6-keto PGF1 Alpha Research Articles

Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type.

1. Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H2 (PGH2) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined. 2. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE2, thromboxane B2 or 6-ketoPGF1 alpha by radioimmunoassay. 3. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) each at 10 ng ml-1) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 microM; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 microM) and cyclohexamide (10 micrograms ml-1; added 30 min before the cytokines). 4. Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1 beta, TNF alpha, IFN gamma each at 10 ng ml-1 for 24 h) caused the accumulation of PGE2 and 6-keto-PGF1 alpha. 5. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1 beta in combination with TNF alpha caused a similar release of PGE2 to that when the three cytokines were given in combination. 6. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 microM for 15 min) after which PGE2 was measured. IL-1 beta and TNF alpha increased COX-2 activity and an additional small increase was produced by the three cytokines in combination. 7. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

Immunosuppressive therapy for recurrent aborters with positive antiphospholipid antibodies and alteration of 6ketoPGF1 alpha/TXB2 ratio.

Twelve women (13 pregnancies) with antiphospholipid antibodies (APA) who had suffered from two or more recurrent spontaneous abortions or fetal deaths and had successful pregnancy outcomes after immunosuppressive therapy were studied. APA titers were determined by enzyme-linked immunosorbent assay against cardiolipin, phosphatidyl serine and phosphatidyl inositol. Plasma levels of 6ketoprostaglandin F1 alpha (6ketoPGF1 alpha) and thromboxane B2 (TXB2) were determined by radioimmunoassay. All of the 13 pregnancies resulted in term delivery. None of the 13 patients suffered from pregnancy-induced hypertension, and only one showed intrauterine growth retardation. A significant decrease of APA titer was observed after immunosuppressive therapy. The 6ketoPGF1 alpha/TXB2 ratios before the therapy, after it and at the 1st, 2nd and 3rd trimesters of pregnancy were 0.62 +/- 0.398, 0.88 +/- 0.106, 0.84 +/- 0.550, 1.25 +/- 0.834 and 0.67 +/- 0.413, respectively. The ratio at the 2nd trimester was significantly higher than that before the therapy (P < 0.05, paired t-test, n = 9). The results indicate that the immunosuppressive therapy affected the physiological balance between thromboxane A2 and prostacyclin, and improved clinical symptoms such as recurrent fetal wastage.

The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity.

1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 micrograms ml-1) and interferon gamma (IFN gamma, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 microM) for 6 h. This treatment induced a significant production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF1 alpha, with an IC50 of 20 microM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF1 alpha in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 microM) for 30 min; this also induced a significant production of 6-keto-PGF1 alpha and MEG (1 microM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF1 alpha. Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF1 alpha production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 microM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins.5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF1alpha production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF1alpha production in the HUVECs. 6.The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33microM and 36microM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3mM) decreased COX-1 activity only, while L-NMA (up to 3mM) had no effect on the activity of either enzyme. 7.These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

Prostaglandin production after experimental discectomy.

This study ascertained the effects of discectomy on prostaglandin synthesis. The purpose of these novel experiments was to measure the levels of two prostaglandins in lumbar epidural fluid obtained from an area subjected to discectomy. For comparison, lumbar epidural fluid from a site not disturbed by discectomy and fluid from a subcutaneous site were analyzed for the prostaglandins. Previous studies have shown that nuclear material obtained from degenerative discs manifests an extraordinarily high level of phospholipase A2 activity. Others have hypothesized that the known inflammatory effects of phospholipase A2 are due to the release of arachidonic acid, which is converted to various eicosanoids, including several algesic prostaglandins (PGI2 and PGE2). No previous study has continuously measured prostaglandin levels in epidural fluid or assessed the effect of discectomy on prostaglandin production. An ultrafiltrate of lumbar epidural fluid of dogs was obtained from indwelling catheters located adjacent to spinal areas that were and were not subjected to discectomy as well as from subcutaneous tissue. The fluid was collected daily for 14 days and analyzed for PGE2 and 6-keto PGF1(alpha) (the stable metabolite of PGI2) by radioimmunoassay. The concentration of 6-keto PGF1(alpha) and PGE2 in fluid collected during the first 24 hours was significantly higher in the area of discectomy than in the epidural region that was not subjected to discectomy and significantly higher than in fluid obtained from the subcutaneous site. The high level of these prostaglandins at the discectomy site fell rapidly, so that by the end of 48 hours the differences in values between spinal fluid from the discectomy and nondiscectomy regions were not statistically significant. The concentration of the prostaglandins in epidural fluid decreased with time and became minimal within the second week. The removal of normal discs is accompanied for 24 hours by a marked rise in the synthesis of two prostaglandins known to produce pain. Because the concentration of prostaglandins in epidural fluid decreased rapidly thereafter, the initial surge obtained appears to be associated more with chemical factors such as phospholipase A2 than with wound healing.

