6-ketoprostaglandin F1 Alpha Research Articles

Panax Notoginseng Saponins Combined with Dual Antiplatelet Drugs Potentiates Anti-Thrombotic Effect with Alleviated Gastric Injury in A Carotid Artery Thrombosis Rat Model

ObjectiveTo observe the combination effects of Panax notoginseng saponins (PNS)and dual antiplatelet drugs (DAPT), and to explore the mechanism via cyclooxygenase /prostaglandin pathway. MethodsRight carotid artery thrombosis was induced in Wistar rats by infiltration with 70% FeCl3, and the animals were randomly divided into sham group, model group, DAPT group and PNS + DAPT group, intragastrically treated for 4 weeks. The cerebral pia mater microcirculation was observed in vivo after anesthetizing by anatomical microscope. The wet weight of carotid artery thrombosis was measured. Gastric mucosal injury was observed by hematoxylin and eosin staining. Platelet aggregation rate was detected with adenosine diphosphate -induced turbidimetry. Platelet CD62p expression was detected by flow cytometry. Concentrations of 6-Ketoprostaglandin F1 alpha, prostaglandin E2 in gastric mucosa and thromboxane B2, 6-Ketoprostaglandin F1 alpha, tissue plasminogen activator, plasminogen activator inhibitor, and fibrin fragment D in the plasma were measured by radioimmunoassay. ResultsPNS and DAPT increased the blood flow volume of cerebral pia mater and decreased erythrocyte aggregation and leukocyte adhesion of model rats. Compared to DAPT, PNS and DAPT further reduced the weight of carotid artery thrombosis with enhanced inhibition of platelet aggregation, increased tissue plasminogen activator levels and decreased fibrin fragment D levels. PNS and DAPT alleviated gastric injury induced by dual antiplatelet drugs and upregulated the expression of 6-Ketoprostaglandin F1 alpha in the gastric mucosa compared with DAPT. ConclusionsPNS combined with DAPT increased anti-thrombosis effects of DAPT and mitigated DAPT-related gastric injury. The underlying mechanisms may be associated with enhanced antiplatelet aggregation and activation of the fibrinolytic system and up-regulation of 6-Ketoprostaglandin F1 alpha expression in gastric mucosa.

The Hydroxysteroid (17β) Dehydrogenase Family Gene HSD17B12 Is Involved in the Prostaglandin Synthesis Pathway, the Ovarian Function, and Regulation of Fertility.

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B+/- females gave birth less frequently than wild-type females, and the litter size of HSD17B12+/- females was significantly reduced. Interestingly, we observed meiotic spindle formation in immature follicles, suggesting defective meiotic arrest in HSD17B12+/- ovaries. The finding was further supported by transcriptome analysis showing differential expression of several genes related to the meiosis. In addition, polyovular follicles and oocytes trapped inside the corpus luteum were observed, indicating a failure in the oogenesis and ovulation, respectively. Intraovarian concentrations of steroid hormones were normal in HSD17B12+/- females, whereas the levels of AA and its metabolites (6-keto prostaglandin F1alpha, prostaglandin D2, prostaglandin E2, prostaglandin F2α, and thromboxane B2) were decreased. In conclusion, our study demonstrates that HSD17B12 enzyme plays an important role in female fertility through its role in AA metabolism.

