Effect of glucocorticoids on arachidonic acid metabolism and prostaglandin secretion by cultures of newborn rat heart cells

Summary We described that oxygen deprivation induced in cultures of heart muscle cells, biochemical events similar to those described in ischemic tissue: arachidonic acid liberation, loss of membrane phospholipids and increase in neutral lipids. Since glucocorticoids have been described to inhibit phospholipase activity and to exert beneficial effects during myocardial infarction, we studied in our experimental model the action of dexamethasone on the metabolism of arachidonic acid and on the synthesis of immunoreactive prostaglandins. Our results show that heart muscle cells produce prostaglandin E₂ and 6-keto-prostaglandin-F₁. This synthesis, inhibited by dexamethasone (70% inhibition), decreased after oxygen-deprivation (–45%). The effect of oxygen deprivation and dexamethasone (–60%) are not additive. Moreover, steroid treatment failed to counteract the loss of polyunsaturated fatty acids from the phospholipids, the increase in neutral lipids and the liberation of arachidonic acid induced by oxygen deprivation in muscle cells. These results may indicate that the cardiovascular effects of glucosteroids are not the consequence of a direct effect on heart metabolism at cellular level.     

Modulation of rat serosal mast cell biochemistry by in vivo dexamethasone administration

To examine steroid-induced biochemical alterations in the mast cell secretory process, rats were injected with intramuscular dexamethasone or saline for 4 days, and serosal mast cells and lung tissue were obtained from each group. Radioligand binding studies utilizing 1-[propyl-1,2-3H]dihydroalprenolol (3H-DHA) demonstrated a 23.1 +/- 0.8% increase in rat lung beta-adrenergic receptors in steroid-treated rats, but the mast cell beta-adrenergic receptors were unaffected. Neither resting mast cell cyclic adenosine 3':5'-monophosphate (cAMP) levels nor the degree of cAMP augmentation induced by isoproterenol were changed by steroid administration. Mast cells from rats treated with dexamethasone released only 48.6 +/- 8.9 and 58.8 +/- 6.0% of the beta-hexosaminidase released from saline-treated rat mast cells when sensitized with anti-dinitrophenyl (DNP) IgE and challenged with DNP-bovine serum albumin antigen or the calcium ionophore A23187, respectively. [3H]serotonin release in cells from steroid-treated rats was 41.8 +/- 7.9 and 87.6 +/- 2.6% of control release stimulated by antigen or A23187, respectively. [14C]arachidonic acid incorporation into mast cell phospholipids followed by antigen or A23187 challenge revealed that cells from dexamethasone-treated rats release 61.3 +/- 15.6% and 62.1 +/- 11.8% of labeled metabolites, respectively, compared to controls. The addition of exogenous arachidonic acid 5 min prior to antigen challenge caused a similar decrease in mediator release in cells from saline- and steroid-treated rats (36.7 +/- 6.1 and 38.4 +/- 0.9%, respectively). When arachidonic acid was added to sensitized cells after specific antigen, no significant changes in beta-hexosaminidase release were noted in either group. Chronic in vivo dexamethasone administration markedly decreases mast cell mediator release without changing resting cAMP levels. The release of arachidonic acid metabolites is reduced in steroid-treated cells, possibly through the inhibition of phospholipases. Exogenous arachidonic acid cannot overcome this inhibition, suggesting that an earlier step in phospholipid metabolism, perhaps involving phospholipase C, may be important.     

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.     

Effect of salicyclic acid on gluccorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma.

The binding activity of [3H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [3H]dexamethasone of 3.64 - 10(-8) M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 - 10(5). Total binding activity to [3H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid was at 37 degrees C. The maximum enhancement was seen at the concentration of 10(-3)M and in 3h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicyclic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin,also enhanced the total binding activity to [3H]dexamethasone of the cytoplasmic fraction at the concentration of 2 - 10(-5) M and 2 - 10(-7) M, respectively.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Effects of dexamethasone on prostacyclin biosynthesis in rabbit mesothelial cells

