High Ratios of C20:4n-6/C20:5n-3 and Thromboxane B2 /6-Keto-Prostaglandin F1α in Placenta Are Potential Risk Contributors for Neural Tube Defects: A Case-Control Study in Shanxi Province, China.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations. Folate supplementation can reduce the risk, but cannot prevent all NTDs, suggesting other reasons for folate-resistant NTDs. The present study assesses placental fatty acid composition, eicosanoids, and cytokines as risk factors for NTDs in a Chinese population with highly incident NTDs. METHODS: Seventy-seven aborted fetuses with NTDs during the third trimester were cases and 142 healthy newborns were controls. Placental fatty acid composition, eicosanoids, and cytokines were determined by standard methods. RESULTS: The placental C20:4n-6/C20:5n-3 and thromboxane B2 (TXB2 )/6-keto-prostaglandin F1α (6-keto-PGF1α ) ratios were significantly higher for cases than controls (p < 0.001 and 0.05, respectively). For the top versus the lowest tertiles of placental C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α , odds ratios for NTD occurrence were 3.79 (95% confidence interval, 1.60-8.96) (p for trend < 0.01) and 5.52 (95% confidence interval, 2.07-14.74) (p for trend < 0.001), respectively, adjusted for fetal sex as well as maternal age, occupation, parity, smoking, passive smoking, periconceptional folate supplementation, conception season, and tea drinking. The C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α ratios were positively correlated (r = 0.14; p < 0.05). The proportions of C18:2n-6, C18:3n-6, C20:3n-6, C18:3n-3, C20:3n-3, C20:5n-3, and C22:5n-3 were significantly lower in cases than controls, and all negatively associated with NTD occurrence (tertile-specific odds ratios); after adjustment for the potential confounders, these associations remained significant (p for trend < 0.05) except for C20:3n-3. CONCLUSION: High placental ratios of C20:4n-6/C20:5n-3 and TXB2 /6-keto-PGF1α are risk factors for neural tube defects.Birth Defects Research 109:550-563, 2017.© 2017 Wiley Periodicals, Inc.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle.

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM(22-52), a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP(8-37), a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with N(G)-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K(+) channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K(+) channels), and apamin (Ca(2+)-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K(+) channels.

Comparison of UHPLC and HPLC methods for the assay of prostanoids: "are the methods equivalent in terms of accuracy and precision?".

BACKGROUND: As new methods are developed to increase efficiency and higher analytical performance, it is necessary to evaluate their quality in comparison to standard methods. To understand how the analytical performance changes between methods, it is common to compare the validation parameters; sensitivity, linearity, accuracy and precision. Here, we compare an UHPLC-UV method to the HPLC-UV method (reference method) for the simultaneous determination of seven prostanoids. Though the basic chromatography theory is the same for HPLC and UHPLC, the instrumentation has been modified to accommodate higher pressures, lower flow rates and smaller sample size. The differences in analytical instrumentation and procedures can give rise to method inequivalencies. Our approach evaluates the UHPLC and HPLC methods and poses the question: are the methods equivalent? To answer this question a statistical comparison of the analytical performance and method parameters is necessary. RESULTS: Statistical comparisons were performed using the t-test, F-test, regression analyses (ordinary linear regression and Deming regression) and Bland-Altman analyses. Statistical comparison of the results, suggested that the precision (amount of variability) is different (p < 0.05) for the HPLC and UHPLC methods. Whereas, the accuracy (method bias and the means) is similar (p > 0.05) for 8-isoprostane, 11-dehydro TXB2, PGE2 PGF(2α), PGD2 and 15-deoxy Δ¹²,¹4 PGJ2. DISCUSSION: Ordinary linear regression shows that the methods are well correlated for all compounds. The Deming regression, which assumes error in both the methods, suggests the existence of a proportional and constant bias for 11-dehydro TXB2 and only proportional bias for 8-isoprostane, PGF(2α), PGD2 and 15-deoxy Δ(12,14) PGJ2 between the two methods. According to Deming regression, the two methods are statistically similar for 6-keto PGF(1α) and PGE2. The Bland-Altman analyses indicate the two methods are commutable.

Gross-total hematoma removal of hypertensive basal ganglia hemorrhages: a long-term follow-up.