Abstract 3368: Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma

Abstract Purpose: Catecholamine mediated stress effects are known to induce production of various pro-inflammatory cytokines. However, the mechanism and functional effect of adrenergic signaling in driving inflammation via pro-inflammatory metabolites is currently unknown. Here we address the functional and biological consequences of adrenergic-induced Cox2/PGE2 axis activation in ovarian cancer metastasis. Methods: We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite PGE2 in vitro and in vivo. To study tumor-specific effects on catecholamine-derived expression of PTGS2, we used a novel DOPC delivery system of PTGS2 siRNA. Results: Our results revealed that levels of PGs were significantly increased in patients with high depressive scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression and protein levels in Skov3 and HeyA8. PGE2 ELISA confirmed that upon treatment with NE, PGE2 levels were increased in conditioned medium from Skov3 and HeyA8 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES as well as PGE2 levels in supernatant. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. ChIP analysis showed enrichment of Nf-kB binding to the promoter region of PTGS2 and PTGES by 2.4 and 4.0 fold respectively when Skov3ip1 cells were treated with NE. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 cells in the presence of NE. Importantly, in the Skov3-ip1 restraint stress orthotopic model, silencing PTGS2 abrogated stress mediated effects and decreased tumor burden by 70% compared to control siRNA with restraint stress. Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu mediated by prostaglandins that drives tumor progression and metastasis in ovarian cancer. Citation Format: Archana S. Nagaraja, Piotr Dorniak, Nouara Sadaoui, Guillermo Armaiz-Pena, Behrouz Zand, Sherry Y. Wu, Julie K. Allen, Rajesha Rupaimoole, Cristian Rodriguez-Aguayo, Sunila Pradeep, Lin Tan, Rebecca A. Previs, Jean M. Hansen, Peiying Yang, Garbiel Lopez-Berestein, Susan K. Lutgendorf, Steve Cole, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2015-3368

Abstract 158: Sustained adrenergic signaling activates pro-inflammatory networks in ovarian carcinoma

Abstract Purpose: To determine the role of adrenergic activation in driving tumor inflammation through COX-2/Prostaglandin E2 (PGE2) axis as PGE2 is known to play an important role in the induction of a pro-inflammatory signature in the tumor microenvironment that promotes tumor growth and metastasis. Methods We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study mechanism at gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite levels (all prostaglandins, PGs) in vitro and in vivo. Results Our results revealed that levels of PGs were significantly increased in patients with high CESD scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression in adrenergic receptor positive cell lines. Mass-spectroscopy analyses confirmed that upon treatment with NE and arachidonic acid (substrate), PGE2 levels were increased in conditioned medium from Skov3 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 and HeyA8 cells in the presence of NE. Importantly, silencing PTGS2 in a restraint stress orthotropic model decreased tumor burden by 70% and metastasis (tumor confined to the ovary) in Skov3-ip1 orthotopic mouse model as compared to control siRNA with restraint stress. Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu that drives tumor progression and metastasis. Citation Format: Archana S. Nagaraja, Guillermo Armaiz-Pena, Julie Allen, Nouara C. Sadaoui, Behrouz Zand, Peiying Yang, Lin Tan, Steve Cole, Susan Lutgendorf, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory networks in ovarian carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2014-158

Human C-reactive protein induces endothelial dysfunction in biobreeding diabetic rats

C-reactive protein (CRP) levels in diabetes predict cardiovascular events. Also, human CRP (hCRP) exacerbated the proinflammatory, pro-oxidant and procoagulant states in a spontaneous model of type 1 diabetes mellitus (T1DM), the biobreeding (BB) rat. Since there is a paucity of data examining the role of CRP on endothelial dysfunction in animal models of diabetes, we tested this hypothesis in the diabetic BB rat. Diabetic BB rats (n = 4 per group) were injected with human serum albumin (HSA) or hCRP [hCRP = 20 mg/kg body weight; intraperitoneal (IP)] for three consecutive days. The rats were euthanized on day 4. Biomarkers that were assayed included endothelin-1 (ET-1), soluble intracellular adhesion molecule-1 (sICAM-1), Von Willebrand factor (vWF) and 6-keto prostaglandin F1-alpha (6-keto PGF1-α) in plasma. hCRP administration resulted in a significant increase in plasma levels. Furthermore, hCRP-treated rats had significantly increased circulating levels of ET-1 (1.12 ± 0.6 pg/mL versus 0.4 ± 0.21 pg/mL), vWF (45 ± 2.4 ng/mL versus 34 ± 7 ng/mL) and sICAM-1 (41 ± 3 ng/mL versus 34 ± 3.4 ng/mL) compared to HSA-treated rats (p < 0.05). There was no significant effect on 6-keto PGF1-α levels. Hence, in this preliminary report, we make the novel observation that hCRP induces endothelial dysfunction in a spontaneous model of T1DM, and this could have implications for the vascular complications in diabetics.