Effect of cytokines on prostaglandin E2 and prostacyclin production in primary cultures of human myometrial cells.

The objective of this study was to characterize interleukin-1, -6, and -8 (IL-1-, IL-6-, and IL-8)-induced prostacyclin (PGI2 as 6-keto PGF1 alpha) and prostaglandin E2 (PGE2) production in primary cultures of human myometrial cells. Prostaglandins (PGs) released into the culture media were quantitated by specific radioimmunoassays. IL-1, but not IL-6 or IL-8, caused a dose- and time-dependent increase in the production of both PGI2 and PGE2. Half-maximally stimulating doses (EC50) of IL-1 were about 0.1 ng/ml, and maximal responses were observed at 1-10 ng/ml, amounting to 15- to 23-fold increases over unstimulated controls. The action of IL-1 was greatly potentiated by the protein kinase C-activating phorbol ester, TPA, and inhibited by actinomycin D and cycloheximide. IL-1-induced PG production was also suppressed by dexamethasone, by the natural IL-1 receptor antagonist (IL-1ra), and by transforming growth factor1 beta (TGF1 beta). It is concluded that IL-1 is a potent agonist of PG synthesis in human myometrial cells, acting by a mechanism dependent on the synthesis of new proteins, presumably key enzymes (phospholipase A2 and/or cyclo-oxygenase-2). This study has added further support to the notion that the myometrium serves as a target for the inflammatory cytokine, IL-1, and thereby may be affected directly, thus promoting preterm labor associated with intrauterine infection.

Nitric oxide-mediated cyclooxygenase activation. A key event in the antiplatelet effects of nitrovasodilators.

We have evaluated the contributions of nitric oxide (NO) and prostacyclin (PGI2) in the in vivo antiplatelet effects of clinically useful nitrovasodilators. In rats, intravenous infusion of three NO donors, glyceryl trinitrate, sodium nitroprusside, or 3'-morpholinosydnonimine, the stable metabolite of molsidomine, released 6-keto PGF1alpha (the stable metabolite of PGI2) and inhibited ex vivo human platelet aggregation to adenosine diphosphate by at least 80%. In in vitro studies, glyceryl trinitrate, sodium nitroprusside, and 3'-morpholinosydnonimine, at clinically attainable concentrations, increased cyclooxygenase activity in endothelial cells (EC), which resulted in a four- to sixfold release of 6-keto PGF1alpha. Pretreatment of the EC with hemoglobin which binds to and inactivates the biological actions of NO, but not by methylene blue (MelB), attenuated the NO-mediated PGI2 from the EC by at least 70%. Release of 6-keto PGF1alpha by the NO donors increased the ability of these compounds to inhibit thrombin-induced human platelet aggregation by at least 10 times; this potentiation was inhibited by hemoglobin but not by MeB. MeB blocked the direct anti-platelet effect of the NO donors in the absence of EC. In summary, we have demonstrated that NO, directly as well as together with an NO-driven cyclooxygenase activation (and hence PGI2), release contributes to the marked anti-platelet effects observed after the in vivo administration of clinically used nitrovasodilators.

Calcium channel blockers in experimental acute pancreatitis: effect on tissue prostanoids and oxygen free radicals.

