Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.     

Dexamethasone-induced stimulation of arachidonic acid release by U937 cells grown in defined medium

Since the presence of serum in culture media has been shown to alter prostaglandin production, as well as to interfere with the action of anti-inflammatory drugs, we have studied the effect of dexamethasone, a potent steroidal anti-inflammatory drug, on the metabolism of arachidonic acid by human monocyte-like cells (U937) grown in a fully defined medium. Under these culture conditions, dexamethasone (10(-6) M, 24 h) induced a marked stimulation of the release of unmetabolized arachidonic acid into the culture medium. The steroid also induced an inhibition of cell proliferation which became significant only after 48 h of treatment. The accumulation of arachidonic acid in the medium after steroid treatment was associated with a significant inhibition of cell acyltransferase activity, suggesting that steroids may also act upon arachidonic acid metabolism at sites other than those of phospholipase activity.     

Relaxin inhibits prostacyclin release by the rat pregnant myometrium

Porcine relaxin (30 μg/ml) when incubated with separated myometrial tissue from 20 day pregnant rats inhibited basal prostacyclin output by 50%. However, relaxin did not inhibit the increased prostacyclin output observed when myometrial tissue was incubated with the prostaglandin precursor, arachidonic acid (10 μg/ml). When prostacyclin release was stimulated by incubation with oxytocin (10 mU/ml), however, relaxin completely inhibited the increased output. The results suggest that relaxin interferes with basal and oxytocin-stimulated prostacyclin formation in pregnant myometrial tissue by inhibiting the action of the enzyme phospholipase A₂ which is responsible for liberating the precursor arachidonic acid endogenously.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E2 and F2 alpha, 6 keto PGF1 alpha and thromboxane B2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10(-7)M - 10(-13)M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E2 and F2 alpha, and to a lesser extent, 6 keto PGF 1 alpha, but TxB2 production was only slightly inhibited by the drug in the absence of arachidonic acid and markedly increased in its presence. Colchicine (10(-7)M-10(-3)M) had the opposite effect, causing an inhibition of TxB2 and stimulating PGE2 and 6 keto PGF1 alpha production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A2 and cyclo-oxygenases, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO4 labeling methodology. The results showed that at the concentrations tested (10(-5)M to 10(-7)M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO4 incorporation into newly synthesized proteoglycan molecules both in the presence (10(-6)M) and absence of exogenous arachidonic acid. In the same concentration range chloroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.     

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.     

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.     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro

Preovulatory follicles isolated from immature rats, treated $\text{in vivo}$ with pregnant mare's serum gonadotropin, were incubated $\text{in vitro}$ and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A₂, capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C₃H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A₂ provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A₂ stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA₂, respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA₂ induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors $\text{in vivo}$ by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.     

On the synthesis of prostaglandins by human gastric mucosa and its modification by drugs.

1. Specific radioimmunoassays for the prostaglandins E2, A2 and F2alpha were used to study the synthesis of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric corpus mucosa. 2. Both prostaglandin E2 and prostaglandin F2alpha were found to be synthesized from arachidonic acid by themicrosomal fraction of human gastric mucosa. The synthesis of prostaglandin E2 exceeded that of prostagladin F2alpha by a factor of about 10. 3. Synthesis of prostaglandin A2 or prostaglandin B2 was not observed under the same incubation conditions. 4. Indometacin effectively inhibited synthesis of both prostaglandin E2 (ID50 4.2 microng/ml) and prostaglandin F2alpha (ID50 1.8 microng/ml) by human gastric mucosa, while paracetamol even in a concentration of 310 microng/ml did not influence prostaglandin synthesis. The anti-ulcer agent carbenoxolone, which has been shown to inhibit prostaglandin inactivation, at the same concentration only slightly inhibited (about 20%) prostaglandin synthesis. 5. The results support the hypothesis that the gastro-intestinal effects or side effects of several drugs are mediated by an influence on the enzymes of prostaglandin synthesis or inactivation.     

Inhibitors of protein synthesis, puromycin and emetine, inhibit prostaglandin E2 release by skeletal muscle

Addition of 1 microM puromycin or 1 microM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E2 release by 51-77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E2 synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E2 production from arachidonic acid added into the incubation medium.     

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Inhibitors of protein synthesis,puromycin and emetine,inhibit prostaglandin E2 release by skeletal muscle

Addition of 1μM puromycin or 1 μM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E₂ release by 51–77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E₂ synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E₂ production from arachidonic acid added into the incubation medium.     

