Inhibitors Of Arachidonic Metabolism Research Articles

Antiplatelet therapies: An overview

The role of blood components including platelets, in initiating inflammation, endothelial dysfunction, atherosclerosis, thrombus formation, thrombus growth, and acute vascular ischemic events, is well established. Given this recognized role played by platelets, there is a considerable interest in understanding the physiology and function of platelets, as well as in the development of novel platelet function-inhibitory drugs. The generation of the second messengers, calcium mobilization, shape change, adhesion, aggregation, contraction, release of granule contents, thrombus development, thrombus growth, and formation of hemostatic plug at the injured vessel surfaces, in brief, constitute platelet activation. Some of the known compounds that inhibit platelet activation include inhibitors of arachidonic metabolism (cyclooxygenase-1 inhibitors; aspirin, ibuprofen, etc.), adenosine diphosphate receptor antagonists (P2Y 12 inhibitors), adenylyl and guanylyl cyclase stimulators, calcium antagonists, and GP11b/111a receptor antagonists. Since platelets have multiple mechanisms of achieving in vivo activation, it is difficult to design a novel drug that offers total protection for developing acute ischemic vascular events, without compromising coagulation mechanisms. Given this complexity, any aggressive antiplatelet therapy results in increased bleeding episodes. Having said that, we feel that there is a great window of opportunity for developing novel antiplatelet therapies. There is also scope for the development of fixed-dose combinations for the primary and secondary management of chronic diseases such as hypertension, heart disease, and type-2 diabetes.

Identification of an endogenous inhibitor of arachidonate metabolism in human epidermoid carcinoma A431 cells

Identification of an endogenous inhibitor of arachidonate metabolism in human epidermoid carcinoma A431 cells

Mechanisms of cytokine-induced death of cultured bovine luteal cells

Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-gamma. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-beta on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A(2) inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-beta prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-beta may confer protective effects against cytokine-induced cell death in bovine luteal cells.

Mechanisms of cytokine-induced death of cultured bovine luteal cells

Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-gamma. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-beta on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A(2) inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-beta prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-beta may confer protective effects against cytokine-induced cell death in bovine luteal cells.

A Role for 12-lipoxygenase in Nerve Cell Death Caused by Glutathione Depletion

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinson's disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.

The role of macrophage-derived TNFa in the induction of sublethal tumor cell DNA damage

In previous studies we showed that tumor-associated macrophages isolated from murine mammary tumors are mutagenic to bacteria and mammalian cells and thus may contribute to tumor progression. We reported previously, and confirm here, that inflammatory macrophages induce DNA strand breaks in cultured mammary tumor cells co-incubated at a 1:1 ratio for 1 h. This activity is prevented by inhibitors of arachidonate metabolism or the removal of H2O2 with catalase. In the present study, we show that two antibodies to recombinant murine tumor necrosis factor alpha (rMuTNFa)--a hamster monoclonal antibody (TN3-19.12) and a rabbit polyclonal antibody (Genzyme)--partially protect tumor cells from DNA strand breaks induced by elicited but not resident peritoneal macrophages. Antibody protection was reversed upon the addition of excess exogenous rMuTNFa. Purified rMuTNFa alone was unable to induce DNA strand breaks in the absence of macrophages, indicating that TNFa is necessary but not sufficient to mediate damage. Tumor target cells were completely resistant to the cytotoxic effects of rMuTNFa in the absence of actinomycin D and relatively resistant (in comparison to WEHI 164 clone 13 cells) in its presence. The incomplete protection seen with either catalase or anti-TNF suggests that macrophage-released TNFa, in the presence of other factors, induces non-cytotoxic DNA effects in tumor cells.

Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties.

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.

Expression of prostaglandin [formula omitted] synthase (cyclooxygenase) during murine fetal thymic development

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [ 14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF 1α, PGF 2α, PGE 2, and PGA 2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [ 14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A 2, which liberates arachidonic acid from membrane phospholipids, and Cyclooxygenase (prostaglandin G H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI 2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.