The effect of verapamil administration on the changes of prostanoid synthesis, and on free radical production associated with acute pancreatitis, has been evaluated. A necrohemorrhagic model of pancreatitis was induced in Wistar rats by intraductal administration of sodium taurocholate (3.5%). This model is associated with initial increases in prostanoid synthesis and peroxidative damage. Verapamil, administered before pancreatitis induction, prevented initial increases in 6-keto prostaglandin F1alpha (PGF1alpha) and thromboxane B2 (TXB2) but had no effect on PGF2alpha or PGE2 or on lipoperoxidative damage. These results indicate that verapamil administration prevents the increases in pancreatic vasoactive prostanoids (TXB2 and 6-keto PGF1alpha) without affecting the increased levels of PGE2 and PGF2alpha and has no effect on oxygen free radical production in the initial stages of experimental acute pancreatitis.

EFFICACY OF CONVECTIVE REMOVAL OF PLASMA MEDIATORS OF ENDOTOXIC SHOCK BY CONTINUOUS VENO-VENOUS HEMOFILTRATION

Continuous veno-venous hemofiltration (CVVH) has been reported to provide beneficial effects during endotoxic shock. This experiment was designed to determine if selective removal of plasma mediators occurs during CVVH and if plasma concentrations of these mediators are reduced. A swine endotoxic-shock model with three groups was used (lipopolysaccharide (LPS) only (n = 6); LPS followed by CVVH (n = 6); and LPS followed by sham CVVH (n = 4). Plasma and filtrate samples were collected at frequent intervals for 5 h. Lactic acid (LA), eicosanoids [prostacyclin (6-keto PGF1 alpha), thromboxane (TxB2), and prostaglandin E2 (PGE2)] and tumor necrosis factor (TNF) were measured in plasma and filtrate. Plasma concentrations of 6-keto PGF1 alpha, TxB2, TNF, and LA were not significantly different in any group. LA, PGE2, 6-keto PGF1 alpha, and TXB2 concentrations were similar in filtrate and plasma. TNF did not move across the membrane into the filtrate, CVVH, as used in this experiment, did not significantly reduce plasma concentrations of any of these mediators.

The effects of chronic sidestream cigarette smoke exposure on eicosanoid production by tracheal epithelium.

Environmental exposure to sidestream cigarette smoke (SSCS) has been associated with an increased incidence of pulmonary infection and bronchospasm. Chronic exposure to SSCS could modify the release of bronchoreactive eicosanoids by tracheal epithelium, the site of initial contact by lung with inhaled toxins. To assess this possibility, New Zealand white rabbits were placed in an environmental chamber flushed with 3 L of SSCS, 15 min/day for 20 days. Eighteen hours after the last exposure the animals were sacrificed and the tracheas were explanted. At 7 days, the epithelial cell outgrowths were exposed to media containing endotoxin (10 micrograms/mL) or acrolein (50 microM), an aldehyde commonly found in smoke, or to control media. After a 2-h exposure, media were assayed for eicosanoids by radioimmunoassay. PGE2 was produced in epithelium from normal animals (5.7 +/- 1.3 ng/10(6) cells), and was not significantly different in SSCS-exposed epithelium. When incubated in medium containing acrolein, PGE2 production increased significantly in SSCS-exposed epithelium (14.9 +/- 2.5, p < .05) but not in control groups. Endotoxin also increased PGE2 production in SSCS-exposed cells (12.6 +/- 3.3 ng/10(6), p < .05). Baseline production of 6-keto PGF1 alpha was 10.8 +/- 3.2 ng/10(6) cells in non-SSCS controls and did not change significantly in these cells with the addition of endotoxin or acrolein. In acrolein plus SSCS-exposed cells, 6-keto PGF1 alpha increased, in a dose-dependent manner, to 88.1 +/- 26.1 ng/10(6) (p < .05 compared to all normals, SSCS-exposed controls, and SSCS plus LPS). TxB2 release in control, non-SSCS-exposed cells was 13.3 +/- 2.8 ng/10(6) cells and was significantly increased (P < .05) only in the SSCS plus acrolein group (60.7 +/- 16.2 ng/10(6) cells). The results indicate that even brief, recurrent exposure to SSCS can change the production of cyclooxygenase products, particularly PGE2, 6- keto PGF1 alpha, and TxB2. This may reflect an altered ability of SSCS-exposed tracheal epithelium to respond to environmental (e.g., acrolein) or bacterial (e.g., endotoxin) insults.

