Anionic phospholipids stimulate an arachidonoyl-hydrolyzing phospholipase A2 from macrophages and reduce the calcium requirement for activity

Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.     

Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.     

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.     

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity.

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0-8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D.

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.     

Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans.

Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase.     

Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity.

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine.

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.     

No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation

Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.