Sodium molybdate prevents hypertension and vascular prostanoid imbalance in fructose‐overloaded rats

(1) Fructose (F) overload produces elevated blood pressure (BP), hyperglycaemia, hypertriglyceridemia and insulin resistance, resembling human metabolic syndrome. Previously, we found altered vascular prostanoid (PR) production in this model. (2) Sodium molybdate (Mo), as well as sodium tungstate, causes insulin-like effects and normalizes plasma glucose levels in streptozotocin-treated diabetic rats. We studied the effects of Mo on BP, metabolic parameters and release of PR from the mesenteric vascular bed (MVB) in F-overloaded rats. (3) Four groups of male Sprague-Dawley rats were analysed: Control, tap water to drink; F, F solution 10% W/V to drink; CMo, Mo 100 mg kg day(-1) and FMo, both treatments. After 9 weeks, the animals were killed and MVBs removed and the released PRs measured. (4) F increased BP, glycemia, triglyceridemia and insulinemia. Mo treatment prevented the increases in BP and glycemia, but did not modify triglyceridemia or insulinemia. In addition, Mo decreased BP in controls. (5) Prostaglandins (PG) F2 alpha and E2, PG 6-ketoF1 alpha and thromboxane (TX) B2 , as well as inactive metabolites of prostacyclin (PGI2 ) and TXA2 were detected. F decreased the production of vasodilator PRs PGI2 and PGE2 in MVB. Mo prevented these alterations and increased PGE2 in controls. Vasoconstrict or PRs PGF2 alpha and TXA2 release was not modified. (6) Mo treatment, beyond its known lowering effect on glycemia, prevents the reduction in the vascular release of vasodilator PR observed in this model. This could be one of the mechanisms by which Mo avoids the increase in BP caused by F overload in the rat.

Antiatherogenic effect of Radix Salviae Miltiorrhizae and Fructus Crataegi on experimental atherosclerosis in rats

To investigate the antiatherogenic effect and possible mechanisms of the extracts of Radix Salviae Miltiorrhizae (RSM) or Fructus Crataegi (FC), as well as their interaction. Wistar rats were randomly divided into 2 groups: normal group and model group. The atherosclerotic model rats were injected VD3 and ovalbumin, while fed with high cholesterol diet. After the model was determined successfully, all model rats were divided into normal group, model group, Xuezhikang group, RSM group, FC group, mixture of RSM and FC group. Each group was given the corresponding drugs for 4 weeks. After 12 weeks, blood serum were analyzed for total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), superoxide dismutase ( SOD), malondialdehyde (MDA) and nitric oxide (NO). And the blood plasma also analyzed for levels of endothelin (ET), 6-keto prostaglandin F1alpha (6-keto-PGF1alpha), thromboxane B2 (TXB2), C-reactive protein (CRP), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-alpha) and so on. At last, the pathological observation of aorta was carried out. Compared with those in model group, the TC, TG, LDL-C, ET, TXB2 and MDA levels and TXB2/PGF1alpha ratio were reduced, while the HDL-C, the serum SOD, No and 6-keto-PGF1alpha level were raised in the intervention groups. Although the levels of CRP, IL-6 and IL-8 were lower than model group, there was no obvious effect on the releasing of TNF-alpha. RSM and FC could inhibit the atherogenesis formation and development, which might be due to regulating the lipid metabolism, enhancing the antioxidation, and reducing the release of inflammatory factors.

Pravastatin Attenuates Carboplatin-Induced Nephrotoxicity in Rodents via Peroxisome Proliferator-Activated Receptor α-Regulated Heme Oxygenase-1

