Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2-200 microM) stimulated the synthesis of prostacyclin (PGI2), as reflected by the release of 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha), in endothelial cells cultured from bovine aorta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF1 alpha triggered by ADP was rapid and onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI2. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously

The capacity of cultured mesothelial cells to produce prostaglandins from both exogenous an endogenous arachidonic acid has been investigated. Incubations with labelled [1-14C]arachidonic acid and [1-14C]prostaglandin endoperoxide H2 indicated the formation of prostacyclin and prostaglandin E2. Evaluation of the transformation of endogenously released arachidonic acid, however, could only confirm the production of prostacyclin.     

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.     

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O2) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O2 levels respectively, p less than 0.05, compared to 21% O2 exposure values). Release of 3H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 microM for 5 min) or PGH2 (4 microM for 2 min) immediately after exposure. Endothelium exposed to 0% O2, but not to 10% O2, produced significantly less prostacyclin after addition of either arachidonic acid (25 +/- 5% of 21% O2 exposure values, n = 6, p less than 0.01) or PGH2 (31 +/- 3% of 21% O2 exposure values, n = 6, p less than 0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O2 level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O2 exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.     

Dopamine inhibits corticosteroid secretion in frog adrenocortical cells: evidence for the involvement of prostaglandins in the mechanism of action of dopamine

We have investigated the possible involvement of arachidonic acid metabolites in dopamine-induced inhibition of adrenocortical steroidogenesis. Administration of dopamine (5 x 10(-5) M) for 20 min to perifused frog adrenal slices caused a marked reduction of the release of both prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin (PGI2). Dopamine also induced a significant inhibition of corticosterone and aldosterone secretion. A lag period of 20 min was observed between inhibition of prostanoid and corticosteroid releases. Prolonged dopamine infusion did not prevent the stimulatory effect of PGE1, PGE2 or arachidonic acid on corticosteroid secretion. These observations indicate that activation of dopaminergic receptors in adrenocortical cells is linked to an inhibition of arachidonic acid metabolism. Our data also suggest that the inhibitory effect of dopamine occurs at a step preceding arachidonic acid formation.     

Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.     

Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells

The effects of elastase on prostacyclin biosynthesis in cultured rat aortic smooth muscle cells were investigated. Prostacyclin is the major product formed from arachidonic acid by aortic smooth muscle cells. When intact cells were incubated with elastase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident. However, the addition of elastase directly to the cell-free homogenates did not show any effects on prostacyclin biosynthesis. The maximal effect of elastase on the stimulation of prostacyclin biosynthesis without any cellular damage was observed at a concentration of 50 unit/ml elastase. Elastase also caused a marked release of arachidonic acid. At higher concentrations of elastase (75-100 units/ml), the release of arachidonic acid and prostacyclin synthesis was observed, but, at these concentrations of elastase, cells were slightly damaged. On the other hand, the releases of prostacyclin and arachidonic acid were markedly enhanced, when cells were preincubated with elastase (1 unit/ml) for 3 days. These results indicate that elastase, even at low concentrations, causes the releases of arachidonic acid and prostacyclin, especially when aortic smooth muscle cells are pre-treated with elastase.     

The platelet cyclooxygenase metabolite 12-L-hydroxy-5, 8, 10-hepta-decatrienoic acid (HHT) may modulate primary hemostasis by stimulating prostacyclin production

Although HHT accounts for approximately one third of the arachidonic acid (AA) metabolites produced by stimulated platelets, no well defined function has been attributed to this product. We report that HHT stimulates prostacyclin production by endothelial cells, and have identified the mechanism for this effect. In human umbilical venous endothelial cells, HHT (0.5 and 1 microM) stimulated prostacyclin (RIA for 6KPGF1 alpha) by 32 +/- 22% (1SD) and 42 +/- 38% (P less than 0.05 and less than 0.01). Similar changes were observed when the effect of HHT on exogenous [1-14C] AA metabolism in fetal bovine aortic endothelial cells (FBAECs) was studied. Kinetic analyses revealed that HHT affected vascular cyclooxygenase. HHT (1 microM) increased Vmax in test microsomes (706 +/- 21 pmol/mg/min, mean +/- 1SE) when compared to controls (529 +/- 20; P less than 0.02). No concomitant effect on Km was observed. A further effect of HHT on AA release from endothelial cell membrane phospholipids was noted. Prelabeling experiments revealed that HHT (1 microM) increased the ionophore stimulated release of AA from FBAECs (20952 +/- 555 cpm/well control mean +/- 1SE vs 25848 +/- 557 for paired HHT treated cells; P less than 0.05). The effect of HHT on platelet AA metabolism was next studied. Preincubation of washed platelets with HHT (1 microM) did not enhance thrombin or arachidonic acid induced platelet TXB2 formation. In platelets prelabelled with [1-14C]AA, HHT (1 microM) had no effect on AA release post thrombin stimulation. Conversion to cyclooxygenase metabolites was also not enhanced. HHT stimulates vascular prostacyclin without a concomitant effect on platelet AA metabolism. HHT may thus be an important local modulator of platelet plug formation.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Arachidonoyl-Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of terminal differentiation of adipose precursor cells by glucocorticoids.

