Diacylglycerol metabolism in mast cells: a potential role in membrane fusion and arachidonic acid release

Purified rat peritoneal mast cells stimulated with the polycationic histamine-releasing agent compound 48/80 demonstrated a two- to four- fold increase in cellular levels of 1,2-diacylglycerol (DAG) within 1 min as detected by radioactive labeling and direct quantitation experiments. When 2-[1-14C]arachidonoyl-DAG was incubated in the presence of mast-cell homogenates, a rapid conversion to free arachidonate, and to a lesser extent, to monoacylglycerol, triglyceride, and phospholipid was observed. The release of arachidonate was proportional to the amount of broken-cell preparation added and the time of incubation, was prevented by preheating mast-cell preparations, and did not occur when 1-[1-14C]arachidonoyl- phosphatidylcholine was used as substrate, suggesting that the degradation was mediated by an enzyme with Dag-lipase activity. Although much work remains to be done to clarify the precise role of DAG in mast cells, DAG metabolism may be involved in secretion by generating substances which may faciliate membrane fusion and also in arachidonic acid-derived mediator formation by liberating esterified arachidonic acid from mast-cell lipids. Taken together, these studies indicate that the formation of DAG may play a central role in mast-cell function.     

Inhibitors of arachidonic acid metabolism: effects on rat striatal dopamine release and uptake.

The purpose of this study was to investigate the possibility that arachidonic acid metabolites mediate D-2 dopamine (DA) receptor inhibition of striatal DA release. The phospholipase A2 inhibitor p-bromophenacyl bromide (BPB; 10 microM) increased electrically evoked overflow of endogenous DA from rat striatal slices and appeared to partially block the modulatory effects of the D-2 DA receptor agonist N-0437 on this release. However, BPB also increased spontaneous DA outflow in a dose-dependent manner. U-73122 (10 microM), another phospholipase A2 inhibitor, decreased evoked overflow of DA, did not affect the action of N-0437 but also increased spontaneous outflow of DA. In contrast, arachidonic acid (30 microM) produced no effects. In slices prelabeled with [3H]DA, exposure to BPB, U-73122 and nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly increased spontaneous outflow of tritium whereas the cyclooxygenase inhibitors aspirin and indomethacin did not. In low micromolar concentrations, BPB, U-73122 and nordihydroguaiaretic acid, but not aspirin and indomethacin, inhibited uptake of [3H]DA into striatal synaptosomes and binding of [3H]mazindol to the DA transporter. Only U-73122 affected D-2 DA receptor binding. Taken together, these results suggest that it is unlikely that arachidonic acid metabolites mediate the actions of release-modulating D-2 DA autoreceptors in the striatum. However, the results also suggest that certain inhibitors of arachidonic acid metabolism are relatively potent DA uptake blockers/releasers and that this action is unrelated to their inhibition of enzymes in the arachidonic acid cascade. Caution should be used when using BPB and nordihydroguaiaretic acid to study mechanisms involved in DA release, because these compounds may increase DA release and thereby appear to antagonize the effects of activation of presynaptic receptors.     

Endothelium-dependent relaxation is independent of arachidonic acid release.

Endothelium-dependent relaxation is mediated by the release from vascular endothelium of an endothelium-derived relaxing factor (EDRF). It is not clear what role arachidonic acid has in this process. Inhibition of phospholipase A2, and diacylglycerol lipase in cultured bovine aortic endothelial cells caused a marked reduction in agonist-induced arachidonic acid release from membrane phospholipid pools, and complete inhibition of prostacyclin production. EDRF release, assayed by measuring endothelium-dependent cGMP changes in mixed endothelial-smooth muscle cell cultures, was not inhibited under these conditions. In fact, EDRF release in response to two agonists, melittin and ATP, was actually increased in cells treated with phospholipase A2 inhibitors. In addition, pretreatment of rats with high-dose dexamethasone, an inhibitor of PLA2, did not attenuate endothelium-dependent relaxation in intact aortic rings removed from the animals, or depressor responses in anesthetized animals induced by endothelium-dependent vasodilators. In summary, inhibition of arachidonic acid release from membrane phospholipid pools does not attenuate endothelium-dependent relaxation in rats, or the release and/or response to EDRF in cultured cells.     

