Stimulation of microsomal prostaglandin synthesis by a vasoactive material isolated from blood plasma

Stimulation of prostaglandin synthesis by a material with coronary vasoconstrictor activity extracted from blood plasma was examined. The vasoactive material decreased the Km for arachidonate in the overall synthesis of prostaglandins by rabbit renal microsomal preparations but did not change Vmax. Increases in prostaglandin synthesis caused by the vasoactive material and L-tryptophan or L-epinephrine were additive or synergistic, whereas increases produced by the vasoactive material and hemin or hemoglobin were not. However, hemin and hemoglobin stimulated synthesis of all prostaglandins equally whereas the active material increased the synthesis of prostaglandin F_2α at the expense of other prostaglandins, both in the presence and absence of heme compounds. The increase in prostaglandin F_2α with respect to the other prostaglandins occurred in the presence of reduced glutathione. The vasoactive material attenuated inhibition of prostaglandin synthesis induced by indomethacin or aspirin but not that produced by 5,8,11,14-eicosatetraynoic acid. The interaction of the vasoactive material and indomethacin was competitive whereas hemin attenuated the effects of only low concentrations of indomethacin. Epinephrine enhanced indomethacin inhibition. These data indicate that mode of action of the vasoactive material in prostaglandin synthesis is unlike that of glutathione, aromatic amines, or heme containing compounds.     

Inhibition of compensatory renal growth by indomethacin

Renal prostaglandins may be important in the modulation of compensatory renal growth. Reductions in renal mass are associated with increased synthesis of these substances by the remaining kidney, and inhibition of prostaglandin synthesis diminishes renal function in partially nephrectomized animals and in patients with reduced functioning renal mass. We examined the effects of uninephrectomy and treatment with indomethacin on renal prostaglandin E₂ and 6-keto prostaglandin F_1α concentrations in adult male Sprague Dawley rats. The renal content of these prostaglandins was significantly increased in the remaining kidney two days following uninephrectomy (p<0.01). Treatment with 5 mg/kg/day of indomethacin over this period abolished the compensatory increase in renal prostaglandin synthesis and significantly attenuated compensatory increases in renal mass, protein and RNA concentration (p<0.05). No alterations in kidney weight, protein or RNA concentrations were found in intact animals treated with the same dose of indomethacin. These findings suggest renal prostaglandins may participate in the biological events leading to compensatory renal growth.     

Inhibition of compensatory renal growth by indomethacin

Renal prostaglandins may be important in the modulation of compensatory renal growth. Reductions in renal mass are associated with increased synthesis of these substances by the remaining kidney, and inhibition of prostaglandin synthesis diminishes renal function in partially nephrectomized animals and in patients with reduced functioning renal mass. We examined the effects of uninephrectomy and treatment with indomethacin on renal prostaglandin E2 and 6-keto prostaglandin F1 alpha concentrations in adult male Sprague Dawley rats. The renal content of these prostaglandins was significantly increased in the remaining kidney two days following uninephrectomy (p less than 0.01). Treatment with 5 mg/kg/day of indomethacin over this period abolished the compensatory increase in renal prostaglandin synthesis and significantly attenuated compensatory increases in renal mass, protein and RNA concentrations (p less than 0.05). No alterations in kidney weight, protein or RNA concentrations were found in intact animals treated with the same dose of indomethacin. These findings suggest renal prostaglandins may participate in the biological events leading to compensatory renal growth.     

Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

Prostaglandin interaction with the renal effects of plasma angiotensin II in the Pekin duck

The present study was designed to determine whether the responses of the avian kidney to circulating angiotensin II, under different osmotic conditions, involve an interaction with prostaglandins. The renal effects of i.v. infusions of angiotensin II at 10, 30 and 90 ng·kg·min^-1 for 30 min were compared in Pekin ducks given maintenance infusions of either 200 mosmol ·l^-1 NaCl or glucose at 0.5 ml·min^-1, with and without prostaglandin inhibition by indomethacin. Birds infused with glucose without indomethacin responded to the two low doses of angiotensin II with dose-dependent reductions in water and sodium excretion, whilst the same doses of angiotensin II in salineloaded birds caused dose-dependent increases in the renal exeretion of salt and fluid. Indomethacin treatment in the animals given glucose had no effect upon the antidiuretic response to the low doses of angiotensin II but did prevent the antinatriuretic effect. In the birds infused with saline, prostaglandin inhibition reversed the natriuretic/diuretic action of angiotensin II, producing renal salt and water conservation. The highest dose of angiotensin II was consistently diuretic/natriuretic and independent of prostaglandin involvement in each case. The results indicate that the antinatriuretic effect of low doses of angiotensin II in glucose-infused birds involves an interaction with prostaglandins, whereas the antidiuretic effect of angiotensin II under this condition is independent of prostaglandins. In salt-loaded birds the diuretic/natriuretic actions of low doses of angiotensin II are mediated by prostaglandins so that inhibition of prostaglandin formation unmasks the normal salt and fluid-retaining actions of systemic angiotensin II.Abbreviations AII angiotensin II - ECFV extracellular fluid volume - PG prostaglandin - PGE prostaglandin E     

