6-keto PGF1α Research Articles

The effect of trinitrobenzene sulfonic acid on gut‐derived smooth muscle cell arachidonic acid metabolism: role of endogenous prostanoids

The contribution of smooth muscle cells as a potential source of eicosanoid production during inflammatory states remains to be elucidated. We investigated the effect of trinitrobenzene sulfonic acid (TNB), a known pro-inflammatory agent, on jejunal smooth muscle cell eicosanoid production. Human gut-derived smooth muscle cells (HISM) were incubated with TNB for 1 hour. Additionally, some cells were preincubated with either dimethylthiourea, or indomethacin for 1 hour before exposure to identical concentrations of TNB. Incubation with TNB led to significant increases in PGE2 and 6-keto PGF-1α release, but not leukotriene B4 release; responses which were both inhibited by dimethylthiourea and indomethacin treatment. Our results suggest that gutderived smooth muscle cells may represent an important source of proinflammatory prostanoids but not leukotrienes during inflammatory states of the intestine. The inhibition of prostanoid activity by thiourea may be mediated by suppression of cyclooxygenase activity in this cell line.

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE 4 release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F 1α (6-keto PGF 1α) release and enhanced (36%) the quantities of LTE 4 detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC 50 values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE 4 release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF 1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.

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 PGF1α) 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 factor1β (TGF1β). 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. © 1996 Wiley-Liss, Inc.

Relationship between Opioids and Prostaglandins in Hypoxia-Induced Vasodilation of Pial Arteries in the Newborn Pig

Previously, it has been observed that methionine enkephalin and leucine enkephalin contribute to hypoxia-induced pial artery dilation in the newborn pig. It has also been observed that the cyclooxygenase inhibitor indomethacin attenuates hypoxic hyperemia in piglets. The present study was designed to determine the relationship between opioids and prostaglandins in hypoxia-induced pial artery dilation. Newborn pigs equipped with closed cranial windows were used to measure pial artery diameter and collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and prostaglandins. Hypoxia-induced artery vasodilation was mildly attenuated during moderate hypoxia (PaCO2 approximately 35 mm Hg), while this response was blunted during severe hypoxia (PaO2 approximately 25 mm Hg) by indomethacin, 5 mg/kg iv (23% +/- 1 % vs 18% +/- 1% and 33% +/- 2% vs 21% +/- 2% for moderate and severe hypoxia in the absence and presence of indomethacin, respectively). Hypoxic dilation was accompanied by increased CSF prostaglandin E2 (PGE2) concentration (1260 +/- 37 vs 1734 +/- 67 and 1256 +/- 33 vs 2859 +/- 189 pg/ml for moderate and severe hypoxia, respectively). Similar changes in CSF 6 keto PGF1alpha concentration during hypoxia were also observed. Topical PGE2 (10,100 ng/ml) increased CSF methionine enkephalin (874 +/- 35, 1290 +/- 44, and 1791 +/- 143 pg/ml for control, 10 and 100 ng/ml PGE2 respectively). Similar increases in CSF methionine enkephalin concentration were observed for topical PGI2. Additionally, these two prostaglandins also increased CSF leucine enkephalin concentration. Furthermore, while indomethacin had no effect on the release of CSF methionine enkephalin during moderate hypoxia, it attenuated the release of this opioid during severe hypoxia (786 +/- 27 and 2633 +/- 74 vs 781 +/- 51 and 2467 +/- 52; 926 +/- 15 and 3489 +/- 156 vs 898 +/- 11 and 2314 +/- 124 pg/ml for control and moderate/severe hypoxia before and after indomethacin, respectively). Similar effects of indomethacin on hypoxic release of leucine enkephalin were also observed. These data indicate that prostaglandins contribute to hypoxic pial dilation. Additionally, these data show that prostaglandins release the opioids methionine enkephalin and leucine enkephalin. Finally, these data suggest that elevated prostaglandin concentrations during severe hypoxia release opioids which in turn contribute to hypoxic pial dilation.