Evidence for requirement of tyrosine phosphorylation in endothelial P2y‐ and P2u‐ purinoceptor stimulation of prostacyclin release

1. The release of prostacyclin (PGI2) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P2-purinoceptors. Here we investigate the hypothesis that tyrosine protein phosphorylations are involved in this response. 2. The use of Western blots with anti-phosphotyrosine antibodies showed that 30 microM 2MeSATP (selective for P2Y-purinoceptors), 300 microM UTP (selective for P2U-purinoceptors) and 300 microM ATP (effective at both these purinoceptors), each stimulate the tyrosine phosphorylation of proteins in bovine cultured aortic endothelial cells. Each of these agonists also stimulates 6-keto PGF1 alpha accumulation in the medium (an index of PGI2 release) in these cells in the same period. 3. The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGF1 alpha response with the same concentration-dependency (1-100 microM) as the tyrosine phosphorylation response. 4. Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 microM) of the 6-keto PGF1 alpha response. 5. Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF1 alpha response to ionomycin. 6. These results show that the regulation of vascular endothelial cells by ATP acting at both P2Y- and P2U-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production.

Role of prostaglandins and histamine in hyperemic response to superficial and deep gastric mucosal injury and H+ back-diffusion in cats.

This study was undertaken to examine the role of prostaglandins and histamine in the hyperemic response to gastric mucosal damage followed by H+ back-diffusion. Cat stomachs were exposed to 2 mol/liter NaCl for 10 min followed by luminal perfusion at pH 1. Hypertonic saline caused extensive (microscopic) damage to the surface epithelium, increased gastric mucosal blood flow, and increased release of histamine, PGE2, and 6-keto PGF1 alpha (prostacyclin) into portal venous blood. The effect of indomethacin and histamine blockers (H1 + H2) on the hyperemic response to acid back-diffusion was related to the depth of the mucosal injury and the region of the stomach. In the corpus, indomethacin enhanced mucosal injury. In areas with superficial damage, the hyperemia was inhibited by indomethacin and antihistamines and eliminated by the combination of both. In corpus areas with indomethacin-induced deep lesions, the blood flow was very high, and this hyperemia was partly inhibited by antihistamines. In the antrum the hyperemic response was reduced by antihistamines. Indomethacin increased the release of histamine into portal venous blood (baseline recordings) and reduced basal gastric mucosal blood flow.

The possibility of clinical application of the thromboxane A2 synthase inhibitor, ozagrel, for the treatment and prevention of preeclampsia: a preliminary report.

This study was performed to investigate whether the TXA2 synthase inhibitor, ozagrel, was effective in the treatment and prevention of pre-eclampsia. Ozagrel was administrated therapeutically to 4 severely pre-eclamptic women, and prophylactically to 5 pregnant women with histories of severe preeclampsia and complications. The therapeutic administration (TA) of ozagrel improved hypertension and proteinuria. Two patients delivered appropriate-for-date (AFD) infants, whereas the other 2 patients delivered light-for-date (LFD) infants. Mean plasma concentrations of TXB2 (plasma TXB2) decreased, whereas plasma 6-keto PGF1 alpha were almost unchanged. The prophylactic administration (PA) of ozagrel prevented the occurrence of preeclampsia in 3 of the 5 patients. All delivered AFD infants. The duration of pregnancy was prolonged more than that of previous pregnancies in all patients. Plasma TXB2 decreased, whereas plasma 6-keto PGF1 alpha increased. PA prevented preeclampsia and intrauterine growth retardation, whereas TA improved only maternal symptoms. These results might justify a large prospective study to determine whether ozagrel is an effective prophylactic.

Diurnal rhythm of prostanoid secretion from bone/marrow organ in the rat.