Suppression of secondary plaque-forming cell responses by rat splenic adherent cells: evidence for dependence on prostaglandin production.

Rat spleens normally contain adherent suppressor cells with macrophage-like characteristics. This paper presents data indicating that the activity of these cells, as measured by decreased secondary in vitro PFC responses to heterologous erythrocytes, is dependent upon the production of prostaglandins. PFC can be restored in vitro by the use of indomethacin and aspirin—different drugs which both inhibit prostaglandin synthesis among other cellular functions. An experimental drug, Ro3-1314, which has as its only known function the inhibition of prostaglandin synthesis, also acts similarly. The suppression relieved by these drugs can be restored by the addition of PGE².     

Effect of indomethacin on the adrenal renin response to nephrectomy in the rat

The role of prostaglandins in the control of adrenal renin in vivo was evaluated in nephrectomized rats. Nephrectomy increased adrenal renin from 13.2 ± 1.37 ng angiotensin I/mg protein/hr to 166.5 ± 17.3 ng angiotensin I/mg protein/hr. Indomethacin treatment significantly suppressed the adrenal renin response to nephrectomy. (47.8 ± 5.22 ng angiotensin I/mg protein/hr). Adrenal aldosterone was also suppressed by indomethacin. Adrenal prostaglandin E₂ increased after nephrectomy and decreased after indomethacin.Plasma corticosterone and serum potassium did not change after indomethacin. These data indicate that inhibition of prostaglandin synthesis by indomethacin partially blocks the adrenal renin response to nephrectomy, suggesting that prostaglandins may play a role in the adrenal response to nephrectomy.     

Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.

In this study, using rat carrageenin-induced pleurisy, we found that treatment of rats with either indomethacin or NS-398 suppressed the pleurisy at 2 h but significantly exacerbated this reaction at 48 h. Exacerbated inflammation was associated with reduced prostaglandin D(2) levels, decreased heat shock factor 1 (HSF1) activation, reduced hsp72 expression and increased activation of nuclear factor kappaB (NF-kappaB). Replacement of cyclopentenone prostaglandins by treating rats with either prostaglandin J(2) or prostaglandin D(2) reversed the exacerbating effects of cyclooxygenase inhibitors leading to the resolution of the reaction. In conclusion, we demonstrate that cyclopentenone prostaglandins may act as anti-inflammatory mediators by inducing in inflammatory cells HSF1-dependent hsp72 expression and NF-kappaB inhibition, two crucial events for the remission of inflammation.     

Block of oxytocin-induced parturition and oviposition by prostaglandin inhibitors

The possibility that oxytocin-induced labor is mediated, at least in part, by endogenous release of prostaglandins was investigated in two animal models using inhibitors of prostaglandin synthesis. Term pregnant rabbits failed to deliver after an i.v. oxytocin challenge (100 mU) when they were pretreated with an oral dose of indomethacin (10–25 mg) 45–60 minutes prior to oxytocin, while control rabbits began to deliver within a few minutes. Similarly, oxytocin- but not prostaglandin E₁-induced oviposition in the coturnix quail was inhibited by indomethacin. 5,8,11,14-Eicosatetraynoic acid, another inhibitor of prostaglandin synthesis, was also effective in blocking oxytocin-promoted oviposition. Based on these observations on two quite diverse species, it is suggested that prostaglandins may play a universal role in the expulsion of the uterine content.     

Influence of prostaglandins on electrical and mechanical activities of gastric muscles of Bufo marinus

1. Study was performed to compare the role of prostaglandins in regulating gastric contractile activity in an amphibian model, Bufo marinus, with mammalian models. 2. The prostaglandin synthesis inhibitor, indomethacin, had little effect on spontaneous mechanical activity, but increased the force and frequency of contractions stimulated by acetylcholine. 3. PGE2 reversed the effects of indomethacin and reduced the force and frequency of contractions. These effects were concentration-dependent. 4. Intracellular measurement of membrane potential demonstrated that the effects of PGE2 could be explained by basic effects on membrane potential and slow wave activity. 5. The data shown that many similarities exist between amphibian and mammalian gastric muscles in terms of the regulatory role played by endogenous PGs. It also appears that the mechanisms of PGE2 action are similar.     