EFFECTS OF CONTINUOUS INFUSION OF FENTANYL CITRATE (FC) ON CSF AND CEREBROVASCULAR PROSTANOID CONCENTRATIONS IN NEWBORN PIGLETS. † 1370

FC is most commonly used for routine, ongoing analgesia and sedation in newborns exposed to repeated pain and prolonged stress in the NICU. The hypothesis that FC exert effects on cerebrovascular prostaglandins was determined in 14 newborn pigs (1-3 days old), treated with either: a loading dose of FC (30 μg/kg i.v.) over 15 minutes, followed by a continuous infusion of FC (10 μg/kg/hr) for 6 hours (n=6); or 5% dextrose at 6 mg/kg/minute (placebo group, n=8). CSF from the cisterna magna was collected at 0 (pre-dose) and 6 hrs, and blood from the sagittal sinus vein was collected at 0 (pre-dose), 0.5, 2, 4 & 6 hrs during infusion for measurements of PGE2, 6-keto PGF1α and TXB2, by enzyme immunoassay. Our data show that in the FC group, CSF levels of PGE2 fell by 75% (p<0.001), and CSF levels of 6-ketoPGF1α and TXB2 increased by 150% and 80%, respectively, at 6 hrs of infusion. In addition, sagittal sinus plasma 6-keto PGF1α and TXB2 increased substantially at 0.5 hr and 2 hrs, with negligible effect on PGE2 levels. Values are mean±SD(pg/ml). *p<0.01, **p<0.001. We speculate that such marked changes in the levels of cerebrovascular prostanoids may contribute to adverse neurophysiologic effects, in newborns treated with FC.Table

CEREBROVASCULAR PROSTANOID PRODUCTION IN NEONATAL GROUP B STREPTOCOCCAL MENINGITIS. † 1777

Group B Streptococcal (GBS) meningitis impairs cerebral blood flow (CBF) autoregulation in newborn piglets. We determined whether the early impairment could be related to increased prostaglandin (PG) production and if dexamethasone and ibuprofen alter PG production in the early phase (1st 6 hrs) of GBS meningitis. Net cerebrovascular PG production (calculated as total CBF× (sagittal-arterial plasma PG concentration) of 6-keto-PGF1α (stable metabolite of prostacyclin), TXB2(thromboxane B2) and PGE2 were measured in 4 groups of piglets given either 1) saline 0.5 ml intracerebroventrically (icv, control grp, n=9), 2) GBS alone (109 cfu/0.5 ml GBS icv, n=7), or 3) GBS icv plus dexamethasone (DEX) 1 mg/kg intraarterial, ia, n=8) or 4) GBS icv plus ibuprofen (IBU) 30 mg/kg ia,n=7) during periods of normotension (MABP= 50-90 mmHg), hypotension (MABP 90mmHg). MABP was randomly adjusted by balloon tipped catheters in the aortic root and descending aorta. CBF was measured by radioactive microsphere technique and PG's were measured by radioimmunoassay. Results show that within each treatment group there was a decrease in cerebrovascular production of 6 keto-PGF1α, TXB2 and PGE2 (p < 0.05) during hypotension possibly due to relative depletion in tissue oxygen for cyclo-oxygenation of arachidonate. However, between treatment groups (Control, GBS alone, GBS+DEX, and GBS+IBU) PG production did not change with BP adjustments except for increased PGE2 during cerebral hypertension(mean±SD, PGE2=250±25 ng/min/100 g brain tissue) compared to hypotension in GBS alone (PGE2=325±30 ng/min/100 g brain, p< 0.05). Irrespective of MABP, cerebral PG production is highest for PGE2, followed by 6-keto PGF1α with no production of TXB2. Since cerebral PG production is minimally influenced by GBS with or without DEX or IBU over a wide range of cerebral perfusion pressures, we suggest that vasoactive mediators other than PG's may mediate CBF impairment in early meningitis. Thus, treatment directed to PG's may not prevent CBF impairment in neonatal GBS meningitis.

