Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Profiles of eicosanoid production from 14C-arachidonic acid and prostaglandin metabolism by rabbit esophageal mucosa and muscularis

In an attempt to elucidate the possible involvements of eicosanoids in esophageal functions and disorders, we have investigated the formation of both cyclooxygenase and lipoxygenase metabolites from 14C-arachidonic acid by rabbit esophageal tissues. Homogenates of rabbit esophageal mucosa and muscularis were incubated with 14C-arachidonic acid and after ether extraction eicosanoids were separated and quantified by reverse phase high performance liquid chromatography. The predominant cyclooxygenase products were 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 for mucosa and 6-keto-PGF1 alpha, and PGE2 for muscularis. The formation of these products was inhibited both by indomethacin and the dual pathway inhibitor, nordihydrogualaretic acid (NDGA). In mucosa the major eicosanoid was 12-HETE (12-hydroxyeicosatetraenoic acid) which was inhibited by NDGA but not by indomethacin which on the contrary enhanced its formation. Additionally four polar products were synthesized which appeared to be lipoxygenase-dependent as their formation was inhibited by NDGA but not by indomethacin. Muscularis produced as a minor lipoxygenase product only 12-HETE, which was inhibited by NDGA but unchanged in the presence of indomethacin. In addition, both tissues, but mucosa more than muscularis, possessed large prostaglandin catabolizing capacity. The present findings indicate that rabbit esophageal tissues can convert 14C-arachidonic acid into lipoxygenase as well cyclo-oxygenase products which may have a role in esophageal physiology and pathophysiology.     

Lipoxygenases in rat embryo tissue

It has previously been reported that rat embryonic tissue produces various prostanoids. This report demonstrates that rat embryo homogenates synthesized various lipoxygenase metabolites, including 12-hydroxyeicosatetraenoic acid (12-HETE) as the major metabolite, 5-HETE, and 15-HETE. The cyclooxygenase product 11-HETE was also formed. Product identification was based on radioimmunoassay and comparison of reverse-phase- and straight-phase-high-pressure liquid chromatography retention times with authentic standards. Additional evidence was the observation that the lipoxygenase inhibitor nordihydroguaiaretic acid inhibited HETE formation. It appears that, under the same (though not necessarily optimal) experimental conditions, lipoxygenase metabolites predominate quantitatively over cyclooxygenase pathway products and that 11-day embryonic tissue produces more HETEs than either 12-day or 13-day embryo homogenates.     

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.     

A rapid miniature thin layer chromatography system for analysis of prostaglandins an lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.     

A rapid miniature thin layer chromatography system for analysis of prostaglandins and lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid

Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.     

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore: Suppression by inhibitors of fatty acid lipoxygenase

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [³H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5–2 μg/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.     

Accumulation of Arachidonic Acid Cyclo- and Lipoxygenase Products in Rat Brain During Ischemia and Reperfusion: Effects of Treatment with GM1-Lactone

The aim of our study was to investigate the changes of various biochemical parameters (concentrations of lactate, free arachidonate, cyclo- and lipoxygenase products) in rat brain after ischemia and reperfusion and the effects of pretreatment with the ganglioside derivative GM1-lactone on the same parameters. Ischemia was induced by reversible occlusion of common carotid arteries for 20 min, which included a final 5 min of respiration of 5% oxygen in nitrogen. Reperfusion was obtained by removing the occlusion. Pre-ischemic conditions were obtained on sham-operated animals. Animals were killed by microwave irradiation of their heads. Brain levels of lactate and of free arachidonate were markedly increased after ischemia and returned to normal values at 5 min of reperfusion. Levels of the cyclooxygenase metabolites prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2 were increased after ischemia, whereas levels of the lipoxygenase metabolite leukotriene C4 (LTC4) did not change. After reperfusion, a very marked increase of the cyclooxygenase products occurred but not of LTC4. Treatment with GM1-lactone prevented the elevation of cyclo- and lipoxygenase metabolites especially during reperfusion, with limited effects on lactate and free arachidonate levels.     

Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate.

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.     

Arachidonate metabolism in the mouse thyroid implication of the lipoxygenase pathway in thyrotropin action

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.     

The production of arachidonic acid metabolites in rat testis.

There is growing evidence that arachidonic acid is oxygenated enzymatically in every cell type and that the oxygenated metabolites regulate a variety of pathological and physiological processes including reproduction. In the present study, the metabolism of arachidonic acid in the testis via cyclooxygenase and lipoxygenase pathways was analyzed. Testicular microsomes showed substantial cyclooxygenase activity as measured by the polarographic method. Analysis of the products on TLC revealed PGF2 alpha (79.5%) as the main product followed by PGE2 (20.3%) and PGD2 (0.17%). At higher substrate concentrations (150 microM), however, 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, was observed in substantial quantities. Maximum activity of lipoxygenase was observed at pH 6.4 in both microsomes and cytosol, the activity being higher in cytosol. Analysis of lipoxygenase pathway products with arachidonic acid as the substrate, revealed the presence of 12-HPETE as the major product both in cytosol and in microsomes. Besides this, 15- and 5-HPETEs were also observed in substantial quantities.     

Effects of protein deficiency on the metabolism of arachidonic acid by rat pleural polymorphonuclear leukocytes

The effects of protein deficiency on the biosynthesis of metabolites of arachidonic acid by rat pleural polymorphonuclear leukocytes stimulated with calcium ionophore were investigated. The major products of metabolism by lipoxygenase in these cells were leukotriene B4 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas the major cyclooxygenase products were thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. At high substrate concentrations (100 microM), the formation of all products by polymorphonuclear leukocytes was lower for protein-deficient rats than for controls. Similar results were obtained when products synthesized from endogenous substrate were measured, except that there was no change in the amount of 5-hydroxy-6,8,11,14-eicosatetraenoic acid formed. The biosynthesis of prostaglandins E2 and F2 alpha by homogenates of rat kidney medulla was reduced as a result of protein deficiency. Acetylsalicylic acid inhibited the formation of cyclooxygenase products and stimulated the formation of lipoxygenase products by polymorphonuclear leukocytes. Protein deficiency did not alter the effects of acetylsalicylic acid on the biosynthesis of these products, although at any given concentration the amounts of products formed were less with protein-deficient rats than with rats fed control diets.     

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.     

Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10⁻⁴M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate ⁸⁶Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or ⁸⁶Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10⁻⁵M indomethacin. Eicosatetraynoic acid (2×10⁻⁵M) inhibited ⁸⁶Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10⁻⁴M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands.In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of ³²PO₄ into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9).There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17).The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.