p-Hydroxymercuribenzoic acid inhibits arachidonic acid esterification into two distinct pools and stimulates insulin release in intact rat islets

Activation of an islet phospholipase A2 may contribute to glucose-induced insulin release. In order to simulate the accumulation of the resultant hydrolytic products (arachidonic acid, AA; its lipoxygenase-derived oxygenation product 12-hydroxyeicosatetraenoic acid; and lysophospholipids) without many of the other concomitants of beta cell activation, we studied the effects on intact rat islets of p-hydroxymercuribenzoic acid (PHMB), which inhibits the reacylation of lysophospholipids with AA in other cell types. PHMB inhibited in a dose-responsive fashion (-90% at 500 microM) the incorporation of [3H]AA into a "basal" pool or pools whose release and reuptake mechanisms appeared to be largely energy- and Ca++-independent (resistant to inhibition by mannoheptulose, antimycin A or CoCl2); reciprocally, islets prelabeled with [3H]AA accumulated an increased amount of [3H]-12-hydroxyeicosatetraenoic acid (twice basal at 200 microM PHMB and three times basal at 500 microM) when reacylation of any [3H]AA released basally at 1.7 mM glucose was inhibited. PHMB also blocked (by up to 99% at 500 microM) the incorporation of [3H]AA into a functionally defined, glucose-stimulated compartment of fatty acid (tightly coupled to the islet 12-lipoxygenase) whose release and reuptake required metabolic energy and Ca++. It was also demonstrated that PHMB inhibited the esterification of [3H]AA (at low or high glucose concentrations) into specific phospholipids in islet membranes. In parallel with these alterations in lipid metabolism, PHMB caused rapid, potent and reversible increments in insulin release with a threshold concentration (about 25 microM) identical to that inhibiting AA fluxes. PHMB both initiated release (at 1.7 mM glucose) and potentiated the effects of islet fuels (16.7 mM glucose or 15 mM alpha-ketoisocaproic acid). Thus, pharmacologic manipulation of the AA reuptake mechanism is a new approach to unmask potential roles in insulin release of phospholipid hydrolysis products from different lipid pools and in the absence or presence of phospholipase A2 activation.     

Compartmentalized regulation of macrophage arachidonic acid metabolism

We show that downregulation of arachidonic acid (20:4) metabolism which occurs following i.p. injection of C. parvum can occur in a single, localized macrophage population, and is therefore unlikely to be mediated solely by a systemic factor.     

P-450-dependent arachidonic acid metabolism in rabbit olfactory microsomes.

Microsomes isolated from rabbit olfactory epithelium catalyzed the conversion of arachidonic acid to several metabolites at a rate of 692 +/- 106 pmol/min/nmol P-450. The major metabolite was the omega-hydroxylated metabolite, 20-hydroxyeicosatetraenoic acid, accounting for 57% of the total metabolite produced. A putative omega-1 hydroxylated metabolite was also formed to a lesser extent. Epoxyeicosatrienoic acids were not detected with microsomal incubations, although metabolites corresponding to the dihydroxyeicosatrienoic acids were observed and represented about 20% of the total metabolite produced. The metabolism of arachidonic acid was also studied in a reconstituted system with six purified P-450 isoforms that are known to be expressed in rabbit olfactory mucosa. These included P-450NMa, P-450 2G1 (NMb), P-450 1A2, P-450 2B4, P-450 2E1, P-450 3A6 and a partially purified preparation of P-450 4A isoforms. The P-450 4A forms were the only enzymes to produce significant amounts of the omega-hydroxylated metabolite of arachidonic acid. The other isoforms were either inactive (P-450NMa and P-450 3A6) or produced metabolites other than the 20-hydroxyeicosatetraenoic acid and, thus, cannot account for the majority of the miscrosomal metabolism of arachidonic acid. Immunoblot analysis with goat anti-rat P-450 4A1 identified one major and a second minor protein of the P-450 4A gene family in olfactory microsomes. The same antibody identified two proteins in rabbit renal tissue that were significantly induced by pretreatment with clofibric acid and were present in a partially purified preparation of P-450 4A from rabbit renal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of arachidonic acid metabolism by macrophage activation