Chemotactic response of rat macrophages is enhanced by two diastereoisomers of LTB 4

We demonstrated that rat peritoneal cells synthesize, and release into the culture medium, substances that elicit a chemotactic responsiveness of rat macrophages. These substances were identified by HPLC analysis as two diastereoisomers of LTB 4, precisely Δ-6- trans-LTB 4 and Δ-6-epi- trans-LTB 4. Peritoneal cells release, also, other lipoxygenase metabolites such as LTD 4 and LTB 4, which were shown not to be active in eliciting chemotaxis of rat macrophages, in agreement with data of other authors. Quantitative assays for the measurement of PMNs or macrophage chemotaxis were carried out in Boyden chambers. Release of leukotrienes by rat peritoneal cells into the culture medium was strongly enhanced upon stimulation with calcium ionophore A23187, an agent known to increase cytoplasmic Ca 2+ concentration, thus leading to the activation of Ca 2+-dependent phospholipase and a consequent increase in the amount of endogenous free arachidonic acid available for biotransformation. The action of several inhibitors of arachidonate metabolism at concentrations more selective for the lipoxygenase than the cycloxygenase pathway, was also studied. α-Tocopherol and ASA were shown to be the most selective in inhibiting the synthesis of both the LTB 4 diasteroisomers active as enhancers of rat macrophage chemotaxis.

Effects of anti-inflammatory agents on lymphocyte migration stimulated by the interferons, tumor necrosis factor and cutaneous inflammation

Our goal was to examine the effect of anti-inflammatory agents on the migration of lymphocytes to cutaneous inflammatory sites and in response to cytokines. The accumulation of i.v. injected 111In-labelled peritoneal exudate lymphocytes in skin sites of rats injected with KLH to induce DTH reactions, LPS, poly I:C and the cytokines, IFN-γ, IFN-α/β and tumor necrosis factor was determined. Systemic dexamethasone (DEX) treatment strongly inhibited the migration of lymphocytes in response to all of the stimuli, however, the effective dose of DEX varied widely with the different recruiting agents. The lowest ED 50 was observed with LPS, while IFN-α/β was the least inhibited. DEX treatment increased lymphocyte accumulation in the bone marrow and spleen. Pretreatment of lymphocytes with DEX had no effect on their migration, while local i.d. hydrocortisone inhibited migration into the skin. Cyclosporin A treatment had no effect on lymphocyte recruitment in response to any of the cytokines or LPS but significantly inhibited migration to DTH reactions and poly I:C. Treatment of rats with indomethacin, ASA and BW755C produced only a marginal inhibition of lymphocyte migration in response to some of the stimuli tested. DEX is a potent inhibitor of lymphocyte migration to inflammation. In addition to suppressing cytokine production, it can suppress migration to cytokines, probably through inhibiting the effects of these agents on the vascular endothelium. Cyclosporin A decreases lymphocyte accumulation only through its ability to suppress lymphokine production, while inhibitors of arachidonate metabolism have little direct effect on lymphocyte migration.

Interleukin-1 alpha modulates collagen gene expression in cultured synovial cells.

The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level.

Blood coagulation and bone metabolism: some characteristic of the bone resorptive effect of thrombin in mouse calvarial bones in vitro

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1–7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [ 3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, β-glucuronidase and β- N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal diesease and rheumatoid arthritis.

Effects of antiinflammatory drugs on the release from porcine synovial tissue in vitro of interleukin 1 like cartilage degrading activity.

The effects were studied of antiinflammatory drugs possessing inhibitory effects on (a) cyclooxygenase (CO), (b) cyclooxygenase plus 5-lipoxygenase (CO-LO), (c) 5-lipoxygenase (5-LO) and (d) phospholipase A2 (PLA2) activities, for their actions on the release from porcine synovial tissue in culture of cartilage-degrading ("catabolin" or interleukin-1 like) activity (= CDA). CDA was reduced by CO inhibitors, enhanced by CO-LO and PLA2 inhibitors, and unaffected by 5-LO inhibitors. These results suggest that (a) antiinflammatory drugs may affect synovial production of CDA ascribable to IL-1, according to their properties as inhibitors of arachidonate metabolism, and (b) products of arachidonic acid metabolism may influence production of synovial CDA.