The aim of this study was to explore the molecular mechanisms underlying the protective effect of pravastatin against carboplatin-induced nephrotoxicity in rodents. We exposed rat NRK-52E renal tubular epithelial cells to carboplatin, with or without pravastatin. Pravastatin decreased production of reactive oxygen species, increased expression of heme oxygenase-1 (HO-1), cyclooxygenase-2, and 6-keto prostaglandin F1alpha, enhanced nuclear translocation of peroxisome proliferator-activated receptor-alpha (PPARalpha), and increased HO-1 promoter and peroxisome proliferator response element (PPRE) activities. We found interaction of PPARalpha with PPRE on the HO-1 promoter in nuclear extracts from pravastatin-treated NRK-52E cells and by chromatin immunoprecipitation. We pretreated mice with pravastatin and then administered a single intraperitoneal injection of carboplatin. Effects on renal function, morphology, apoptosis, and survival were assessed. In response to carboplatin injection, mice developed acute renal failure, with elevated activated caspase-3, increased apoptotic bodies, and decreased survival. Pretreatment with pravastatin significantly ameliorated renal dysfunction and apoptosis and improved renal morphology and survival. Injection of pravastatin also induced overexpression of PPARalpha and HO-1 in wild-type mice, and HO-1 expression was significantly attenuated in PPARalpha-knockout mice. These results indicate that pravastatin up-regulates HO-1 and protects against carboplatin-induced renal dysfunction and apoptosis via a PPARalpha-dependent pathway.

Effect of Sodium Depletion on the Renin-Angiotensin-Aldosterone System and Renal Prostaglandins in Acromegalic Patients

The effects of 5 days low sodium diet and diuretic were studied in 7 normotensive acromegalics, 8 hypertensive acromegalics and 12 normal subjects. Plasma renin activity was significantly lower in both groups acromegalics, compared with that of normal subjects. Plasma aldosterone was similar in normotensive acromegalics and healthy controls. The hypertensive acromegalics showed increased plasma aldosterone levels and blunted responses of plasma renin activity and plasma aldosterone to sodium depletion. Urinary prostaglandin E2 was significantly lower in hypertensive acromegalics before and after sodium depletion in comparison with normal subjects. Urinary 6-keto prostaglandin F1 alpha and thromboxane B2 were similar in all groups studied. The decreased production of PGE2 could contribute to the development of arterial hypertension in actomegaly.

Association of GLu461ALa polymorphism of prostacyclin synthase gene with myocardial infarction in Uigur population

To investigate the association between the polymorphism of prostacyclin synthase gene (CYP8A1) and myocardial infarction (MI) in Uigur population. Totally 210 patients with MI and 206 healthy control subjects were detected by polymerase chain reaction and restriction fragment length polymorphism. The serum 6-keto-PGF(1alpha) was detected with radioimmunoassay kit in all subjects. The frequencies of CC, AC and AA were 0.024 (5/210), 0.124 (26/210) and 0.852 (179/210) in MI group while ones those 0.010 (2/206), 0.073 (15/206) and 0.917 (189/206) in the controls. There was no significant difference in frequencies of CC, AC and AA genotypes between controls and MI cases (chi(2) = 0.782, P > 0.05), but the frequency of CC + AC genotype in MI group [0.14 (31/210)] was higher than that in the controls [0.083 (17/206)] giving significant difference (chi(2) = 4.321, P = 0.031). The C allele frequency in the MI group [0.086 (36/420)] was higher than that in the controls [0.046 (19/412)] showing significant statistical difference (chi(2) = 5.284, P = 0.021). There was significant difference (t = 6.255, P < 0.01) in serum 6-keto-PGF(1alpha) level between MI group [(17.40 +/- 4.56) pg/ml] and control group [(20.34 +/- 5.02) pg/ml]. In the cases and control group, the serum 6-keto-PGF(1alpha) level of the persons with CC + AC genotype [(14.30 +/- 3.31) pg/ml, (18.31 +/- 4.62) pg/ml] was lower than those of AA genotypes [(19.34 +/- 5.51) pg/ml, (25.10 +/- 5.00) pg/ml], and the statistical significance was also observed (t' = 6.934, P < 0.05; t = 5.393, P < 0.01). Logistic regression analysis showed that the C allele of the CYP8A1 gene was an independent risk factor for MI (OR = 1.77; 95% CI: 1.06 - 2.05). The C allele of CYP8A1 might be a risk factor of MI in Uigur population, and be resulting from the decrease of serum 6-keto-PGF(1alpha) level for gene variation.