Levels of zymosan-induced arachidonic acid (20:4) metabolism by peritoneal macrophages elicited with inflammatory agents and resident macrophages were similar. Thyioglycollate (THIO)-elicited macrophages represented the exception; however, the diminished metabolism by these cells was reproduced by exposing resident cells to 5 mg/ml THIO broth in vitro. In contrast, reduced prostaglandin synthesis by macrophages from mice variously treated with the immunologic agents, Corynebacterium parvum or Bacille Calmette Guerin (BCG), closely correlated with enhanced antitoxoplasma activity, one measure of macrophage activation. This relationship, although not causative, suggested that the capacity for 20:4 metabolism is a function of the macrophage activation state. Modulation of macrophage 20:4 metabolism in vivo apparently required factors in addition to lymphocyte-derived products. Treatment of resident macrophages in vitro with BCG lymphokine was without effect on 20:4 release or prostaglandin synthesis. Activated macrophages from animals inoculated i.p. with C. parvum exhibited reduced 20:4 release and also failed to metabolize 70% of the 20:4 released in response to a zymosan stimulus. Consequently, the quantities of 20:4 metabolites formed were significantly less than expected from 20:4 release. These activated macrophages displayed greatly reduced synthesis of prostacylcin and leukotriene C compared with other 20:4 metabolites. It appeared that factors that regulate macrophage 20:4 metabolism influence the level of the inducible phospholipase and synthetic enzymes for specific 20:4 oxygenated products.     

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.     

Effect of homocysteine on arachidonic acid release in human platelets

BACKGROUND: It has been suggested that homocysteine is implicated in the risk of atherosclerosis and thrombosis. The pathogenic mechanism has not been clarified, but oxygen-free species produced by the homocysteine metabolism and auto-oxidation could have a role. DESIGN: We have studied the effect of homocysteine on arachidonic acid release in human platelets. Two important products of arachidonic acid metabolism - thromboxane B2 (TXB2) and reactive oxygen species (ROS) - have been assayed. RESULTS: Results indicate that homocysteine induces arachidonic acid release that is partially inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA). Platelet incubation with homocysteine significantly increases basal levels of TXB2 and ROS. The effect is time- and dose-dependent. The TXB2 formation is strictly correlated with the arachidonic acid release. Moreover, ROS accumulation is largely inhibited by ETYA and partially reduced by diphenyleneiodonium (DPI), suggesting the involvement both of enzymes metabolising arachidonic acid (cyclooxygenase, lipooxygenase, cytochrome P450 monooxygenase) and of NAD(P)H oxidase. CONCLUSION: Homocysteine induces oxidative stress in human platelets in vitro. The unbalance in platelet redox-state and the increased TXB2 formation may generate hyperactivation, contributing to a thrombogenic state leading to cardiovascular diseases.     

Rifampin inhibits prostaglandin E2 production and arachidonic acid release in human alveolar epithelial cells

Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogeneses of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E(2) (PGE(2)) production in human alveolar epithelial cells stimulated with interleukin-1beta. Rifampin caused a dose-dependent inhibition of PGE(2) production. At doses of 100, 50, and 25 microg/ml, it inhibited PGE(2) production by 75%, 59%, and 45%, respectively (P < 0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25, and 10 mug/ml, it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (P < 0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A(2) or the expression of cyclooxygenase-2. The inhibition of PGE(2), and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis and may explain some of its adverse effects.     

Human complement in the arachidonic acid transformation pathway in platelets

Arachidonate-mediated release of 14C serotonin and thromboxane B2 (TXB2) is significantly enhanced in the presence of complement. Only purified complement components C5, C6, C7, C8, and C9 are required for this reactivity. No known activating mechanism of the classical or alternative pathway is required, nor is C3. In the absence of exogenously added complement, platelet membrane-bound complement components play an essential role in modulating arachidonate-mediated serotonin release. Incubation of platelet membranes with arachidonate and C5--C9 led to the production of dimers of the membrane attack complex (C5b--9) on the platelet surface. These macromolecular complexes were eluted from the platelet membrane and were identified physicochemically and morphologically. The possibility arises that C3 in association with C5--C9 is required for mobilization of the arachidonic acid from the phospholipid of the platelet membrane. Once the arachidonic acid is mobilized, C3 is no longer required, C5--C9 being sufficient to modulate this pathway leading to enhanced production of TXB2.     

