Dependent Phospholipase Research Articles

Characterization of a platelet activating factor acetylhydrolase from rat adipocyte

Platelet activating factor-acetylhydrolases (PAF-AHs) are a family of enzymes with the common property of hydrolyzing and inactivating PAF and thus regulating its levels. In the course of studying the role of PAF in rat adipocytes and its possible implication in body weight regulation and immune response, conditions in which adipocytes are involved, we investigated the existence of PAF-AH in these cells. We detected PAF-AH activity in rat adipocytes which is mainly distributed in the cytosol. The behaviour of the enzyme during hydrophobic chromatography, together with the fact that part of the enzyme activity was found in the fat cake of adipocyte homogenate suggests the hydrophobic nature of rat adipocyte PAF-AH. The enzyme activity was distinct from the Ca 2+-dependent and independent phospholipase A 2, the lysophospholipase, the lecithin:cholesterol acyltransferase (LCAT) and from non-specific acetylhydrolases. We partially purified PAF-AH from rat adipocyte cytosolic fraction and the purified enzyme revealed a major protein band of 66 kDa and a minor one of 37 kDa on SDS/PAGE. The purified PAF-AH has an apparent K m value of 4.9 μM and the enzyme activity was inactivated by PMSF and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and moderately stimulated by dithiothreitol (DTT). Furthermore, in this study we identified PAF in rat adipocytes and determined its concentration.

The Effects of Aliphatic (n-Nonane), Naphtenic (1,2,4-Trimethylcyclohexane), and Aromatic (1,2,4-Trimethylbenzene) Hydrocarbons on Respiratory Burst in Human Neutrophil Granulocytes

This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)), and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes. The free radical formation was measured as 2,7-dichlorofluorescein diacetate-amplified (DCF) fluorescence, by electron paramagnetic resonance (EPR) spectroscopy and by hydroxylation of 4-hydroxybenzoate. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included as positive controls. DCF fluorescence was elevated in a concentration-dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed the strongest effect on respiratory burst and was therefore selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca2+, fluorescence in response to TMCH and fMLP was reduced by 77 and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the DCF fluorescence stimulated with TMCH, fMLP, and PMA by 82, 56, and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP-activated DCF fluorescence by 87 and 76%. In addition, the TMCH- and fMLP-induced DCF fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83 and 52%, respectively. The importance of protein kinase C, phospholipase C, and phospholipase D for elevation of respiratory burst was also demonstrated by the EPR experiments using the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of DCF fluorescence in response to TMCH, fMLP, and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. The hydrocarbons stimulated formation of mainly the superoxide (O•−2) adduct of DEPMPO (DEPMPO–OOH) but also small amounts of the hydroxyl adduct (•OH) (DEPMPO–OH). Using 4-hydroxybenzoate as a hydroxyl radical trap confirmed formation of •OH after stimulation with the hydrocarbons. In conclusion, our findings indicate that TMCH-activated respiratory burst is dependent on the Ca2+-dependent phospholipase C, phospholipase D, and protein kinase C prior to activation of the NADPH oxidase.

Hydrogen Peroxide Activation of Ca2+-Independent Phospholipase A2 in Uterine Stromal Cells

In rat uterine stromal cells (UIII cells), an oxidative stress induced by H2O2 caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca2+ concentration and was not inhibited by Ca2+-dependent phospholipase A2 (cPLA2) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H2O2 treatment did not impair AA esterification but significantly increased Ca2+-independent PLA2 (iPLA2) activity. Since iPLA2 specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H2O2, we conclude that iPLA2 activity represents the major mechanism by which H2O2 increases the availability of non-esterified AA in UIII cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H2O2, suggesting a regulatory mechanism of iPLA2 by PKC that remains to be clarified.

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation.

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis.

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.

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 A2 Activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca2+-dependent cytosolic phospholipase A2 (cPLA2) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca2+-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 μM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 μM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca2+-chelator 1,2-bis[2-aminophenoxy]ethane-N,N,N′,N′-tetraacetic acid (10 μM) abolished fMLP-mediated Ca2+ signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 μM) or the inhibitor of cPLA2 arachidonyl trifluoromethyl ketone (AACOCF3; 10–30 μM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca2+ ionophore A23187 (10 μM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 μM). This effect was inhibited by either Ro 318220 or AACOCF3. Accordingly, a translocation of cPLA2 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 Ca2+ signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by ≈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 μM), decreased fMLP-mediated AA release by ≈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 cPLA2 pathway, but may originate from activation of PC-PLC and PLD.

Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1 beta by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1 beta and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1 beta processing was not due to perturbation of the export machinery for pro-IL-1 beta and IL-1 beta or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1 beta processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1 beta maturation.

Mechanism of fat-induced attenuation of glucose-induced insulin secretion from mouse pancreatic islets.

In order to investigate the mechanism behind fat-induced inhibition of glucose-induced insulin secretion a selection of enzymes that may participate in regulation of pancreatic islet glucose oxidation was studied in islets isolated from mice that had been fed on a laboratory chow diet or on a high-fat diet for 10-12 weeks. At 20 mmol/L glucose production of (14)CO(2) from [U-(14)C]-glucose was decreased 50% in islets from fat-fed mice. At 3.3 mmol/L glucose the glucose oxidation rate was similar in the two groups. The fatinduced decrease in glucose oxidation rate was correlated with a 35% decrease in the maximal glucokinase activity. The K(m) for glucose was unchanged. No differences between the diet groups were found in the activities of hexokinase, phosphofructo-1-kinase, glucose 6-phosphatase or mitochondrial glycerophosphate dehydrogenase. After preincubation with 20 mmol/L glucose the activity of cytosolic Ca(2+)-independent as well as Ca(2+)-dependent phospholipase A(2) was unchanged by fat-feeding. However, the activity of lysophospholipase was significantly increased by fat feeding, which may result in lowered concentrations of islet lysophosphatidylcholine (lysoPC). It is concluded that in fat-induced diabetic animals a decrease in islet glucokinase may contribute considerably to the decrease in islet glucose oxidation rate. Furthermore, the study raises the possibility that changes in islet lysoPC may contribute to the fat-induced attenuation of glucose-induced insulin secretion.

Regulatory effects of HDL on smooth muscle cell prostacyclin release.

One mechanism by which high density lipoproteins (HDLs) exert their protective effect against coronary artery disease could be related to the induction of prostacyclin (PGI(2)) release in the vessel wall. We have recently shown that HDL increases PGI(2) production in rabbit smooth muscle cells (RSMCs) and that this increase is dependent on cyclooxygenase-2 (Cox-2). Here we analyze the mechanism by which rabbit HDL induces PGI(2) release in RSMCs. Our results show that although HDL(2) and HDL(3) share a similar capacity to induce Cox-2 protein levels, HDL(3) stimulates a higher PGI(2) release than does HDL(2), probably because of their relative arachidonate contents. Acetylsalicylic acid pretreatment (300 micromol/L, 30 minutes) significantly reduced the HDL-induced PGI(2) release, suggesting that both preexisting and induced Cox-2 activities were involved in the HDL effect. Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) and Cox-1 protein levels were not altered by HDL. Dexamethasone (2 micromol/L), which also inhibited the HDL-induced PGI(2) release, reduced significantly both Cox-2 mRNA and protein levels without affecting cPLA(2) and Cox-1 protein levels. In addition, methylarachidonyl fluorophosphonate, a potent inhibitor of cPLA(2), did not produce any effect on HDL-induced PGI(2) release. In the presence of cycloheximide, Cox-2 mRNA levels were induced by HDL and inhibited by dexamethasone, suggesting that HDL and dexamethasone work in the absence of de novo protein synthesis. These results indicate an early effect of HDL on PGI(2) biosynthesis, specifically increasing Cox-2. PD98059, an inhibitor of mitogen-activated protein kinase kinase, completely inhibited HDL-induced PGI(2) release, whereas GF109203X, a protein kinase C inhibitor, had no effect. Thus, HDL induces PGI(2) synthesis by a mechanism dependent on the mitogen-activated protein kinase pathway but independent of protein kinase C.

Changes in calcium and iron levels in the brains of rats during kainate induced epilepsy

Epilepsy is a recurrent disorder of cerebral function characterised by sudden brief attacks of altered consciousness, motor activity or sensory phenomena, and affects approximately 1% of the population. Kainic acid injection induces neuronal degeneration in rats, is associated with glial hypertrophy and proliferation in the CA3–CA4 fields of hippocampal complex, and is a model for temporal lobe epilepsy. In this study we have applied Nuclear Microscopy to the investigation of the elemental changes within the hippocampus and the cortex areas of the rat brain following kainate injection. Analyses of unstained freeze dried tissue sections taken at 1 day and 1, 2, 3 and 4 weeks following injection were carried out using the Nuclear Microscopy facility at the Research Centre for Nuclear Microscopy, National University of Singapore. Quantitative analysis and elemental mapping indicates that there are significant changes in the calcium levels and distributions in the hippocampus as early as 1 day following injection. Preliminary results indicate a rapid increase in cellular calcium. High levels of calcium can activate calcium dependent proteins and phospholipases. Activation of phospholipase A2 can be harmful to surrounding neurons through free radical damage. In addition to observed increases in calcium, there was evidence of increases in iron levels. This is consistent with measurements in other degenerative brain disorders, and may signal a late surge in free radical production.