Leukotriene B&lt;sub&gt;4&lt;/sub&gt; Production in Normal Rat Glomeruli

The production of the lipoxygenase metabolite of arachidonic acid, leukotriene B4 (LTB4) by normal rat glomeruli has been studied. Isolated glomeruli from saline-perfused rat kidneys were incubated in Kreb's buffer for 15 min at 37 degrees C. The concentrations of LTB4 and other eicosanoids in cell-free supernatants were determined by direct radioimmunoassays (RIA). Mean basal syntheses of eicosanoids were: thromboxane B2 (TXB2) 0.77 +/- 0.13, prostaglandin (PG) E2 0.40 +/- 0.05, 6-keto-PGF1 alpha 0.38 +/- 0.06 ng/mg glomerular protein (mean +/- SEM n = 8). In these assays immunoreactive LTB4 synthesis was 0.12 +/- 0.02 ng/mg. In samples incubated with BW755C, 50 micrograms/ml, an inhibitor of arachidonate metabolism via both the cyclo-oxygenase and lipoxygenase pathways, there was more than 80% inhibition of the synthesis of TXB2, PGE2 and 6-keto-PGF1 alpha. However, immunoreactive LTB4 was only reduced by 44%, suggesting the presence of other materials which cross-react in the RIA. The presence of authentic LTB4 in the supernatants was confirmed after extraction and high-pressure liquid chromatography (HPLC). This material represented 25% of the original material detected by RIA. Although the physiological role of LTB4 in the normal state is unknown, its chemotactic activity may be of great significance during glomerular inflammation.

Nafazatrom, an arachidonate metabolism inhibitor, decreases prolactin and GH release

Nafazatrom, an inhibitor of arachidonate metabolism by the lipoxygenase enzymes, decreases basal prolactin and growth hormone release in a concentration-dependent manner without significantly affecting the synthesis of either hormone. It is required that nafazatrom be incubated with pituitary cells for approximately 2 h for this effect to become evident; the blockade increases in magnitude for up to 4 h. Nafazatrom blocks the increase in prolactin release caused by the releasing factors TRH and angiotensin II and the increase in prolactin and growth hormone release due to the calcium channel activator maitotoxin. These data suggest that the lipoxygenase products of arachidonate metabolism may be important mediators in basal and secretagogue-induced release of prolactin and growth hormone.

Blockade of N1E-115 murine neuroblastoma muscarinic receptor function by agents that affect the metabolism of arachidonic acid

Inhibitors of arachidonate metabolism and perturbants of the oxidation-reduction state of the cell were employed to develop a pharmacologic profile for muscarinic receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Several lipoxygenase inhibitors [eicosatetraynoic add (ETYA), nordihydroguaiaretic acid (NDGA), FPL 57231, FPL 55712, BW755c, propylgallate, and AA861] blocked the elevation of [ 3H]cyclic GMP induced by muscarinic receptor activation. The cyclooxygenase inhibitors indomethacin and ibuprofen were two orders of magnitude less potent in blocking the muscarinic receptor-mediated [ 3H]cyclic GMP response than in blocking cyclooxygenase in other systems. ETYA and NDGA did not affect the muscarinic inhibition of the prostaglandin E 1-mediated increases in [ 3H]cyclic AMP levels in N1E-115 cells. ETYA did not have a reproducible effect on the muscarinic receptor-induced release of inositol phosphates. Thus, these lipoxygenase inhibitors appeared to be selective for the effector system coupled to the low-affinity muscarinic agonist-receptor conformation, i.e. that which induces cyclic GMP formation. Other effective inhibitors of the cyclic GMP response were methylene blue, catalase, bromphenacyi bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant α-tocopherol in the concentration range of 100 μM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response ( ic 50 = 45 μM). Linoleic acid and oleic acid were less potent ( ic 50 = 130 and 190μM, respectively), and stearic acid was ineffective. When arachidonic acid was air-oxidized, its inhibitory potency was increased 10-fold. Most but not all of the spontaneously-produced oxidative metabolites, separable by reverse-phase high pressure liquid chromatography, were inhibitory to the receptor response. Enzymatically synthesized 12-hydroxyeicosatetraenoic add and 15-hydroxyeicosatetraenoic add inhibited the muscarinic receptor [ 3H]cyclic GMP response, with ic 50 values of 17 and 8 μM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response ( ic 50 = 5μM) while having no effect on either the muscarinic receptor-induced inositol phosphate release or the reduction of cyclic AMP levels. Thus, the effector system for increasing cyclic GMP in these cells displays many of the expected characteristics for the involvement of a lipoxygenase or a related enzyme that oxidatively metabolizes arachidonate in order to activate the guanylate cyclase. A number of pharmacologic agents differentiate between this effector system and those to which the muscarinic receptor couples in order to effect cyclic AMP decreases or the release of inositol phosphates.