The secretion of prostanoids from an adult diaphysial rat bone organ was assessed throughout a 24 h period at 4 h intervals as well as the 24 h activities of bone alkaline phosphatase isoenzyme and serum corticosterone levels. Femurs were removed from 16 rats at each interval and incubated in the absence or presence of indomethacin (100 micrograms). The levels of prostaglandin E2 (PGE2) prostaglandin F2 alpha (PGF2 alpha), and the stable metabolites of thromboxane and prostacyclin, thromboxane B2 (TxB2), and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) were measured by radioimmunoassay (RIA). The patterns for each of the variables was subjected to cosinor analysis for predetermined various periods. The chronograms obtained indicated that PGF2 alpha, PGE2, and TxB2 showed 24 h rhythms with computed peak hour acrophases at 1700, 1800, and 2200 h, respectively, and prostacyclin demonstrated a 19 h rhythm with a peak secretion at 1100 h. Corticosterone levels and bone alkaline phosphatase isoenzyme activity in the serum were at peak at 1630 and 2200 h, and at nadir at 0500 and 1000 h, respectively, both exhibiting 24 h rhythms similar to those of PGF2 alpha, PGE2, and TxB2. Bone alkaline phosphatase isoenzyme activity in the femurs' incubation media showed a 12 h diurnal rhythm with peaks at 1230 and 2330 h and nadirs at 0600 and 2330 h (p < 0.01). In summary, this study demonstrated for the first time a 24 h rhythm of prostanoid secretion from diaphysial bone.(ABSTRACT TRUNCATED AT 250 WORDS)

N‐methyl‐D‐aspartate‐evoked release of cyclo‐oxygenase products in rabbit hippocampus: An in vivo microdialysis study

In vivo microdialysis of the rabbit hippocampus was used to study the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on dialysate concentrations of thromboxane B2 (Tx B2)- and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha)-immunoreactive materials that are stable metabolites of biologically active thromboxane A2 and prostacyclin. All pharmacological substances were applied in the dialysis medium. The application of 1 mM NMDA for 20 min resulted in five- and eightfold increases in Tx B2 and 6-keto PGF1 alpha concentrations, respectively. An increase in NMDA concentration to 2.5 mM did not potentiate a peak eicosanoid release, but significantly prolonged this effect. Either 10 microM MK-801 or the extrusion of Ca2+ from the dialysis medium inhibited the release by about 50%. Quinacrine, a phospholipase A2 inhibitor (250 microM), decreased the NMDA-evoked eicosanoid release by 30%, whereas 10 microM indomethacin, a cyclo-oxygenase inhibitor, completely suppressed the release. One hundred micromolar furegrelate, an inhibitor of thromboxane synthase, reduced by 75% Tx B2 release with concomitant 100% increase in 6-keto PGF1 alpha formation. Thus, stimulation of NMDA receptors induces calcium-dependent formation of thromboxane A2 and prostacyclin in the hippocampus, which may have pathophysiological implications. The neuronal site of their formation seems probable, although a transcellular mechanism of their synthesis should be also considered.

Regulation of prostaglandin production by nitric oxide; an in vivo analysis.

1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively. 3. The intravenous infusion of the NO donors sodium nitroprusside (SNP) or glyceryl trinitrate (GTN)increased prostaglandin production in normal animals (for instance urinary PGE2 excretion was increased from 96 +/- 10 to 576 +/- 12 pg min-1 and 400 +/- 24 pg min-1 in the presence of GTN or SNP respectively).4. Proteinuria was measured in order to evaluate the roles of NO and PG in renal damage associated with the in vivo injection of LPS. Interestingly, dexamethasone and the NOS inhibitors attenuated proteinuria in the LPS-treated rats. The COX inhibitors had no effect. It therefore appears that NO and not PG contributes to the LPS-induced renal damage; these findings support the potential use of NOS inhibitors in the treatment of renal inflammation.5. This study demonstrates the regulatory contribution of NO on the in vivo production of prostanoids and suggests that in inflammatory diseases that are driven by both NO and the prostaglandins, NOS inhibitors may act to reduce inflammation by the dual inhibition of cytotoxic NO and pro-inflammatory PG.