Protein kinase C stimulates PtdIns-4,5-P2-phospholipase C activity

The tumour promoter, phorbol ester 12,13-dibutyrate (PDBu), acts on rectal palisadic epithelial cells and mimics the effects of neuroparsin, an antidiuretic neuronal hormone isolated from nervous lobes of the African locust corpora cardiaca. PDBu stimulated Ca 2+-dependent phospholipase C (PLC) activity resulting in inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) production, increased cytosolic free calcium (monitored with the probe indo-1) and rectal fluid resorption. A 15-min pre-treatment with polymyxin B (PMXB), a protein kinase C (PKC) inhibitor acting at the phosphatidylserine (PS) binding site, suppressed PDBu stimulatory effects on free calcium entry and fluid resorption but not on phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) breakdown. On the contrary, bisindolylmaleimide Ro 32-0432 (which inhibits PKC at its ATP binding site) abolished entirely PDBu-stimulated PLC activity. It was concluded that two PKC are involved in transduction of the antidiuretic signal of neuroparsin. One PKC is PMXB sensitive and stimulates biological response after cytosolic free Ca 2+ increase, while another PKC, insensitive to the PKC inhibitor, regulates the processes induced by the former PKC. Since PMXB-insensitive PKC exerts a stimulatory effect on PtdIns-4,5-P2-PLC production, this original mechanism may be considered as a new signalling pathway under control of PKC.

Characterization of Ca2+-dependent Phospholipase A2 Activity during Zebrafish Embryogenesis

We have developed a simple fluorescent assay for detection of phospholipase A2 (PLA2) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA2 inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca2+-dependent cytosolic PLA2 (cPLA2) subtype. Embryonic cPLA2 activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA2, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.

Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes

Phosphatidylcholines (1- O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1- O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl- rac-glycero-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with K i-values up to 83 ( K irev) and 177 ( K i(inact)) μm. The compounds exhibit only marginal inhibition of Ca 2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1- O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-methyl- rac-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8°C, the compound accumulated in the membranes of U937 cells, at 37°C it was internalized into intracellular compartments. Structure–activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the ( sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the ( sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.

Dexamethasone-Induced Thymocyte Apoptosis: Apoptotic Signal Involves the Sequential Activation of Phosphoinositide-Specific Phospholipase C, Acidic Sphingomyelinase, and Caspases

AbstractGlucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Dexamethasone-Induced Thymocyte Apoptosis: Apoptotic Signal Involves the Sequential Activation of Phosphoinositide-Specific Phospholipase C, Acidic Sphingomyelinase, and Caspases

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Role of type IIA secretory phospholipase A2 in arachidonic acid metabolism.

Recent recognition of the rapidly growing sPLA2 family has led to a suggestion that some of the previously described functions of sPLA2-IIA need to be reevaluated, since studies based upon enzyme activities and using inhibitors or antibodies against sPLA2-IIA may not discriminate these sPLA2s. Our present studies reconfirm the involvement of sPLA2-IIA in biological responses, demonstrated significant crosstalk between the two Ca(2+)-dependent PLA2s (cPLA2 and sPLA2) where one enzyme is required for the induction of the other, and revealed segregated coupling of discrete PLA2 and COX enzymes in the different phases of PG biosynthesis. Based upon the analysis of cells derived from sPLA2-IIA "natural knock-out" mice, it is apparent that sPLA2-IIA is not essential for the initiation of delayed PGE2 biosynthesis. However, it is capable of contributing to the delayed response as an enhancer when appropriately induced by proinflammatory stimuli, leading to optimal COX-2-dependent PGE2 generation. Importantly, in order for sPLA2-IIA (or related sPLA2 isozymes) to attack the biological membranes, so-called "membrane rearrangement" should take place in activated, but not resting, cells. Membrane rearrangement also occurs when cells are undergoing apoptosis, during which acidic phospholipids, the preferred substrates for sPLA2-IIA, are exposed on the outer leaflet of the plasma membranes. Nonetheless, in view of the dramatically elevated levels of sPLA2-IIA in inflamed or ischemic sites, it is likely that this extracellular isozyme participates in the expansion of chronic tissue disorders by augmenting generation of proinflammatory eicosanoids or lysophospholipids, depending upon the states of the inflammatory response.

