On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [¹⁴C]arachidonic acid release and the release of histamine both required Ca²⁺ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [¹⁴C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D₂. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [¹⁴C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.     

Eicosanoid generation from antigen-primed mast cells by extracellular mammalian 14-kDa group II phospholipase A2.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.     

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.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release.

Indomethacin augmented the release of histamine and SRS-A but abolished synthesis of TxB2. Compound CLI that inhibited both cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism did not augment release of anaphylactic mediators. 13-HPLA enhanced mediator release from lungs in which arachidonic acid metabolism was blocked by compount CLI. Thus, it is concluded that 13-HPLA enhances mediator release not by altering the balance of arachidonic acid metabolites, e.g. by inhibiting synthesis of prostacyclin, but by a direct effect on lung mast cells. A corollary to this conclusion is that the fatty acid hydroperoxide (HPETE) formed by lipoxygenase from arachidonic acid may also augment the release of anaphylactic mediators. Thus, the enhancement of mediator release by indomethacin may be attributed to increased synthesis of HPETE following inhibition of cyclo-oxygenase.     

Effects of inhibitors of eicosanoid synthesis on insulin release by neonatal pancreatic islets

In the pancreatic islet, eicosanoids may arise from both cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid. The inclusion of inhibitors of selective steps in these pathways indicated that in cultured neonatal rat islets, arachidonic acid may be metabolised through both pathways, concurrent with insulin release stimulated by D-glucose, D-glyceraldehyde and 2-ketoisocaproate. The effects of the inhibitors suggested that the products of the lipoxygenase pathway were necessary for the stimulatory effects of nutrients to be observed. In contrast to glucose, where insulin release was stimulated in the presence of inhibitors of cyclooxygenase, the stimulatory action of D-glyceraldehyde, 2-ketoisocaproate and melittin was only minimally affected by these inhibitors, although it was inhibited by lipoxygenase inhibition. These findings support a major stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in nutrient-induced secretion, and a negative or modulatory role of cyclooxygenase pathway products on glucose-stimulated insulin release in the neonatal islet.     

Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Inhibition of COX activity by NSAIDs or ascorbate increases cAMP levels and enhances differentiation in 1alpha,25-dihydroxyvitamin D3-induced HL-60 cells

Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 uM ionophore, MC9 cells produce 270 ± 43 pmoles 5-HETE, 74 ± 40 pmoles 5,12 di HETEs and 65 ± 31 pmoles LTC₄/10⁶ cells from 37 uM exogenously added [1-¹⁴C]arachidonic acid in two minutes. 5-HETE and 5,12-di HETES, including LTB₄ were identified by GC/MS whereas LTC₄ was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD₂ and TxB₂ (8.5 ± 2.4 and 5.4 ± 1.2 pmoles/10⁶ cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca⁺² and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 ± 3% cellular histamine) and continued for 30 minutes (37 ± 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process.

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14-kDa group-II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast-cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase-A2 inhibitors suppressed histamine release in a concentration-dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non-immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14-kDa-group-II-phospholipase-A2-specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone-marrow-derived mast cells. When bone-marrow-derived mast cells were activated, they secreted both a soluble and an ecto-enzyme form of 14-kDa group-II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transiently. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was decreased by treatment with 14-kDa-group-II-phospholipase-A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14-kDa group-II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.     

Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80

Rat peritoneal mast cells respond to various secretagogues, such as ionophore A23187, concanavalin A (Ig E receptor cross-bridging) and compound 48/80 (membrane perturbing), to secrete histamine and to liberate arachidonic acid. Arachidonic acid release was made identifiable by pretreatment with BW755C, an inhibitor of both lipoxygenase and cyclo-oxygenase. The extent of arachidonic acid release varied among these three secretagogues. A23187 appeared to be most potent, whereas compound 48/80 was weakest. The sources of released arachidonic acids may be different depending on the types of stimulants. The stimulation with A23187 released arachidonic acid mainly from phosphatidylcholine and triacylglycerol. After treatment with concanavalin A and compound 48/80, in addition to phosphatidylcholine, phosphatidylinositol also appeared to serve as a donor of arachidonic acid.     

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures

Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.     

Influence of arachidonate metabolism on enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages

We examined whether arachidonate metabolism exerted any influence on the enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages. Enhancement of the intracellular transglutaminase activity was observed on stimulation of macrophages with normal sheep red blood cells (SRBC) or immunoglobulin G (IgG)-coated SRBC, and was inhibited by inhibitors of phospholipase A2 and cyclooxygenase. Moreover, prostaglandin E2 (PGE2), a main product of the cyclooxygenase pathway, leukotriene B4 (LTB4), a product of 5-lipoxygenase, and arachidonic acid also could directly induce high levels of intracellular transglutaminase activity without stimulation of macrophages by SRBC or IgG-coated SRBC, but leukotriene C4, prostaglandin D2, and prostacyclin were unable to induce high activity of the enzyme. Enhancement of transglutaminase activity induced by LTB4 was inhibited by cyclooxygenase inhibitor, but the enzyme activity induce by PGE2 was not inhibited. Furthermore, the quantity of PGE2 released into the culture medium of macrophages stimulated with SRBC or IgG-coated SRBC correlated well with the activity of intracellular transglutaminase in macrophages. Moreover, enhancement of transglutaminase activity by treatment of macrophages with SRBC or IgG-coated SRBC was partially suppressed by sodium benzoate, which is a scavenger of hydroxy radical. These findings suggest that arachidonate metabolism, in particular the cyclooxygenase pathway, plays an important role in the enhancement of intracellular transglutaminase activity.     

Differential alteration of lipoxygenase and cyclooxygenase metabolism by rat peritoneal macrophages induced by endotoxin tolerance

Altered macrophage arachidonic acid metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e. endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT)C4/D4 and prostaglandin (PG)E2 production by tolerant cells was greater than that by non-tolerant controls (p less than 0.001). However, A23187-stimulated i-6-keto-PGF1 alpha levels were lower in tolerant macrophages compared to controls. Stimulation of prostaglandin and thromboxane (Tx)B2 synthesis by endotoxin or glucan was significantly less in tolerant macrophages compared to controls (p less than 0.05). iLTC4/D4 production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in the non-tolerant cells. Synthesis of iLTB4 by control macrophages was stimulated by endotoxin (p less than 0.01). These results demonstrate that arachidonic acid metabolism via the lipoxygenase and cyclooxygenase pathways in macrophages is differentially altered by endotoxin tolerance.     

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells.

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.     

Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid

Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5-lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4.     

Piroxicam, a structurally novel anti-inflammatory compound. Mode of prostaglandin synthesis inhibition

Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.