The effects of interleukin-1, arachidonate and inhibitors of arachidonate metabolism on the activity of preoptic thermosensitive neurons

The effects of interleukin-1, arachidonate and inhibitors of arachidonate metabolism on the activity of preoptic thermosensitive neurons

Possible role of endogenous arachidonic acid metabolites in stimulated release of insulin and glucagon from the isolated, perfused rat pancreas.

Enhancement of arachidonic acid metabolism results in increased insulin secretion. To determine which pathways of arachidonic acid metabolism were involved in this stimulation, we studied the effects of various inhibitors of arachidonate metabolism on arginine-induced insulin and glucagon secretion in the isolated, perfused rat pancreas. The release of PGE2 from the pancreas was monitored to document the efficacy of the inhibitory drugs. p-Bromophenacyl bromide, a phospholipase A2 inhibitor, diminished PGE2 release and significantly inhibited both the early and late phases of insulin and glucagon release in response to arginine. Flurbiprofen, a specific cyclooxygenase inhibitor, decreased the early phase of insulin release and inhibited both phases of arginine-stimulated glucagon secretion; these decreases were concurrent with a large inhibition of PGE2 release. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, at a dose of 10(-5) M did not affect PGE2 release, inhibited the early phase of insulin release, and did not modify glucagon secretion. The combination of flurbiprofen and nordihydroguaiaretic acid, although the most potent in inhibiting PGE2, lowered only the early phase of insulin and had no effect on glucagon secretion. We conclude that: (1) endogenous cyclooxygenase-derived metabolites of arachidonic acid promote insulin and glucagon release, (2) endogenous lipoxygenase products preferentially stimulate insulin release, and (3) phospholipase A2 activity has an intrinsic modulatory effect on insulin and glucagon secretion.

Arachidonic acid metabolism in isolated pancreatic islets: I. Identification and quantitation of lipoxygenase and cyclooxygenase products

The metabolism of arachidonic acid by pancreatic islets has been studied with purified populations of large numbers of islets isolated from the rat. Sequential high-performance liquid chromatographic analyses of islet-derived metabolites of 3H-labeled arachidonate in both reversed and normal phases with 14C-labeled internal standards have demonstrated synthesis by the islets of the cyclooxygenase products prostaglandin E 2, prostaglandin F 2α, thromboxane B 2 and 12-hydroxyheptadecatrienoic acid as well as the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE). Islet synthesis of these compounds was suppressed with appropriate inhibitors of arachidonate metabolism. Synthesis of the identified metabolites from endogenous arachidonate has also been quantitated with the use of deuterated internal standards, capillary column gas chromatographic analyses, and negative ion-chemical ionization mass spectrometric measurements. The relative abundances of metabolites derived from exogenous, radiolabeled arachidonate versus endogenous precursor differed considerably, and 12-HETE was by far the most abundant of these metabolites synthesized from endogenous arachidonate. Platelets contaminating the isolated islet preparations have been excluded as the source of the identified arachidonate metabolites. These studies establish that cells intrinsic to pancreatic islets synthesize a clearly characterized profile of arachidonate lipoxygenase and cyclooxygenase products. The sensitive and specific mass spectrometric methods for quantitation of these compounds permit detailed evaluation of their possible participation in insulin secretion from isolated islets.

Effects of lipoxygenase and cyclooxygenase inhibitors on glucose-stimulated insulin secretion from the isolated perfused rat pancreas

Some of the metabolites of arachidonic acid formed in the lipoxygenase and cyclooxygenase pathways stimulate insulin release. We studied the relative importance of each of these pathways in the modulation of glucose-induced insulin release by using inhibitors of arachidonate metabolism. Perfusion of the isolated rat pancreas with two chemically different inhibitors of cyclooxygenase, flurbiprofen and sodium salicylate, markedly inhibited prostaglandin E2 release, but had little effect on glucose-induced insulin release or on potentiation of insulin release caused by prior exposure to glucose. On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, not only inhibited both phases of glucose-induced insulin release but also abolished the potentiation effect. These effects of NDGA prevailed, when it was administered together with flurbiprofen, which caused profound inhibition of prostaglandin E2 release. We conclude that 1) lipoxygenase pathways play a dominant role in glucose-stimulated insulin release, and 2) endogenous lipoxygenase metabolites influence the potentiating effect of glucose on the release of insulin in response to a subsequent stimulation.