A Novel Inhibitory Effect on Prostacyclin Synthesis of Coupling Factor 6 Extracted from the Heart of Spontaneously Hypertensive Rats

The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10(-6) M)-induced prostacyclin synthesis and [3H]arachidonic acid (AA) release in human umbilical vein endothelial cells in a dose-dependent manner (10(-9)-10(-7) M). Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10(-7) M recombinant coupling factor 6. Base-line and bradykinin-induced [3H]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 at 40 microM, and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine at 1 microM nor bromoenol lactone at 1 microM affected AA release. Preincubation (1 min) with 10(-7) M recombinant coupling factor 6 had no influence on adenosine diphosphate- and collagen-induced platelet aggregations. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic phospholipase A2, although it is unclear whether coupling factor 6 functions in normal conditions or only in pathophysiological states.

Acetylcholine activates intracellular movement of insulin granules in pancreatic beta-cells via inositol trisphosphate-dependent [correction of triphosphate-dependent] mobilization of intracellular Ca2+.

Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.

Ortho-Substituted Polychlorinated Biphenyls Activate Respiratory Burst Measured as Luminol-Amplified Chemoluminescence in Human Granulocytes

The effect of polychlorinated biphenyls (PCBs) on the activation of respiratory burst measured as luminol-amplified chemoluminescence in human granulocytes is elucidated here. Chemoluminescence was stimulated in a concentration-dependent manner (ED50≈ 10 μM) byortho-substituted PCB congeners, whilemeta- andpara-substituted congeners had no significant effect. Twoortho-substituted PCB congeners were chosen for the mechanistic studies, namely 2,2′,4,4′-TeCB and 2,2′-DCB, since they have been used in previous studies by others. In the absence of extracellular calcium, the respiratory burst in response to 2,2′-DCB and 2,2′,4,4′-TeCB was reduced by 63% and 82%, respectively. Bisindolylmaleimide, which inhibits protein kinase C, reduced activated chemoluminescence by 2,2′-DCB, 2,2′,4,4′-TeCB,N-formyl-methionyl-leucyl-phenylalanine, and phorbol 12-myristate 13-acetate. Neomycin, which inhibits phospholipase C, had a slight, but significant, effect on the 2,2′,4,4′-TeCB-activated chemoluminescence but had a more pronounced effect on the 2,2′-DCB-activated chemoluminescence. 2,2′-DCB and 2,2′,4,4′-TeCB significantly increased phospholipase D (PLD) activity measured as the amount of14C-phosphatidylbutanol formed. Ethanol (1%), a phospholipase D modulator, reduced the response to 2,2′-DCB and 2,2′,4,4′-TeCB by 72% and 75%, respectively. Furthermore, wortmannin (25 nM), a phosphatidylinositol 3-kinase, and genistein, a more unspecific tyrosine kinase inhibitor, reduced chemoluminescence in response to PCB. In conclusion, our results indicate that PCB-activated chemoluminescence is dependent on the Ca2+-dependent phospholipase D or phospholipase C, phosphatidylinositol 3-kinase, and protein kinase C activation prior to activation of the NADPH oxidase. Defects in neutrophhil functions upon exposure to PCB may render a greater susceptibility in the host to invading microorganisms or evoke inappropriate inflammatory responses leading to tissue injury.

Cloning and expression of a group IV cytosolic Ca 2+-dependent phospholipase A 2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca 2+-independent phospholipase A 2

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet β-cells express low molecular weight secretory phospholipases A 2 (PLA 2) and a Group VI, Ca 2+-independent PLA 2 (iPLA 2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca 2+-dependent PLA 2 (cPLA 2). To further examine the possible expression of cPLA 2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA 2 sequence, and isolated a full-length cPLA 2 cDNA. The rat islet cPLA 2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA 2. Transfection of COS-7 cells with cPLA 2 cDNA in an expression vector induced expression of Ca 2+-dependent PLA 2 activity and of a protein recognized by anti-cPLA 2 antibody. Comparison of recombinant islet cPLA 2 and iPLA 2 activities expressed in transfected COS-7 cells indicated that iPLA 2 but not cPLA 2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA 2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA 2. RT-PCR experiments with RNA from purified islet β-cells and from an α-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA 2 mRNA is more abundant in the β-cell population. Immunoblotting analyses indicate that islets express cPLA 2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA 2 is thus one of at least three classes of PLA 2 enzymes with distinct properties expressed in β-cells.