We investigated whether glucocorticoids reduce the formation of arachidonic acid metabolites in a non myeloid cell type, the mesothelial cell, which is functionally and embryologically related to the vascular endothelial cell and which forms almost exclusively prostacyclin from arachidonic acid. Preincubation of rabbit mesothelial cells with 2.5 microM dexamethasone suppressed basal as well as bradykinin- or thrombin-stimulated prostacyclin biosynthesis. In further experiments bradykinin was selected as stimulus. The inhibition by dexamethasone was dose-dependent between 0.025 and 2.5 microM. The minimum contact period required for expression of this effect was 30 min and after a contact period of 60 to 120 min the inhibition reached a maximum, but was never complete. After 240 min, sufficient activity was secreted in the extracellular medium for inhibition of the prostacyclin formation in untreated cells. Experiments with cycloheximide were somewhat confused by its direct effects on prostacyclin biosynthesis, but still suggested that the anti-prostacyclin effect of dexamethasone required de novo protein biosynthesis. Our experiments indicate that glucocorticoids induce the formation of lipocortin-like factor(s) in non-phagocytic mesothelial cells, thereby suppressing the formation of prostacyclin, their main arachidonic metabolite.     

Regulatory effects of epidermal growth factor and retinol on the glucocorticoid receptor level in cultured chick embryonic skin

When undifferentiated skin from 13-day-old chick embryos was cultured in a chemically defined medium, glucocorticoid specifically decreased the dexamethasone-binding activity of the epidermal cytosol after 1 day of culture, 3 days before it induced formation of a cornified layer over the intermediate cells of the epidermis. The binding activity reappeared after removal of the steroid from the medium. This reappearance was inhibited by epidermal growth factor (EGF, 100 ng/ml). The Addition of 2 microM retinol resulted in a 3-fold increase in specific dexamethasone binding in the epidermal cytosol within 12 h with no change in the binding affinity. The inhibition of glucocorticoid-induced keratinization by retinol is due a to mechanism other than inactivation of the glucocorticoid receptor.     

Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.     

Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells

Dexamethasone inhibited the stimulus-induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet-activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone-induced formation of a protein which regulates phospholipase A2. The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only. Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet-activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone. These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes.     

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.     

Conceivable difference in the anti-inflammatory mechanisms of lipocortins 1 and 5

Human recombinant lipocortins (LCT) 1 and 5 have been expressed in a yeast secretion vector and purified by ion exchange chromatography. The action of the proteins has been investigated in two models of experimental acute inflammation in the rat: carrageenin induced paw oedema and zymosan induced pleurisy. The effects of the proteins on PGE(2) release in vitro by rat macrophages stimulated with zymosan and on rat neutrophil chemotaxis induced by FMLP have also been assessed. LCT-1 significantly inhibited both paw swelling in carrageenin oedema and leukocyte migration in zymosan pleurisy. Moreover it showed a dose dependent, inhibitory effect on PGE(2) release. Neutrophil chemotaxis was only weakly affected by LCT-1. Conversely LCT-5 did not reduce carrageenin oedema and slightly inhibited PGE(2) release, but showed profound, dose dependent inhibitory activity on leukocyte migration in zymosan pleurisy and on neutrophil chemotaxis. These data suggest that LCT-1 acts mainly by interfering with arachidonic acid metabolism via the inhibition of phospholipase A(2). The anti-inflammatory activity of LCT-5, at variance with LCT-1, may be due to a direct effect on cell motility in addition to the interference with arachidonic acid metabolism.     

Prostacyclin production by human endothelial and bovine smooth muscle cells in culture. Effect of repeated stimulation with arachidonic acid, thrombin and ionophore A23187

Prostacyclin (prostaglandin I2) is the major product of arachidonic acid metabolism in vascular cells. Its physiological role may be linked to the ability of the cells to respond continuously with prostaglandin I2 production to a variety of stimuli. We report that human endothelial cells or bovine smooth muscle cells in culture respond with prostaglandin I2 synthesis to a first but not to a second stimulation with arachidonic acid. The development of this refractoriness was independent of the arachidonic acid concentration used (6.6-25 microM) and lasted for about 6 h. The same time was required for the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. Neither cis-polyunsaturated fatty acids (linoleic or oleic acids) nor stearic acid (a long-chain saturated fatty acid) prevented the generation of prostaglandin I2 by arachidonic acid. Similarly to arachidonic acid, thrombin and ionophore A23187 could elicit vascular prostaglandin I2 synthesis only once. Pretreatment of the cells with arachidonic acid rendered the cells unresponsive to any other stimulus. These results indicate that the mechanism of the refractoriness induced by arachidonic acid was different from that induced by the other stimuli. It is proposed that vascular cells cannot be stimulated continuously to produce prostaglandin I2, but this process is regulated by different feedback mechanisms.     