Prostaglandin synthesis in rabbit graafian follicles in vitro. Effect of luteinizing hormone and cyclic AMP

We have demonstrated the capacity of isolated follicles from estrous rabbits to synthesize prostaglandins in vitro and to respond to gonadotropins added to the incubation medium with an increased accumulation of these lipids. The increase in both prostaglandins became apparent only after 5 hours of incubation. The effect was specific for hormones with LH activity and the threshold dose of LH appeared to be 0.005 μg/ml. In addition, we have shown that cyclic AMP (0.02 M) added to the incubation medium also increased prostaglandin in this in vitro system and appears to be a likely mediator of this action of LH.     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles

Preovulatory follicles isolated from immature rats, treated with pregnant mare's serum gonadotropin, were incubated and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

Effects of dibutyryl cyclic AMP and prostaglandin E1 on cultured human glioma cells

Dibutyryl cyclic AMP (db-cAMP) and prostaglandin E₁ (PGE₁) induced morphological alterations in cultured human glioma cells (138 MG). Cells in serum-free medium, treated with db-cAMP (1 mM) or PGE₁ (10μg/ml), within 1–3 h showed multiple thin processes resembling those of normal glial cells. These processes increased in size during a 24 h incubation. In serum-containing medium the appearance of cells with multiple processes was delayed. The induced morphological alterations were reversible upon exchange with fresh serum-containing but not with serum-deprived medium. Actinomycin D (5 μg/ml) did not prevent the changes induced by PGE₁ or db-cAMP. Inhibition of protein synthesis with cycloheximide (10 μg/ml) did not arrest the initial (1–3 h) changes in morphology but blocked further growth of the processes on prolonged incubation. Vinblastine sulphate (0.1 μg/ml) completely inhibited the alterations induced by PGE₁ or db-cAMP.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.     

Inhibition of the prostaglandin-liberating activity of bradykinin in the rat]

In the rat, hydrocortisone, dexamethasone and chloroquine do not inhibit the hypotensive activity of arachidonic acid, which is due to endogenous prostaglandin synthesis. They reduce or suppress the secondary hypotensive phase induced by bradykinin. These substances inhibit the prostaglandin releasing activity of bradykinin but do not interfere with prostaglandin synthesis.     

The effect of oxytocin,estrogen, calcium ionophore A23187 and hydrocortisone on prostaglandin F2α and 6-oxo-prostaglandin F1α production by cultured human endometrial and myometrial explants

Normal human endometrium (classified by histology and date after last menstrual period) was cultured for 72h, and the output of prostaglandin F₂α and 6-oxo-prostaglandin Fla detected by radioimmunoassay. Hormones/stimuli were added to the culture during the second day of culture for 5h and 19h periods.

• 1.1) The output of prostaglandin F₂α from cultured endometrium was significantly higher (p<0.05) at the beginning (d4–8) and end (d25–30) of the menstrual cycle, compared to mid-cycle (d13–24) endometrium. Significantly more prostaglandin F₂α was released from proliferative than from secretory phase endometrium (p<0.02).
• 2.2) Prostaglandin F₂α release was rapidly stimulated by sodium arachidonate (20–300 μg/ml), and by calcium ionophore A23187 (5 μg/ml) at an extracellular calcium ion concentration of 1.8mM.The ionophore stimulation was greater in mid-cycle endometrium than in endometrium from the beginning or the end of the menstrual cycle.
• 3.3) Estradiol-17β (10 ng/ml) gradually increased the output of prostaglandin F₂α from secretory phase endometrium, and this stimulation was observed in the post-incubation period after hormone had been removed from the incubation medium.
• 4.4) Oxytocin (1 × 10⁻⁵U/ml caused a more rapid stimulation of prostaglandin F₂α output from secretory phase tissue (p<0.05 during the first 5h incubation period with hormone).
• 5.5) Oxytocin (1 × 10⁻⁵ U/ml) and estradiol (long/ml) together significantly stimulated prostaglandin F2a production by proliferative as well as secretory phase endometria.
• 6.6) A high dose of hydrocortisone (loo μg/ml) inhibited the output of prostaglandin F₂α from proliferative and secretory phase endometrium and also from ionophore-stimulated endometrium. However, this dose of hydrocortisone did not inhibit the synthesis of prostaglandin F2a from exogenous arachidonic acid, or the estradiol-induced increase in prostaglandin F₂α production.
• 7.7) Co-culture of endometrium with myometrium did not modify the output of prostaglandin F₂α or of 6-oxo-prostaglandin Fla from cultured tissues.
• 8.8) These experiments suggest that arachidonic acid supply to the cyclooxygenase enzyme may vary during the menstrual cycle: and indicate a gradual increase in prostaglandin synthesising capacity in response to estrogen, more rapid control via oxytocin, and an interaction between estrogen and oxytocin to modulate prostaglandin F2a synthesis in human endometrium.