Indomethacin inhibits the uptake of sodium by ovine trophoblastic tissue in vitro

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of ²²sodium ([²²Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF_2α and 13,14-dihydro-15-keto-PGF_2α (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37°C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin mM reduced (P<.01) trophoblastic release (ng/μg DNA) of PGF_2α from

Full-size image (<1K)

, reduced PGFM from

Full-size image (<1K)

, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [²²Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [²²Na], which is an indirect measure of the transport of water across epithelia, from 3680 ± 1118 to 934 ± 248 cpm/μg DNA (P<.03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts.     

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.     

Effects of indomethacin on renal inner medullary plasma flow

The effects of the prostaglandin system on renal hemodynamics were studied by treating rats with a single intraperitoneal dose of indomethacin, an inhibitor of prostaglandin synthesis. Medullary plasma flow was significantly reduced 30–45 minutes after indomethacin, but was elevated 3–6 hours after indomethacin. These changes in medullary plasma flow correlated well with circulating levels of prostaglandins A and E. Total renal blood flow decreased following indomethacin treatment, but returned to normal levels within an hour. These results indicate that the inhibition of prostaglandin synthesis following a single intraperitoneal dose of indomethacin is short-lived and is followed by a significant elevation in prostaglandin synthesis. It is likely that prostaglandin levels play an important role in the control of renal medullary plasma flow.     

Prostaglandins antagonize fibroblast proliferation stimulated by tumor necrosis factor

Tumor necrosis factor (TNF) is known to be a mitogen for human diploid FS-4 fibroblasts. We have shown in an earlier study (Hori et al. (1989) J. Cell. Physiol. 141, 275-280) that indomethacin further enhances the cell proliferation stimulated by TNF. Since indomethacin inhibits the activity of cyclooxygenase, the role of prostaglandins in TNF-stimulated cell growth was examined. Cell growth stimulated by TNF and indomethacin was inhibited by exogenously added prostaglandins (PGE2, PGF2 alpha, and PGD2), among which PGE2 caused the greatest inhibition of cell growth. Treatment of FS-4 cells with 10 ng/ml TNF resulted in the release of prostaglandins (PGE2, 6-keto-PGF1 alpha, PGA2, PGD2, and PGF2 alpha) 2 to 4 fold over that of untreated cells. The amount of all these prostaglandins increased in a time-dependent manner over 6 h after treatment. In both TNF-treated and control cells, PGE2 was released as the predominant prostaglandin. Furthermore, when PGE2 production and DNA synthesis were determined in FS-4 cells treated with increasing doses of indomethacin, these two cellular responses were inversely affected by indomethacin. These data show that prostaglandins induced by TNF antagonize growth stimulatory action of TNF.     

The extracellular matrix and cytokines regulate microglial integrin expression and activation

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-alpha promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the alpha(4)beta(1) and Mac-1 integrins. In contrast, TGF-beta1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the alpha(4)beta(1), alpha(5)beta(1) and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-beta1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.     

Optimum time for administration of indomethacin to inhibit ovulation in the rabbit

The antiinflammatory agent, indomethacin, inhibits ovulation in mammals by interfering with the synthesis of prostaglandins in preovulatory follicles. To determine the optimum time to administer this inhibitor, indomethacin was given at specific intervals from 10 h before, and up to 9 h after, the ovulatory process had been initiated by hCG (50 I.U./kg). The drug dosage ranged from 1.25 mg/kg to 40 mg/kg. The optimum time to give indomethacin was at 7–8 h after hCG (i.e., 2–3 h before expected rupture of the follicle) at which time the minimum effective dose was 2.5 mg/kg. Since a significant elevation in prostaglandin synthesis occurs as early as 3–5 h after hCG stimulation of rabbit follicles (1), these results reveal that nonsteroidal antiinflammatory agents can interrupt the ovulatory process even the follicle has begun producing substantial amounts of prostaglandins. The data suggest that prostaglandins need to be produced continuouly in the follicle up to the time of actual rupture, or else that indomethacin is interfering with some other aspect of the ovulatory process which transpires after the elevation of prostaglandins.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. IV. Evidence for participation of prostaglandins of the E series in the early events.

The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1^α, enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10^?4) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.     

Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [¹⁴C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E₂, D₂, and F_2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E₂. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E₂ or D₂ alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F_2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.     

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.