent prostaglandin production. Our results support the proposal that prior production of AA by cPLA2 in cytokine-stimulated cells destabilizes the cellular membranes, thereby rendering them more susceptible to subsequent hydrolysis by sPLA2-IIA.     

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.     

Signalling events mediating the activation of protein kinase C by interleukin-2 in cytotoxic T cells.

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP3) and intracellular Ca2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A2 (PLA2) since the PLA2 inhibitors oleoyloxyethyl phosphocholine and AACOCF3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.     

Phorbol ester plus calcium ionophore induces release of arachidonic acid from membrane phospholipids of a human B cell line

Binding of LA350, a lymphoblastoid human B cell line, by phorbol myristate acetate (PMA) plus a calcium ionophore, either ionomycin or A23187, produced unique alterations in the release of arachidonic acid (AA) from cellular phospholipids. After equilibrium labeling of cells with radioactive fatty acids, [14C]AA demonstrated a selective enhanced release from the cells in response to the binding of PMA plus calcium ionophore as compared to the release of [14C]stearic acid (STE), [3H]oleic acid (OLE) and [3H]palmitic acid (PAL). The major phospholipid sources of the released [14C]AA were shown to be phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The participation of protein kinase C (PKC) in the enhanced synergistic release of [14C]AA was demonstrated by the inhibition of the release by the PKC inhibitor, staurosporine. Approximately 2-6% of the labeled AA liberated was converted to 5-hydroxyeicosatetraenoic acid by an endogenous 5-lipoxygenase. Therefore during cell activation the B cell is capable of liberating AA via a PKC-dependent mechanism, implicating AA and/or its metabolites in signal transduction.     

The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.     

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.     

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.     

Activation of phospholipase D in a rat mast (RBL 2H3) cell line. A possible unifying mechanism for IgE-dependent degranulation and arachidonic acid metabolite release.

RBL 2H3 cells (a model of mast cell function) were sensitized with anti-TNP IgE (0.5 micrograms/ml) and triggered to secrete both histamine and arachidonic acid (AA) metabolites by the addition of TNP-OVA (0 to 100 ng/ml). After a 3-min delay, the release of both groups of mediators proceeded in a parallel manner. In cells labeled with [14C]-AA, TNP-OVA produced a rapid increase in phosphatidic acid (PA), and subsequently, 1,2-diacylglycerol (DAG) and intracellular AA levels. Concurrently, there was a decrease in [14C]-AA labeled phosphatidylcholine. The release of labeled AA from phosphatidylcholine in response to TNP-OVA was paralleled by a liberation of free choline but no evidence of liberation of phosphorylcholine. When ethanol (0.05 to 2% v/v) was included in the culture medium, phosphatidylethanol was synthesized at the expense of PA and DAG, with a resulting inhibition of secretion. D,1 propranolol, an inhibitor of PA phosphohydrolase, inhibited the IgE-dependent production of [14C]-DAG, and [14C]-free fatty acid but not [14C]-PA. The IgE-dependent release of both histamine and AA metabolites was completely inhibited by pretreatment with propranolol. Taken together, the above results suggest that phospholipase D is activated upon cross-bridging of IgE receptors on the surface of RBL 2H3 cells and that this may be a pivotal step in the signal transduction cascade leading to the release of both presynthesized and de novo synthesized mediators.     

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.     

Group V phospholipase A(2)-mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D(1) macrophages

P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.     

Regulation of diacylglycerol levels in carbachol-stimulated pancreatic acinar cells: relationship to the breakdown of phosphatidylcholine and metabolism to phosphatidic acid.