BACKGROUND AND PURPOSE: Hypertensive basal ganglia hemorrhage (HBGH) accounts for 35%-44% of cases of hypertensive intracranial hemorrhage (ICH), which is one of the most devastating forms of cerebrovascular disease. In this study, intracerebral hematoma was evacuated with a burr hole craniectomy. The relationships of residue hematoma volume to brain edema, inflammation factors and the long-term prognosis of HBGH patients were studied. METHODS: One hundred and seventy-six patients with HBGH were randomly divided into gross-total removal of hematoma (GTRH) and sub-total removal of hematoma (STRH) groups. The pre-operative and post-operative data of the patients in the two groups were compared. The pre-operative data included age, sex, hematoma volume, time from the ictus to the operation, Glasgow Coma Scale (GCS) scores, and the European Stroke Scale (ESS) scores. The post-operative information included edema grade, level of thromboxane B2 (TXB2), 6-keto-prostaglandin F1a (6-K-PGF1a), tumor necrosis factor-a (TNF-a) and endothelin (ET) in hematoma drainage or cerebral spinal fluid (CSF), ESS and Barthel Index (BI). RESULTS: There was no statistical difference between the two groups (P>0.05) in the pre-operative data. The levels of TXB2, 6-K-PGF1a, TNF-a and ET in the GTRH group were significantly lower than those in the STRH group at different post-operative times. The ESS in the GTRH group increased rapidly after the operation and was higher than that in the STRH group. There was a significant difference between the two groups (P<0.05). The post-operative CT scan at different times showed that the brain edema grades were better in the GTRH group than in the STRH group. The BI was higher in the GTRH group than in the STRH group (P<0.05). CONCLUSIONS: GTRH is an effective method to decrease ICH-induced injury to brain tissue. Such effect is related to decreased perihematomal edema formation and secondary injury by coagulation end products activated inflammatory cascade.

Quantitative characterization of prostaglandins in the uterus of early pregnant cattle.

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography-tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF(1alpha) (stable metabolite of PGI(2)) was predominant followed by PGF(2alpha)>PGE(2)>PGD(2) approximately TXB(2) (stable metabolite of TXA(2)). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF(2alpha)/PGE(2) ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF(1alpha) (6.4 ng/ml) followed by PGF(2alpha) (1.1 ng/ml) and PGE(2) (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI(2) and PGF(2alpha) receptors were abundantly expressed by the trophoblast, abundant amounts of PGI(2) and PGF(2alpha) in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE(2) in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice.

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H(2) (PGH(2)) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC(Min/+) mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH(2) into PGE(2), surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p<0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p<0.0005). No deviation regarding the expression of other PGE(2) related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE(2) levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH(2) derived prostanoids were generally enhanced, being most prominent for TxA(2) and PGD(2). Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE(2) during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.

Predominant basal directional release of thromboxane, but not prostacyclin, by placental trophoblasts from normal and preeclamptic pregnancies.

OBJECTIVE: To investigate apical and basal releases of thromboxane (TX) and prostacyclin (PGI2) by trophoblasts (TCs) from normal and preeclamptic (PE) placentas. METHODS: TCs isolated from normal and PE placentas were incubated in cell culture inserts for 48h. Medium from the upper (apical) and the lower (basal) chambers were then collected separately and measured for TX and PGI2 by their stable metabolites of TXB2 and 6-keto PGF1alpha by ELISA. Apical and basal releases of TX and PGI were also examined with apical exposure of TCs to arachidonic acid (AA)+/-aspirin at different concentrations. Villous tissue expressions for PGI synthase, TX synthase and TX (TP) receptor were examined by immunohistochemistry. RESULTS: (1) TXB2, but not 6-keto PGF1alpha, concentrations were significantly higher in the lower than in the upper chambers with both normal and PE TCs (p<0.01); (2) apical exposure of TCs to AA resulted in a significant increase in TX release towards both the upper and the lower chambers in normal TCs (p<0.01), but only a significant increase in the upper chamber in PE TCs (p<0.01); (3) aspirin could attenuate AA-induced TX release both in the upper and the lower chambers in normal, but not in PE, TCs (p<0.01), respectively; (4) there were no differences in 6-keto PGF1alpha productions both in normal and PE TCs treated with AA+/-aspirin; (5) intense staining of TX synthase and TP receptor was seen in syncytiotrophoblast layer, villous core vessels and stromal cells in preeclamptic placental tissue sections. CONCLUSION: Predominant basal release of TX together with intense staining of TX synthase and TP receptor in trophoblasts, stromal cells and villous core vessels are found in placentas from PE. We speculate if predominant basal release of TX by TCs occurs in vivo as we found in our in vitro culture condition, basal released TX may play a significant role in increased placental vasoconstriction such as in PE.

Involvement of prostaglandin E receptor EP3 subtype and prostacyclin IP receptor in decreased acid response in damaged stomach.