ROLE OF NITRIC OXIDE (NO) IN pH-DEPENDENT MODULATION OF VASOACTIVE RESPONSES IN NEWBORN PULMONARY ARTERIES (PA). • 1933

Pulmonary vascular responses to hypoxia are modulated by changes in pH(Δ pH). We examined the effect of acidosis (pH 6.8) and alkalosis (pH 7.7) on bradykinin (BK)-mediated relaxation in newborn piglet PA rings precontracted with the thromboxane A2 analogue, U46619 (0.1 μM). Responses to changes in pO2 and pH were compared to the response of that same vessel in 95% O2/5% CO2 (pH 7.4). Responses to 4 consecutive exposures to U46619 in 95% O2/5% CO2 (pH 7.4) varied by <10%. Hypoxia (pO2 <30 mm Hg) increased contraction to U46619 by 28 ± 4%. Alkalosis reversed, while acidosis restored the enhanced contraction in the presence of hypoxia. In 95% O2/5% CO2, BK(0.1 μM) caused 80 ± 10% relaxation. Hypoxia diminished by 50% the relaxation response to BK. Alkalosis partially reversed the inhibitory effect of hypoxia; acidosis plus hypoxia had a greater inhibitory effect on BK-mediated relaxation than hypoxia alone. To identify mechanisms by whichΔ pH may modify BK-mediated relaxation, we measured NO and prostacyclin(PGI2) production by EIA for cGMP and 6-keto PGF1α, respectively. PA endothelial cells (EC) and mixed EC/VSMC co-cultures were exposed to pH 6.8, 7.4 and 7.7 ± BK at high and low pO2. BK increased both PGI2 and cGMP production at all pH levels. At pH 7.4, hypoxia had no affect on BK-stimulated PGI2 release but decreased cGMP levels. Basal cGMP was depressed at pH 6.8. BK-stimulated PGI2 and cGMP was enhanced at pH 7.7. Indomethacin (0.01 mM) blocked the increase in PGI2 induced by BK but had no effect on BK-mediated relaxation in PA rings. NG-nitro-L-arginine (0.1 mM) completely abolished both the BK-mediated increase in cGMP and the relaxation response at all pH levels. We conclude that in the presence of hypoxia, pH-dependent alterations in pulmonary vascular responses to BK are modulated by an effect of pH on NO production. (Supported by ALA/ATS RG-080-N)

Rapid Quantitation of a Large Scope of Eicosanoids in Two Models of Inflammation: Development of an Electrospray and Tandem Mass Spectrometry Method and Application to Biological Studies

Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 μl with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2(TxB2) to 10 pg for 6-keto prostaglandin F1α(6-keto PGF1α). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2(PGE2), TxB2, and 6-keto PGF1α; prostaglandins E1, D2, and F2αand leukotrienes B4and C4were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1α, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.

Relationship between prostaglandin in peritoneal fluid and pelvic venous congestion after sterilization

Pelvic congestion may cause chronic pelvic pain in women. The aim of the study is to elucidate a possible role in this condition for prostaglandin. Prostaglandin levels in peritoneal fluid were measured in 18 women with pelvic pain caused by pelvic congestion following sterilization, 10 women without pain following sterilization, and 10 normal healthy women. Peritoneal fluid was aspirated by a silastic catheter from the cul-de-sac under laparoscopic direct vision. Concentration of 6-keto-PGF 1α, TXB 2, PGF 2α and PGE 2 were measured with the standard radioimmunoassay method in all samples. Results showed that 6-keto-PGF,. levels in peritoneal fluid from patients with pelvic congestion were markedly higher than those from two control women (P < 0.05); 6-keto-PGF 1α/TXB 2 in pelvic congestion and control groups were markedly different (P < 0.05); the total amounts of peritoneal fluid was higher in pelvic congestion than that in two control groups (P < 0.001). These data suggested that 6-ketoPGF 1α is increased in peritoneal fluid of women with pelvic congestion and the change might play an important role in attack of this disease.