Mechanism of enhanced fibroblast arachidonic acid metabolism by mononuclear cell factor.

Chronic inflammation is associated with an infiltration of mononuclear cells, fibroblast proliferation, and elevated levels of prostaglandin (PG) E2. Mononuclear cell conditioned factor (MNCF) medium (5%) stimulated a 100-fold increase in basal human dermal fibroblast PGE2 release over 48 h as compared with fibroblasts that were incubated with control medium (conditioned medium prepared without cells). The MNCF-induced PGE2 production was suppressed by protein synthesis inhibitors. Fibroblasts pretreated with control medium released PGE2 only modestly in response to 1 nM bradykinin for 1 h (basal, 50 +/- 7 pg PGE2/micrograms protein; stimulated, 104 +/- 12 pg PGE2/micrograms protein), whereas cells that had been pretreated with MNCF showed a greatly facilitated bradykinin-induced release of PGE2. (basal, 297 +/- 59 pg PGE2/micrograms protein; stimulated, 866 +/- 85 pg PGE2/micrograms protein). The exaggerated agonist response is not specific for bradykinin because platelet-derived growth factor elicits a similar response. Exogenous arachidonic acid conversion to PGE2 was also facilitated (two- to threefold) by MNCF pretreatment as compared with control. Both the enhanced agonist-stimulated and exogenous arachidonic acid-induced PGE2 release from the MNCF pretreated cells were inhibited by actinomyin D or cycloheximide. A kinetic study of microsomal cyclooxygenase prepared from fibroblasts pretreated with MNCF showed a threefold increase in the maximum velocity (Vmax) but the same Michaelis constant (Km) as control-treated cells. This augmented arachidonic acid metabolism and subsequent enhanced PGE2 production may play an important role in macrophage-fibroblast interactions at sites of inflammation.     

A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells.

We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects. Cell incubation with AA (0.3-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry. Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA. Two epoxyeicosatrienoic acids derived from AA were also identified. Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects. Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets. Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions. The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.     

Glucose-induced protein kinase C activity regulates arachidonic acid release and eicosanoid production by cultured glomerular mesangial cells.

Changes in glomerular eicosanoid production have been implicated in the development of diabetes-induced glomerular hyperfiltration and glomerular mesangial cells (GMC) are major eicosanoid-producing cells within the glomerulus. However, the mechanism for the effect of diabetes mellitus on glomerular mesangial eicosanoid production is unknown. The present study therefore examined whether elevated glucose concentrations activate protein kinase C (PKC) in GMC and whether this PKC activation mediates an effect of elevated glucose concentrations to increase the release of arachidonic acid and eicosanoid production by GMC. The percentage of [3H]arachidonic acid release per 30 min by preloaded GMC monolayers was significantly increased after 3-h exposure to high glucose (20 mM) medium (177% vs control medium) and this increase was sustained after 24-h exposure to high glucose concentrations. 3-h and 24-h exposure to high glucose medium also increased PGE2, 6-keto-PGF1 alpha, and thromboxane (TXB2) production by GMC. High glucose medium (20 mM) increased PKC activity in GMC at 3 and 24 h (168% vs control). In contrast, osmotic control media containing either L-glucose or mannitol did not increase arachidonic acid release, eicosanoid production, or PKC activity in GMC. Inhibiting glucose-induced PKC activation with either H-7 (50 microM) or staurosporine (1 microM) prevented glucose-induced increases in arachidonic acid release and eicosanoid production by GMC. These data demonstrate that elevated extracellular glucose concentrations directly increase the release of endogenous arachidonic acid and eicosanoids by GMC via mechanisms dependent on glucose-induced PKC activation.     

The effect of irradiation on platelet arachidonic acid metabolism

Irradiation of platelets in vitro to 2000 rads did not alter arachidonic acid metabolism through the cyclooxygenase pathway. Production of platelet thromboxane B2 and 12-hydroxy-5,8-10-heptadecatrienoic acid remained similar in control and paired irradiated platelets. In paired samples the platelet lipoxygenase metabolite 12-hydroxy-5,8,10,14-eicosatetraenoic acid was significantly increased postirradiation to 42 +/- 2.8 percent when compared to a control value of 34.1 +/- 3.6 percent (p less than 0.02). Although this latter product has been demonstrated to be chemotactic for neutrophils, its role in platelet function has not been specifically determined.     