Ganglioside GM1 preventsN-methyl-d-aspartate neurotoxicity in rabbit hippocampus in vivo

Microdialysis was used to apply 1 mM N-methyl-D-aspartate (NMDA) for 20 min to the hippocampus of rabbits, control and pre-treated with GM1 ganglioside (im injections of 30 mg/kg for 3 d, twice a day). Concentrations of ionized Ca2+ and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha)-immunoreactive material in the dialyzates and 45Ca and [14C]sucrose efflux from the prelabeled hippocampus were determined. After 24 h, the morphology of the hippocampal neurons was examined. In control animals, the application of NMDA resulted in 25% decrease in Ca2+ concentration and in 1000% increase in 6-keto PGF 1 alpha concentration in the dialyzates. A 30% decrease in 45Ca efflux was accompanied by 20% increase in [14C]sucrose efflux, reflecting a corresponding reduction of the extracellular space volume. A degeneration of CA1 pyramidal neurons in the vicinity of a microdialysis probe was observed. In GM1-treated rabbits the NMDA-induced decrease in Ca2+ concentrations in the dialyzates was not reduced significantly, whereas a 70% stimulation of 45Ca efflux was noted, with a concomitant 40% reduction of 6-keto-PG F1 alpha release. NMDA-evoked increase in [14C]sucrose efflux did not differ from the control. In these animals CA1 neurons were well preserved. These results indicate that the pretreatment with GM1 results in activation of calcium extrusion from the NMDA-stimulated rabbit hippocampal neurons that alleviates destabilization of calcium homeostasis and reduces NMDA-evoked neuronal injury.

Role of renal thromboxane in salt-sensitive blood pressure rise in DOCA salt hypertension

Urinary excretion and release of prostaglandins (PG) from isolated glomeruli and renal papilla of deoxycorticosterone acetate (DOCA)-treated rats fed with a normal salt (0.6% NaCl) diet and a high salt (4%NaCl) diet were determined. Mean blood pressure was significantly higher in the high salt diet group than in the normal salt diet group (146.2 +/- 2.3 vs 118.6 +/- 1.9 mmHg, p < 0.01). Urinary excretion of thromboxane (Tx) B2, stable metabolite of Tx A2, and 6-keto PG-F1 alpha, a stable metabolite of prostacyclin, increased significantly in the high salt diet group compared to the values of the normal salt diet group increased significantly by 104%, 55%, and 74% compared to those of the normal salt diet group, respectively. Release of 6-keto-PG F1 alpha from renal papilla of the high salt diet group decreased significantly, but there were no intergroup differences in the release of PG E2 and Tx B2. Stepwise multiple linear regression analysis showed that the significant contributory factors underlying the mean blood pressure were urinary excretion of Na (F = 14.187, p < 0.01) and release of Tx B2 from isolated glomeruli (F = 4.135, p < 0.05). These findings suggest that the manifestation of Tx synthesis in renal glomeruli has a predominant role in the salt sensitive pressor response of DOCA salt hypertension in rats.

DNA synthesis and prostaglandin secretion by bovine endometrial cells as regulated by interleukin-1.

Interleukin-1 (IL-1) is a pleiotropic cytokine which has been implicated in the regulation of endometrial function through alterations in proliferation, protein synthesis, and prostaglandin secretion. The objectives of this study were to determine if recombinant bovine interleukin-1 beta (rbIL-1 beta) stimulated prostaglandin secretion and altered the proliferation of bovine endometrial epithelial and stromal cells. Uterine flushes were also evaluated by enzyme-linked immunosorbent assay (ELISA) to determine if IL-1 beta was present in the bovine uterus. Effects of rbIL-1 beta (1-1000 ng mL-1) on DNA synthesis by endometrial epithelial and stromal cells prepared from cows at Days 11, 14 and 17 after oestrus were determined by monitoring uptake of [3H]thymidine into DNA. Incorporation of [3H]thymidine by epithelial cells was not affected by IL-1, but rbIL-1 beta inhibited [3H]thymidine incorporation by stromal cells. Secretion of prostaglandin E2 (PGE2), PGF2 alpha, and 13,14-dihydro 15-keto PGF2 alpha (PGFM) by epithelial and stromal cells was increased by rbIL-1 beta. Uterine flushes collected on Days 11, 14 and 17 after oestrus were positive for IL-1 regardless of pregnancy status, whereas flushes from pregnant cows on Day 25 and Day 30 did not have any detectable IL-1. In conclusion, IL-1 can be considered as a local regulatory factor of the bovine endometrium because of its presence within the uterus and its ability to alter DNA synthesis and prostaglandin secretion of endometrial cells.