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ 14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F 1α, PGE 2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12( S)-, 15( S)-, and 11( R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11( R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF 1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF 1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.

Role of interferon-α in arachidonic acid metabolism by the rat uteri from spayed animals: Effect of 17β-estradiol

Interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. We attempted to explore the effect of interferon (IFN)-α in the isolated rat uterus from ovariectomized animals on the metabolism of labelled arachidonic acid (AA). The experiments were performed with uterine preparations isolated from spayed rats treated or untreated with 17β-estradiol prior to sacrifice. The pre-incubation of uterine tissue isolated from ovariectomized rats with three different doses (4, 150 and 300 IU/ml) of IFN-α decreased the radioconversion of [ 14C]AA into 6-keto PGF 1α, PGF 2α and PGE 2, whereas the formation of thromboxane B 2 (TXB 2) decreased after treatment with 4 IU/ml of INF-α but was augmented with 150 and 300 IU/ml. The three doses used diminished the synthesis of 5-hydroxyeicosatetraenoic acid (5-HETE) and increased the production of 12-HETE. We also studied the synthesis of uterine products of cyclooxygenase and lipoxygenase in uterine tissue from ovariectomized rats injected with 17β-estradiol and preincubated with IFN-α. We found that the three doses of IFN-α diminished the production of 6-keto-PGF 1α, PGE 2, PGF 2α and TXB 2 as well as 5-HETE and 12-HETE. These findings show that IFN-α modifies the basal synthesis of PGs from uterine tissue. Ovine and bovine trophoblastic protein prevents luteolysis by inhibiting the synthesis of PGF 2α. On the other hand, IFN-α shows 70% homology with ovine trophoblastic protein and binds to the same receptor. The results of the present study suggest that IFN-α may also act in the rat as a luteotrophic agent.

Distribution of cyclooxygenase isoforms in murine chronic granulomatous inflammation. Implications for future anti‐inflammatory therapy

Inhibition of the enzyme cyclooxygenase (COX) is the basis for the mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs). COX exists as a constitutive (COX-1) and a mitogen-inducible (COX-2) isoform. The relative contribution of COX-1 and COX-2 to inflammation is unknown. This study investigated COX activity and the distribution of COX-1 and COX-2 during the development of a murine air pouch model of chronic granulomatous inflammation. COX activity progressively rose and was maximal at day 14. Of the COX metabolites measured, PGE2 was the greatest > 6-keto PGF1a > TXB2 > PGF2a. By day 7, COX-2-labelled fibroblast- and macrophage-like cells were observed and their number and distribution increased with time. At all time points, endothelial cells of venules in the loose connective tissue of the dermis showed immunoreactivity for COX-2. After day 14, labelling of capillaries in the granuloma was also observed. This study is the first to show that COX-2 is the predominant COX isoform in all stages of the inflammatory response. These results suggest that selective inhibition of COX-2 may prove more beneficial, with fewer gastric and renal side-effects, than existing NSAID therapy for the treatment of chronic inflammatory diseases.

Antinociceptive activity of filenadol on inflammatory pain

The novel analgesic filenadol (d,1-erythro-1-(3′,4′-methylenedioxyphenyl)-1-morpholinopropan-2-ol) inhibited phenyl-p-benzoquinone-induced writhing in mice with ID 50 values of 68.8 (p.o.), 1.67 (i.v.) and 0.48 (i.c.v.) mg/kg. Hyperalgesia induced by arachidonic acid, PGE 2 or LTB 4 in this test was also decreased by filenadol ( ID 50 = 24.4, 3.7 and 50.1 mg/kg p.o., respectively). This compound was effective on PGE 2, LTB 4, bradykinin, PAF or IL-1μ-induced decrease in pain threshold in the rat paw pressure model and almost totally suppressed the writhing induced by zymosan in mice, while peritoneal production of 6-ketoPGF 1α was inhibited by 48.5–62 % and only at 100 mg/kg significant inhibition of LTC 4 was achieved. The late phase of formalin-induced pain response in mice was prevented by filenadol, without affecting the oedema. Filenadol is an antinociceptive agent that reduces the hyperalgesic effects of inflammatory mediators besides inhibiting partially the synthesis of eicosanoids.