Effect of indomethacin on arachidonic acid metabolism in human leukocytes stimulated ex vivo.

We had previously shown that inhibition of cyclooxygenase in vitro by indomethacin can cause increased formation of products of the 5-lipoxygenase pathway of arachidonic acid metabolism in leukocytes. To determine if this effect also occurred in vivo, we studied leukocyte arachidonic acid metabolism in 12 volunteers before and after ingestion of 150 mg indomethacin daily for 3 days. Blood was collected before treatment and 2 hours, 2 days, and 5 days after the final dose of indomethacin. Serum thromboxane B2, a measure of platelet cyclooxygenase activity, was profoundly suppressed 2 hours after the final dose of indomethacin but had recovered to control values at 2 days. Mixed leukocyte suspensions and purified neutrophil suspensions were prepared and stimulated with calcium ionophore A23187 and the resultant 5-lipoxygenase metabolites were quantified by HPLC. Two hours after the final dose of indomethacin, the stimulated levels of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and leukotriene C4 were significantly increased to 247% +/- 68%, 135% +/- 14%, and 149% +/- 23% of pretreatment values, respectively. Two days after the final dose of indomethacin, 5-hydroxyeicosatetraenoic acid levels were still significantly elevated. By 5 days all parameters had returned to baseline. Similar effects were not observed in purified neutrophil suspensions, probably because of the loss of indomethacin from the cells during the multiple washing procedures used in their preparation. This is in accord with the reversible nature of the inhibitory effect of indomethacin on cyclooxygenase. We conclude that indomethacin at a commonly used dose increases the ability of circulating leukocytes to produce 5-lipoxygenase products.     

葡萄糖胰岛素释放试验对估价糖尿病的胰岛β细胞功能及其治疗效果探讨

对36例糖尿病人进行葡萄糖胰岛素释放试验(GIRT),结果糖耐量与胰岛素释放曲线与健康人比较明显降低(P<0.01);据患者β细胞功能状态,其胰岛素释放线态呈“胰岛素分泌缺乏型”(DIS)、“胰岛素分泌不足型”(IIS)及“高胰岛素分泌型”(HIS)、三型胰岛素分泌值、各项胰岛素分泌指数、血糖面积与胰岛素面积比较具有重要的统计学意义(P<0.01);治疗后,HIS型与IIS型者上述参数指标可获明显改善。由于提示GIRT对估价糖尿病患者细胞功能状态、疾病诊断分型、治疗效果以及预后判断都有较大实用价值。     

Plasmin inhibition of platelet function and of arachidonic acid metabolism

To study interactions between platelets and the fibrinolytic system, we examined the effects of human plasmin on human platelets washed by gel filtration. Plasmin concentrations that did not affect platelet shape change, release, or aggregation (less than 1.0 caseinolytic units [CU]/ml) caused a dose- and time-dependent inhibition of platelet aggregation in response to thrombin, ionophore A23187, and collagen. Complete loss of aggregation occurred at 0.1-0.5 CU/ml of plasmin. In a parallel dose-dependent manner, plasmin likewise inhibited thrombin, ionophore, and collagen-stimulated thromboxane B2 production. In contrast, neither aggregation nor thromboxane B2 formation induced by arachidonate was inhibited by plasmin pretreatment of the platelets. Plasmin blocked the thrombin-induced release of [3H]arachidonic acid from platelet membrane phospholipids and the thrombin-induced platelet oxygen burst. However, plasmin did not inhibit the arachidonate-induced oxygen burst. Inhibition of arachidonic acid release by plasmin was not mediated by increase in platelet cyclic AMP. These results suggest that plasmin inhibits platelet function, at least in part, by blocking the mobilization of arachidonic acid from membrane phospholipid pools. The effects of plasmin on platelets may contribute to the hemostatic abnormalities seen in pathologic and pharmacologic fibrinolysis.