The role of glucocorticoids on adipose conversion has been studied using confluent Ob1771 mouse preadipose cells maintained in a serum-free culture medium able to support the emergence of early but not that of late markers of differentiation. Under these culture conditions, glucocorticoids play, at physiological concentrations, a permissive role for terminal differentiation, characterized by glycerol-3-phosphate dehydrogenase expression and triacylglycerol accumulation within 12 days, whereas progesterone, testosterone, and estradiol are inactive. Glucocorticoids behave as mitogenic-adipogenic stimuli able to trigger growth-arrested, early marker-expressing cells to enter the terminal phase of the differentiation program and thus appear to mimic the mitogenic-adipogenic activity already described for arachidonic acid and cyclic AMP-elevating agents, especially prostacyclin. When compared to corticosterone alone, exposure of Ob1771 cells to both corticosterone and arachidonic acid leads to an additional increase in the glycerol-3-phosphate dehydrogenase activity and number of differentiated cells; this potentiation is further enhanced when the culture medium is supplemented with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. This suggests indirectly the involvement of prostacyclin as a metabolite of arachidonic acid able to induce cyclic AMP accumulation. In agreement with this hypothesis, it is found that a promoting effect is exerted by corticosterone on the metabolism of arachidonic acid, leading in turn to an increase in the production of prostacyclin. These findings allow a better understanding of the role of glucocorticoids on adipose cell differentiation and explain a posteriori the effectiveness of the combination of dexamethasone-isobutyl-methylxanthine used in innumerable studies.     

Eicosapentaenoic acid utilization by bovine aortic endothelial cells: effects on prostacyclin production

We have investigated whether the presence of other fatty acids in physiologic amounts will influence the effects of eicosapentaenoic acid on cellular lipid metabolism and prostaglandin production. Eicosapentaenoic acid uptake by cultured bovine aortic endothelial cells was time and concentration dependent. At concentrations between 1 and 25 microM, most of the eicosapentaenoic acid was incorporated into phospholipids and of this, 60-90% was present in choline phosphoglycerides. Eicosapentaenoic acid inhibited arachidonic acid uptake and conversion to prostacyclin (prostaglandin I2) but was not itself converted to eicosanoids. Only small effects on the uptake of 10 microM eicosapentaenoic acid occurred when palmitic, stearic or oleic acids were added to the medium in concentrations up to 75 microM. In contrast, eicosapentaenoic acid uptake was reduced considerably by the presence of linoleic, n-6 eicosatrienoic, arachidonic or docosahexaenoic acids. Although a 100 microM mixture of palmitic, stearic, oleic and linoleic acid (25:10:50:15) had little effect on the uptake of 10 or 20 microM eicosapentaenoic acid, less of this acid was channeled into endothelial phospholipids. However, the fatty acid mixture did not prevent the inhibitory effect of eicosapentaenoic acid on prostaglandin I2 formation in response to either arachidonic acid or ionophore A23187. An 8 h exposure to eicosapentaenoic acid was required for the inhibition to become appreciable and, after 16 h, prostaglandin I2 production was reduced by as much as 60%. These findings indicate that the capacity of aortic endothelial cells to produce prostaglandin I2 is decreased by continuous exposure to eicosapentaenoic acid. Even if the eicosapentaenoic acid is present as a small percentage of a physiologic fatty acid mixture, it is still readily incorporated into endothelial phospholipids and retains its inhibitory effect against endothelial prostaglandin I2 formation. Therefore, these actions may be representative of the in vivo effects of eicosapentaenoic acid on the endothelium.     

Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.     

The role of thrombin in the regulation of the endothelial prostaglandin production

Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+-ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Inhibition of prostacyclin synthesis in endothelial cells by methylisobutylxanthine is not mediated through elevated cAMP level

Methylisobutylxanthine (MIX) raised cAMP levels and inhibited prostacyclin synthesis and arachidonic acid release in endothelial cells from both pig aorta and human umbilical vein. These effects were reversible and dose dependent on MIX concentrations. Dibutyryl cAMP (3 mM) alone did not inhibit prostacyclin synthesis or arachidonic acid release. When added with MIX, dibutyryl cAMP did not enhance the inhibition elicited by MIX. MIX inhibited the formation of lysophospholipids, 1,2-diacylglycerol and phosphatidic acid in bradykinin-stimulated pig endothelial cells, suggesting that the inhibition of prostacyclin synthesis resulted from an apparent inhibition of both phospholipase A2 and phospholipase C. Other phosphodiesterase inhibitors, theophylline and mopidamole, also raised cAMP levels and inhibited arachidonic acid release. However, there was no correlation between cAMP levels and these inhibitions. Forskolin, an adenylate cyclase activator, elevated intracellular cAMP levels with no apparent inhibition on prostacyclin synthesis. We conclude that the inhibitory effect of MIX on phospholipase A2 and phospholipase C is probably through mechanisms other than the elevation of the cAMP level.