Neutrophil Regulation of Splanchnic Blood Flow After Hemorrhagic Shock

This study examines the hypothesis that neutrophils impair splanchnic blood flow during resuscitation from hemorrhage by inhibiting the release of the compensatory vasodilator PGI2 from the bowel. Resuscitation from hemorrhagic shock is associated with neutrophil infiltration into the intestine, reduced splanchnic perfusion, and reduced release of PGI2 from the intestine. Sprague-Dawley rats received either vinblastine (VIN) to deplete circulating neutrophils or normal saline (NS). These animals then underwent either hemorrhage and resuscitation (SK + R) or sham operation (SHAM). Superior mesenteric artery flow and splanchnic 6-keto PGF1a (metabolite of PGI2) release were measured. Superior mesenteric artery blood flow was significantly greater in VIN-treated animals sustaining SK + R than in those treated with NS (p < 0.05). Neutrophil depletion preserved 6-keto PGF1a release after SK + R, whereas 6-keto PGF1a release in the NS-treated, SK + R group was significantly reduced (p < 0.05). These data are compatible with the hypothesis that neutrophils may influence splanchnic perfusion after SK + R by inhibiting splanchnic PGI2 release.

Eicosanoid production and levels of newly characterized eicosanoid‐forming enzymes in glomeruli and tubules of rat kidneys

Summary: We examined the production of prostaglandin (P0) E2, 6‐keto PGF1α and thromboxane (Tx) B2, the mass of cyclooxygenase (COX) isoenzymes and the activities of phospholipases A2 and C in glomeruli, cortical tubules and medullary tubules of rat kidneys. Medullary tubules produced significantly greater amounts of PGE2, 6‐keto PGF1α. and TxB2 than glomeruli or cortical tubules. the most abundant eicosanoid in medullary tubules was 6‐keto PGF1α. By contrast, glomeruli and cortical tubules predominantly produced POE2 (glomeruli &gt; cortical tubules). Levels of COX 1 were markedly greater in medullary tubules than in glomeruli or cortical tubules. Glomeruli had significantly greater amounts of COX 1 than cortical tubules. Detectable amounts of COX 2 were not present in the three preparations. the activity of phospholipase (PL) A2 against phosphatidyicholine (PC) was significantly greater in tubules (medullary tubules &gt; cortical tubules) than in glomeruli. By contrast, there was a significant increase in the activity of PLA2 against phosphatidylethanolamine (PE) in glomeruli as compared to tubules (medullary tubules &gt; cortical tubules). the activity of PLC was the Weatest in medullary tubules. Glomeruli had significantly greater activity of PLC than cortical tubules. the order of magnitude for the total activity of the three phospholipases in membranes was medullary tubules&gt; glomeruli&gt; cortical tubules. the total production rate of POE2, 6‐keto PGF1α and TxB2 was in parallel with the amount of COX 1 and the total activity of membranous phospholipases A2 and C in the three preparations. In conclusion, there are differences in the production of PGE2, 6.‐keto PGF1α and TxB2, the ainount of COX 1 and the activities of phospholipases A2 andC among glomeruli, cortical tubules and medullary tubules of rat kidneys; and the different aspects of COX 1 and phospholipases A2 and C have a key role in the control of eicosanoid production in the three preparations.