Rat pancreatic acinar cells prelabeled with [14C]palmitic acid and then exposed to carbachol (CCh) exhibited a time-dependent increase in 1,2-[14C]diacylglycerol ([14C]DAG) levels, which was first detected at 2 min and then continued to rise in a linear manner. There was a concomitant increase in [14C]phosphatidic acid, which plateaued after 2 min and then remained at steady-state levels. CCh also promoted the release of phosphocholine, but not choline, within 60 s and caused a decrease in [14C]phosphatidylcholine in cells prelabeled with [14C]glycerol after 15 min. The inability to detect a rise in [14C]phosphatidylethanol accumulation and a fall in [14C]phosphatidate levels in [14C]palmitate prelabeled cells after exposure to CCh plus ethanol documented the absence of a phospholipase D-mediated pathway. The rapid phosphorylation of diglyceride in homogenates from unstimulated and carbachol-treated cells increased with increasing concentrations of exogenous substrate, thereby affirming that carbachol stimulates the phosphorylation of DAG by promoting the accumulation of the diglyceride. These collective findings provide evidence for the existence of an integrative control mechanism for regulating endogenous DAG levels during pancreatic acinar cell activation involving phosphatidylcholine-specific phospholipase C and DAG kinase.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

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.     

Arachidonoyl transacylase in human platelets. Coenzyme A-independent transfer of arachidonate from phosphatidylcholine to lysoplasmenylethanolamine

Human platelets contain an enzyme that catalyzes CoA-independent release of arachidonic acid from phosphatidylcholine with concomitant incorporation into plasmenylethanolamine. Addition of lysoplasmenylethanolamine (10-80 microM) to a crude membrane preparation of prelabeled platelets (0.24 mg of protein/ml) induces transfer of [3H]arachidonate from endogenous phosphatidylcholine to lysoplasmenylethanolamine (0.8 nmol of arachidonic acid/min/mg of protein). The transacylation reaction occurs in the absence of Ca2+, has a broad pH optimum from 7 to 8, is not affected by excess unlabeled arachidonic acid, and is inhibited by N-ethylmaleimide (0.2 mM) and Triton X-100 (0.1 mg/ml). The enzyme shows a high specificity toward the acyl donor (phosphatidylcholine), transfers fatty acids in the order: arachidonic greater than eicosatrienoic greater than oleic, and preferentially acylates lysoplasmenylethanolamine but also other lysophosphatides (lysophosphatidylethanolamine greater than lysophosphatidylserine greater than lysophosphatidylinositol = 0). Platelet acyltransferase, on the other hand, acylates ethanolamine lysophosphatides with free arachidonic acid in the order: lysophosphatidyl-ethanolamine greater than lysoplasmenylethanolamine. These results suggest that a distinct acylation mechanism exists for introduction of arachidonic acid into plasmalogen phosphatides. In stimulated platelets, the transacylase may play an additional role in the controlled release of esterified arachidonic acid for synthesis of the biologically active oxygenated metabolites.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

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.     

ras-transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin

The results of studies to evaluate the hypothesis that the 21 kDa GTP-binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H-ras transformation on basal, tumor-promoting phorbol ester (TPA)-stimulated, and bradykinin-mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H-ras-transformed MDCK cells (MDCK-RAS) to two non-transformed strains of MDCK cells (MDCK-D1 and MDCK-ATCC). In unstimulated MDCK-RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK-C) activity was decreased, and specific binding of [3H]phorbol ester was reduced in MDCK-RAS relative to the non-transformed MDCK cells suggesting that elevated DAG may activate and thereby down-regulate PK-C. Consistent with this finding in MDCK-RAS, TPA-stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA-stimulated choline phosphate release was increased. Bradykinin receptor-stimulated phospholipid hydrolysis in MDCK-RAS was similar to that of non-transformed cells, suggesting that the ras-derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA-treatment to inhibit bradykinin-stimulated phosphoinositide hydrolysis and enhance bradykinin-stimulated AA release was attenuated in MDCK-RAS. Additionally, in MDCK-RAS the conversion of arachidonic acid to prostaglandin E2 was substantially reduced. We conclude that ras transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down-regulating PK-C and certain responses to TPA. Since activation of PK-C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK-C levels associated with ras transformation may account for the inconsistent effects previously observed in studies evaluating the effect of ras transformation on phospholipases and other signal transduction systems.