Selective COX2 inhibition improves whole body and muscular insulin resistance in fructose‐fed rats

The effects of cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) inhibition on insulin resistance in subjects with the metabolic syndrome remain elusive. Aims of this study were to examine the effects of COX1 and COX2 inhibitors on whole body and muscular insulin resistance in fructose-fed rats, an animal model of the metabolic syndrome. The rats on regular or 60% fructose-enriched diets for 6 weeks were further divided into rats combined with or without piroxicam (a selective COX1 inhibitor) or celecoxib (a selective COX2 inhibitor) treatment for an additional 2 weeks. Euglycaemic hyperinsulinaemic clamp (EHC) with a tracer dilution method was performed at the end of the study. The present result showed that fructose-induced increases in systolic blood pressure and fasting plasma insulin levels were significantly suppressed in rats treated with celecoxib but not piroxicam. In the EHC period, celecoxib significantly reversed fructose-induced decreases in whole body glucose uptake, mainly by glucose storage. Hepatic glucose production and whole body glycolysis were not significantly changed among groups. Celecoxib but not piroxicam significantly reversed fructose-induced decreases in glycogen synthase activities in red and white quadriceps muscles and insulin-stimulated membrane GLUT4 recruitment in soleus muscles. Celecoxib and piroxicam both significantly diminished fructose-induced increases in plasma thromboxane B2 and 6-keto prostaglandin (PG) F1alpha; but only celecoxib treatment significantly attenuated a fructose-induced increase in 8-isoprostane levels. Plasma PGE metabolites were not different among groups. This study demonstrates that a therapeutic dose of celecoxib, but not piroxicam, could significantly attenuate fructose-induced whole body and muscular insulin resistance in rats.

Differential regulation of myometrial prostaglandin production by changes in length

The purpose of this study was to determine whether stretching and shortening of human myometrial strips alters prostaglandin production. We measured 6-keto prostaglandin F1alpha (PGF1alpha; a PGI2 metabolite) and PGF2alpha in the contraction baths of myometrial strips that were obtained at cesarean delivery from 5 term pregnant women. Fluid was collected after the stretching and after the strip lengths were decreased by 4%, 6%, or 8%. Prostaglandin concentrations were measured in triplicate by competitive enzyme immunoassay and compared by Kruskal-Wallis tests. Stretching myometrial strips increased the production of 6-keto PGF1alpha by 31%, but not PGF2alpha. Shortening strips had little effect on 6-keto PGF1alpha production, but increased PGF2alpha production by 158%. Stretching myometrial strips from pregnant women increases synthesis of PGI2; shortening strips increases synthesis of PGF2alpha. Differential prostaglandin synthesis that is related to myometrial stretching and shortening could play a role in uterine quiescence during gestation and increased contractility during parturition.

Alteration of prostaglandin metabolism in alcoholic liver disease: its association with platelet dysfunction after chronic alcohol ingestion

Recently, hepatic microcirculation has been focused on as an important pathogenic factor in progression of alcoholic liver disease (ALD). Therefore, blood levels of several prostaglandins, which are associated with organ microcirculation, were determined in various liver diseases, including ALD. Blood thromboxane B2 (TXB2) level was significantly increased in ALD, when compared to other types of liver diseases, whereas both 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and prostaglandin E were not changed. These consequences resulted in the imbalance of 6-keto PGF1 alpha to TXB2, which might promote platelet aggregation and blood vessel contraction. Indeed, the increase of beta-thromboglobulin and platelet factor 4 in blood was observed in ALD. Furthermore, in ALD, the rate of arachidonate-induced platelet aggregation was prominently enhanced, and malondialdehyde production in platelet, which was well correlated with blood TXB2 levels, significantly increased. Thus, the present study may indicate that, in ALD, hyper-aggregability of platelet is produced, because of the derangement of prostaglandin metabolism and platelet dysfunction.

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.