787-5 Systemic Effect of Ramipril on Endothelin, but not on Elcosanoid Levels in Patients with Coronary Artery Disease

Neurohumoral effects of ramipril (R) alone or in combination with isosorbide dinitrate (ISDN) compared to ISDN or placebo. Placebo-controlled double-blind parallel group trial. 32 patients with coronary artery disease (CAD) received placebo, R5 mg, ISDN 20 mg slow release b.i.d. or R + ISDN for one week. A 24 hour kinetic profile of ramipril and its metabolite, of ace activity (ACE-A) and of related hormones (renin and aldosterone), of endothelin and of prostaglandins (PG), thromboxane B2 (TXB2), PGF2a, 6-keto PGF1a, the stable metabolite of prostacyclin (PGI2-M) was studied after the first dose. Measurements were repeated after 8 days of treatment before and 3 hours after the morning dose. Hormone measurements are presented as means of percent difference of patients treated with R (n = 16) vs. those without ace-inhibitor (n = 16). Time (hrs) 0 1 2 3 4 6 8 24 0–8 3–8 Ramipril (mg/l) 0 8 5 4 3 2 1 0.4 1 7 Ramiprilat (mg/l) 0 6 11 10 8 7 5 1 2 14 ACE-A (%) -6 -78 -95 -98 -98 -97 -96 -80 -82 -98 Aldosteron (%) 9 -5 -31 -34 -21 -27 -23 -34 - - Renin (%) -9 -7 7 19 13 17 53 32 - - Endothelin (%) -17 -13 -25 -19 -16 -15 -4 0 - - TXB2 (%) 4 10 4 5 - 5 0 12 -3 PGF2a (%) 10 17 0 0 - 8 - 2 2 -9 PGI2-M (%) 7 0 -3 8 - -5 9 -1 -7 13 - = not done; significant differences are printed in italics (two-tailed t-test) A single oral dose of 5 mg R reduced ACE activity (p &lt; 0.001), decreased aldosterone and increased renin (p &lt; 0.1). R did not influence plasma levels of the vasoconstricting (TXB2, PGF2a) or vasodilating (PGI2-M) eicosanoid mediators, but decreased endothelin (p &lt; 0.1). R, in a dose that results in significant systemic inhibition of the renin angiotensin aldosteron system, does not induce measurable changes of circulating eicosanoid concentrations, but seems to diminish systemic release of endothelin.

Effect of DP-1904, a Thromboxane A2 Synthase Inhibitor, Administered from the Autologous Phase on Crescentic-Type Anti-GBM Nephritis in Rats

The antinephritic effect of DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid hydrochloride], a thromboxane (TX) A2 synthase inhibitor, was compared with that of OKY-046 and azathioprine, using an experimental model of nephritis, crescentic-type anti-glomerular basement membrane (GBM) nephritis. Test drugs were given p.o. once daily from an autologous phase in which proteinuria was already fully developed. DP-1904 (15 and 45 mg/kg per day) and OKY-046 (20 mg/kg per day), another TXA2 synthase inhibitor, significantly inhibited the development of glomerular alteration as well as the elevation of proteinuria. On the other hand, azathioprine (20 mg/kg per day), an immunosuppressive agent, failed to suppress the proteinuria. A single administration of DP-1904 or OKY-046 inhibited glomerular TXB2 production and increased glomerular prostaglandin (PG) E2 and 6-keto PGF1α production in nephritic rats. Both drugs apparently decreased the depositions of both rabbit immunoglobulin (Ig) G and rat IgG on GBM in nephritic rats, but azathioprine inhibited only the deposition of rat IgG. These results suggest that DP-1904 may be an effective agent for the treatment of proliferative glomerulonephritis and its antinephritic effect may be due to the amelioration of abnormal metabolism of arachidonic acid.