Effects of MK-801 and ganglioside GM1 on postischemic prostanoid release and hippocampal lesion in gerbil brain

In this study Mongolian gerbils were submitted to a normothermic bilateral carotid ligation lasting 5 min. A noncompetitive antagonist of NMDA receptors, MK-801, 0.8 mg/kg, was injected i.p. 30 min before ischemia, or the ganglioside GM1, 30 mg/kg, was given i.p. for 3 days, twice a day. The morphology of the hippocampal CA1 neurones and the brain content of cyclooxygenase metabolites of arachidonic acid: prostaglandin 6-keto PGF1 alpha and thromboxane Tx B2 were studied. Untreated ischemia induced the accumulation in brain of the 6-keto PGF1 alpha and Tx B2 immunoreactive materials, and resulted in a lesion of 70% of CA1 neurones. In the MK-801- and GM1-pretreated groups the postischemic levels of Tx B2 were significantly decreased. However MK-801 and GM1 did not prevent damage to the CA 1 neurones in gerbils normothermic after ischemia, whereas a partial neuroprotection was observed in hypothermic, MK-801 treated gerbils. The results of this study indicate that NMDA receptors may participate in the mechanism of postischemic release of eicosanoids in brain. They also confirm a potential modulatory role of gangliosides. These results are discussed in terms of the involvement of cyclooxygenase metabolites of arachidonic acid in the mechanism of a selective delayed neuronal damage to the hippocampus CA1 after ischemia.

Effects of Dietary Protein on Glomerular Eicosanoid Production in Rats with Bilateral Ureteral Obstruction

Greater protein intake increases glomerular eicosanoid production in rats. Bilateral ureteral obstruction (BUO) also enhances glomerular eicosanoid production in experimental animals. To examine the effects of dietary protein intake on glomerular eicosanoid production in ureteral obstruction, we measured the in vitro production of the vasodilatory prostaglandins, PGE2, and 6-keto PGF1 alpha, and the vasoconstrictor, TxB2, and the mass of cyclooxygenase in glomeruli of sham-operated control (SOC) rats and rats with BUO of 24 hr duration fed a low- (6% casein) or a high- (40% casein) protein diet for approximately 4 weeks. The animals were pretreated or not with the angiotensin converting enzyme inhibitor, enalaprilat, prior to sham-operation or ureteral obstruction. Glomeruli from SOC rats fed a high-protein diet produced significantly greater amounts of PGE2, 6-keto PGF1 alpha, and TxB2, and had substantially increased mass of cyclooxygenase when compared with glomeruli from SOC rats fed a low-protein diet. Pretreatment of animals with enalaprilat prior to sham operation prevented the increase in glomerular eicosanoid production and cyclooxygenase content in SOC rats fed a high-protein diet and the levels observed were similar to those in SOC rats fed a low-protein diet. Both eicosanoid production and cyclooxygenase mass were further increased in glomeruli from rats with BUO fed a high-protein diet when compared with glomeruli of SOC rats fed the same diet. The increased levels of these measurements in BUO rats fed a high-protein diet fell markedly when the rats were pretreated with enalaprilat in vivo. The values were essentially comparable to those of SOC rats fed a low-protein diet. By contrast, there was no substantial increase in the production of PGE2, 6-keto PGF1 alpha, and TxB2 and in the mass of cycloxygenase in glomeruli of BUO versus SOC rats fed a low-protein diet. Enalaprilat did not affect glomerular eicosanoid production or cyclooxygenase content in SOC and BUO rats fed a low-protein diet. Taken together, the present study indicates that dietary protein affects BUO-induced increases in glomerular eicosanoid production by altering the activity of the cyclooxygenase pathway mainly via the reninangiotensin system. Thus, protein content in a diet may modify an alteration in renal hemodynamics caused by BUO by changing the glomerular production of eicosanoids and the activity of the renin-angiotensin system.