Role of vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 in the vasodilator response to vascular endothelial growth factor in the neonatal piglet lung

Vascular endothelial growth factor (VEGF) regulates vascular proliferation and causes vasodilation. In the pulmonary circulation, the vasorelaxing effect of VEGF has been attributed to nitric oxide, whereas in other vascular beds, prostacyclin and other mechanisms are also involved. This vascular effect follows binding to two receptors, VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2), the latter of which is thought to be the main receptor responsible for the vasorelaxing effect of VEGF. The role of VEGFR1 in the neonatal pulmonary vasculature remains to be determined. Prospective randomized laboratory investigation. Animal laboratory. Newborn Yorkshire-Landrace piglets. To determine the mechanisms of action of VEGF in the neonatal pulmonary vasculature, the effect of VEGF (10-10 M) was tested in isolated perfused piglet lungs, alone and in the presence of a VEGFR2 kinase inhibitor, N-nitro-l-arginine (L-NNA), indomethacin (Indo), L-NNA + Indo, and GF109203X, a protein kinase C inhibitor. The effect of a VEGFR1 agonist, placenta growth factor (PlGF), was also studied with or without L-NNA. Perfusate was collected, and cyclic guanosine monophosphate (cGMP), as well as 6-keto prostaglandin F1alpha and thromboxane B2, the stable metabolites of prostacyclin and thromboxane, respectively, was measured. VEGF caused vasorelaxation with a concomitant increase in cGMP. PlGF also decreased vascular tone and increased cGMP. VEGFR2 kinase inhibitor did not prevent the reduction in perfusion pressure seen with VEGF but blocked the increase in cGMP. Pretreatment with L-NNA completely inhibited VEGF and PlGF vasodilation and prevented the increase in cGMP seen with both agonists. Pretreatment with Indo or GF109203X did not reduce the dilator response to VEGF. VEGF vasodilation may follow nitric oxide release in the piglet pulmonary circulation. VEGF vasorelaxation may not only occur through binding to VEGFR2, since PlGF, the specific VEGFR1 agonist, also causes vasodilation. Therefore, vasodilator response to VEGF may involve both types of receptor in the neonatal piglet pulmonary vasculature.

Redirection of Eicosanoid Metabolism in mPGES-1-deficient Macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.

Novel Eicosanoid Pathways: The Discovery of Prostacyclin/6-keto Prostaglandin F&lt;sub&gt;1α&lt;/sub&gt; and the Hepoxilins

This article reviews a lecture I was honored to present at the Leon Wolfe Symposium in Montreal on March 25, 2004. The lecture described my research career, which started with my interaction with Wolfe at the Montreal Neurological Institute as a postdoctoral fellow and research associate and was followed by additional research discoveries after I left Montreal for my first academic position at the Research Institute, The Hospital for Sick Children and University of Toronto. The article consists of two parts. The first part involves the discovery (in Wolfe's laboratory) of a new pathway of arachidonic acid, in which a bicyclic prostanoid structure (later called prostacyclin by John Vane and his group) was described, and its further development in Toronto, which led to the discovery of the conversion of the bicyclic prostanoid into 6-keto prostaglandin F1alpha. The second part deals with the hepoxilin pathway, a pathway I discovered during a sabbatical leave in Japan with Professor Shozo Yamamoto, which was followed by a stay of several months in the laboratory of Professor Bengt Samuelsson in Sweden. I deal with the historical aspects of both pathways and end with interesting novel aspects of hepoxilin stable antagonist analogs in the treatment of solid tumors in experimental animals.

Synergistic action of famotidine and chlorpheniramine on acetic acid-induced chronic gastric ulcer in rats