Corticosteroid induction of Ig+Ia- B cells in vitro is mediated via interaction with the glucocorticoid cytoplasmic receptor

Flow microfluorometry was used to examine the effect of dexamethasone on the expression of surface Ia (sIa) on resting and activated murine B cells. Although dexamethasone resulted in a 50% reduction in sIa expression 12 h after injection, it was significantly less suppressive when injected together with B cell activators. In vitro dexamethasone, but not other related steroid hormones, induced a population of cells that were sIg+sIa-. A 20% reduction in the expression of sIa was noted by 4 h of culture with 10 nM dexamethasone, but maximal inhibition of 70% was not reached until 12 h of culture, and this degree of suppression persisted as long as dexamethasone remained in culture. When the dexamethasone was washed out after 8 h of culture, the maximal reduction was still noted at 12 h, but by 24 h there was re-expression of sIa toward base line levels, indicating it did not induce irreversible lethal alterations in the B cell. The inhibition of sIa expression correlated with a specific reduction in the quantity of messenger RNA for sIa as measured by Northern blot analysis, indicating that this is mediated at least in part by suppression of the steady state levels of Ia mRNA. The corticosteroid receptor antagonist RU486 was able to reverse the suppressive effects of dexamethasone on sIa expression, thus demonstrating that its effect is mediated specifically by binding to its intracellular receptor. Furthermore, when protein synthesis was inhibited during the short period of time that cells were preincubated with dexamethasone, minimal suppression of Ia expression was noted, suggesting that the dexamethasone may be stimulating a protein that has suppressive effects on MHC class II expression. The suppressive effects of dexamethasone in vitro were substantially reduced when B cells were simultaneously activated by stimuli that increase the expression of sIa. These data indicate that the suppressive effects of corticosteroids on immune response Ag are corticosteroid specific; are greater in resting than in activated B cells; are induced via the classical steroid mechanism of action, which is receptor mediated; and may result from the induction of an inhibitory protein that suppresses Ia mRNA.     

Enhancement of renal medulla prostaglandin synthetase activity by dexamethasone treatment in the rat

We studied in rats the effect of dexamethasone (2.5 mg/kg per week) on the conversion of radiolabeled arachidonic acid to prostaglandins by renal medulla slices, microsomes, and homogenates. The steroid did not affect the rate of conversion of arachidonic acid to prostaglandins by renal medulla slices, but significantly increased the rate of conversion by both the microsomes and the 10,000 × g supernatatant of renal medulla homogenates. We conclude (a) that dexamethasone treatment increases the activity of renal medulla prostaglandin synthetase measured in broken cells preparations, and (b) that such a change in enzyme activity is not manifested by augmentation of prostaglandin synthesis in renal medulla slices incubated with exogenous arachidonic acid.     

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

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.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Effects of dexamethasone on the activity of histidine decarboxylase, ornithine decarboxylase, and dopa decarboxylase in rat oxyntic mucosa

Since accelerated turnover of histamine in oxyntic mucosa may be an important factor in the pathogenesis of peptic ulcers, the effect of dexamethasone and other glucocorticoids on the activity of gastric histidine decarboxylase (HDC) was studied in the rat. The activity of HDC in rat oxyntic mucosa increased significantly after dexamethasone was injected s.c. to rats at doses larger than 0.4 mg/kg body weight. The maximum response of the HDC activity to dexamethasone (4 mg/kg) was observed 8 h after the treatment. The activity of ornithine decarboxylase (ODC) increased at 4 h, while that of DOPA decarboxylase showed no significant change throughout the 16-h period following a single injection of dexamethasone. The mucosal levels of histamine, putrescine, and spermidine rose significantly after the steroid treatment, while the spermine levels remained nearly constant. There was no sex difference in these responses to dexamethasone. Betamethasone showed nearly the same effects as dexamethasone on the decarboxylase activities and the mucosal levels of diamines. Serum gastrin levels showed no significant change for the first 4 h and then rose significantly 8 and 16 h after dexamethasone treatment. Pentagastrin (0.5 mg/kg) increased the HDC activity, while it showed no significant effect on either the mucosal ODC activity or levels of polyamines and histamine. These data suggest that dexamethasone influences the metabolism of histamine and polyamines in rat oxyntic mucosa both directly and via stimulation of gastrin release.