Prostanoid production in hypoxic rat isolated atria: Influence of acute diabetes

The effects of hypoxia on prostanoid production were studied in atria from normal, acute diabetic and insulin-treated diabetic rats. Diabetes was induced by intravenous administration of 65 mg/kg of streptozotocin, the rats were killed 5 days later. Hypoxia was performed by incubation of the atria during 60 min in nitrogen-equilibrated glucose free Krebs'solution followed by 15 min of reoxygenation. The prostanoids 6-keto prostaglandin F1α (6-keto PGF1α) and thromboxane B2 (TXB2), stable metabolites of prostacyclin and TXA2, respectively, as well as PGE2, were measured by reversed phase HPLC-UV. In control atria, the production of 6-keto PGF1α was equivalent to that of PGE2, whereas TXB2 was released in a much smaller amount. In diabetic atria, 6-keto PGF1α production was reduced by 65%, whereas TXB2 release was increased by 158% compared to the controls. When the normal atria were exposed to 60 min of hypoxia, the release of 6-keto PGF1α increased by 142% compared to basal values and remained elevated after 15 min of reoxygenation whereas in diabetic and insulin-treated diabetic tissues the 6-keto PGF1α production was not modified by the hypoxia-reoxygenation period. The release of TXB2 was increased after 60 min hypoxia in normal as well as in diabetic and insulin-treated diabetic tissues and remained elevated during the reoxygenation. The PGE2 output increased only after the onset of the reoxygenation in the three groups studied. Since it is well known that prostacyclin exerts cytoprotective actions in cardiac tissues, the present results suggest that short-term diabetic atria could be more susceptible to hypoxia-reoxygenation injuries.

Ancrod-Formed Fibrin Stimulates Prostacyclin Production of Human Umbilical Vein Endothelial Cells via de Novo Synthesis of Cyclooxygenase

Ancrod, a thrombin-like enzyme, has been used as defibrinogenating agent in prevention of venous thrombosis. This study showed ancrod in citrated plasma elevated 6-keto PGF1α production of human umbilical vein endothelial cells; this increment of 6-keto PGF1α was completely inhibited by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The enhanced prostacyclin production, but not basal level of prostacyclin, was inhibited by actinomycin D and cycloheximide. In washed aspirin-pretreated cells, ancrod-formed fibrin induced restoration of prostacyclin production by a cycloheximide- and actinomycin D-sensitive process. Ancrod-formed fibrin stimulated synthesis of cyclooxygenase as probed by Western blotting and this enhancement was blocked by actinomycin D and cycloheximide. In conclusion, we first report that ancrod-formed fibrin stimulates prostacyclin production of human endothelial cells and this event is dependent on de novo synthesis of cyclooxygenase.

N-3 fatty acids reduce in vitro thromboxane production while having little effect on in vitro prostacyclin production in the rat

Male Sprague-Dawley rats were fed diets with 50% of the energy from fat over a 20-day period. The fats used were hydrogenated beef-fat (HBF), or HBF supplemented with ethyl arachidonate A, safflower oil (SO), or a mixture of SO and linseed oil (LO). For comparative purposes, another group of animals was fed a diet providing 50% of the energy as butter-fat. In vitro aortic prostacyclin (PGI 2) and serum thromboxane (TXA 2) levels (from clotting blood) were determined by radioimmunoassay of 6-keto PGF 1α and TXB 2, respectively. The HBF diet had similar AA levels relative to the butter-fed rats but significantly reduced tissue levels of eicosapentaenoic acid (EPA) and this was associated with an increased production of serum TXB 2. Supplementing the HBF diet with AA increased tissue levels of AA while maintaining low levels of n-3 fatty acids. These changes were accompanied by significant increases of both TXB 2 and 6-keto-PGF 1α. LO supplementation to the HBF diet (with constant SO) led to elevated levels of EPA and relatively constant AA levels and this was associated with reduced producton of TXB 2. These results highlight the responsiveness of TXA 2 to n-3 fatty acids in contrast to PGI 2 which was more influenced by the level of AA in the tissue phospholipids in the rat.