To assess the synergistic action of famotidine (FMD) and chlorpheniramine (CPA) on acetic acid-induced chronic gastric ulcer in rats. Chronic gastric lesions were induced in male Sprague-Dawley (SD) rats by serosal application of the acetic acid. Forty SD rats were randomly divided into blank group (n = 8), control group (n = 8), FMD group (n = 8), CPA group (n = 8), and FMD+CPA group (n = 8). Each group was given intraperitoneally (i.p.) 0.5 mL/100 g distilled water, 9 g/L NaCl saline, 4 mg/kg FMD, 10 mg/kg CPA, 4 mg/kg FMD+10 mg/kg CPA, respectively, daily for 10 d. On d 10, ulcer area was determined by planimetry. The level of myeloperoxidase (MPO) in the liver homogenization was determined by biochemical methods and the plasma levels of 6-ketoprostaglandin F1 alpha (6-keto-PGF(1a)) and IL-8 were determined by radioimmunoassay. The synergistic effects of FMD+CPA group on the lesion, IL-8, 6-keto-PGF(1a) and MPO were confirmed. The effect of FMD+CPA group was significantly different as compared to the control and FMD groups. The lesion (mm(2)) was reduced from 40.18+/-2.6 in control group to 6.83+/-2.97 in PMD+CPA group, P<0.01, and from 32.9+/-3.27 in FMD group to 6.83+/-2.97 in PMD+CPA group, P<0.01. The plasma levels of IL-8 decreased from 0.69+/-0.11 ng/L in control group to 0.4+/-0.04 ng/L in PMD+CPA group, P<0.01, and from 0.51+/-0.08 ng/L in FMD group to 0.4+/-0.04 ng/L in PMD+CPA group, P<0.05. The level of 6-keto-PGF(1a) increased from 7.55+/-1.65 ng/L in control group to 16.62+/-0.97 ng/L in PMD+CPA group, P<0.01, and from 13.15+/-1.48 ng/L in FMD group to 16.62+/-0.97 ng/L in PMD+CPA group, P<0.05. The levels of MPO in the liver homogenate decreased from 9.12+/-2.05 u/L in control group to 4.33+/-0.95 u/L in PMD+CPA group, P<0.01, and from 8.3+/-1.29 u/L in FMD group to 4.33+/-0.95 u/L, P<0.01. The synergistic action of FMD and CPA on acetic acid-induced chronic gastric ulcer in rats decreases the incidence of ulcer and also enhances the healing of ulcer.

Early endothelial dysfunction following renal mass reduction in rats.

Endothelial dysfunction has been previously described in severely hypertensive rats with renal mass reduction (RMR) receiving large dietary Na loads. Because hypertension and Na loading reduce endothelium-dependent vasodilation, the effect of renal failure per se is unclear. Responses to acetylcholine in noradrenaline-contracted isolated perfused mesenteric arteries were studied. Vessels were obtained from RMR rats kept on a normal diet, 3 and 10 days after surgery, and the results were compared with those from sham-operated rats (SN). The role of three putative mediators of endothelium-dependent vasodilation was assessed using: L-NAME (10(-4) mol L(-1)); indomethacin (INDO, 10(-5) mol L(-1)); and a mixture of charybdotoxin and apamin (C/A, both 10(-7) mol L(-1)), inhibitors of Ca-activated K-channels to mediate the effects of endothelium-derived hyperpolarizing factor (EDHF). Response to acetylcholine but not that to nitroprusside (endothelium-independent) was decreased in RMR. L-NAME reduced further acetylcholine relaxations in SN but not in RMR. By contrary, INDO decreased acetylcholine vasodilation in RMR but had no effect in SN. C/A had similar effects in the SN and RMR rats. The levels of 6-keto prostaglandin F1alpha were elevated in the urine of the RMR rats and were perfusate from the RMR vessels. Endothelial dysfunction occurs early after RMR, even when systolic blood pressure is only minimally elevated and Na intake is normal. This alteration may be because of decreased availability of nitric oxide, partially compensated by increased prostacyclin production.

Determination of Prostacyclin in Plasma through a Bioluminescent Immunoassay for 6-Keto-prostaglandin F1α: Implication of Dosage in Patients with Primary Pulmonary Hypertension

This work describes a solid-phase immunoassay for 6-keto-prostaglandin F1alpha, the stable hydrolysis product of prostacyclin (prostaglandin I2). Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-prostaglandin F1alpha can be directly correlated with levels of prostacyclin. Therefore, 6-keto-prostaglandin F1alpha, has become the indicator of choice to measure prostacyclin levels. The single-step immunoassay for 6-keto-prostaglandin F1alpha reported here was developed using the bioluminescent protein aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-prostaglandin F1alpha and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-prostaglandin F1alpha toward its antibody and the bioluminescent properties of aequorin were retained in the conjugate, which was then used to generate a dose-response curve for the analyte in a convenient microtiter plate format. The concentration of 6-keto-prostaglandin F1alpha after extraction from plasma showed good correlation with the concentration of 6-ketoprostaglandin F1alpha obtained without prior extraction of the same plasma sample. This measurement demonstrated that the assay allows the measurement of 6-keto-prostaglandin F1alpha directly in plasma without any pretreatment of the samples, which results in a much simpler method with a faster assay time.