We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandin E (PGE) receptor EP1 and EP3 subtypes or prostacyclin IP receptors in the decrease in acid secretion in the damaged mouse stomach. Male C57/BL6 mice, both wild type and animals lacking EP1, EP3, or IP receptors, were used after 18 h of fasting. Under urethane anesthesia, the stomach was mounted on an ex-vivo chamber and perfused with saline, and acid secretion as well as transmucosal potential difference (PD) was measured before and after exposure to 20 mM taurocholate Na (TC) for 20 min. Indomethacin, SC-560 or rofecoxib was given i.d. 30 min before TC. Mucosal exposure to TC in wild-type mice caused a reduction in PD, followed by decrease in acid secretion. Indomethacin attenuated the decrease in acid secretion after exposure to TC in wild-type mice, an effect mimicked by SC-560 but not rofecoxib, yet none of these drugs affected the decrease in PD. An altered acid response after exposure to TC was similarly observed in EP1 (-/-) mice but mitigated in mice lacking either EP3 or IP receptors, although a decrease in PD was observed in all groups. Furthermore, the decreased acid response was also attenuated by prior administration of the EP3- but not EP1- antagonist. Mucosal levels of PGE(2) and 6-keto PGF(1a) increased after exposure to TC in all groups of mice. In conclusion, the decrease in acid secretion in the damaged stomach is mediated by endogenous PGs derived from COX-1, through PGE(2)/EP3 receptors and prostacyclin/IP receptors.

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft.

BACKGROUND: Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha). METHODS: Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1alpha in the culturing solutions were examined by radioimmunology to measure endothelial function. RESULTS: Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234 +/- 29) pg/ml and (428 +/- 98) pg/ml respectively in Group C and (196 +/- 30) pg/ml and (346 +/- 120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P < 0.05). CONCLUSIONS: Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.

Antioxidative and anti-inflammatory actions of docosahexaenoic acid and eicosapentaenoic acid in renal epithelial cells and macrophages.

Oxidative stress due to excessive reactive species (RS) and weakened antioxidant defenses is causally associated with inflammation and inflammatory mediators. To investigate the effects of the major fish oil ingredients, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on oxidative stress-related inflammatory status, we conducted in vitro experiments utilizing rat renal epithelial cells (NRK-52E) and murine macrophages (RAW 264.7) by assessing their effects on the generation of cyclooxygenase (COX)-2-derived and xanthine oxidase (XOD)-derived RS, reduced glutathione (GSH) levels, and antioxidative enzyme activities. Additionally, 6-keto-prostaglandin (PG) F1alpha, PGE2, and nitrite levels were measured in lipopolysaccharide-stimulated RAW 264.7 macrophages. Results showed that the generation of RS from arachidonic acid through the COX-2 and XOD pathways was effectively suppressed by DHA and EPA, while GSH levels and antioxidative enzyme activities were significantly enhanced by DHA and EPA. Furthermore, levels of inflammatory mediators (thromboxane B2, PGE2, and 6-keto-PGF1alpha) and nitrite were effectively down-regulated by DHA and EPA. These results strongly indicate that DHA and EPA exert antioxidative and anti-inflammatory actions by reducing the cellular levels of RS, pro-inflammatory mediators, and nitrite levels and by maintaining higher GSH levels and antioxidative enzyme activities.

Experimental arterial thrombosis regulated by androgen and its receptor via modulation of platelet activation.

OBJECTIVES: The aim of our study is to elucidate whether experimental arterial thrombosis is regulated by physiological doses of androgen and its receptor via modulation of platelet activation. METHODS: Surgical castration was performed in male rats and ferric chloride (FeCl(3)), as a stimulator, induced the experimental arterial thrombosis. Testosterone was measured directly by chemiluminescent immunoassay on the Bayer ADVIA Centaur analyzer. Dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. A platelet aggregometer was used to assess aggregation, and a platelet adherometer was used to measure adhesion. The contents of TXB(2) and 6-Keto-PGF(1alpha) were assayed by radio-immunoassay using commercially available kits. RESULTS: Our data showed that DHT replaced restored circulating DHT of castrated rats to physiological levels, without being altered by treatment with flutamide. Castration caused significant increases in the thrombus area and weight in castrated rats as compared with control group. In PRP diluted with autologous PPP, ADP-induced platelet aggregation rate was only 9.10%. However, in PRP diluted with Tyrode's buffer, 1 microM ADP-induced platelet aggregation rate rose to 63.65%. In PRP diluted with Tyrode's buffer, and pretreated with DHT (1 nM, 2 nM), ADP-induced platelet aggregation was significantly lowered again. Platelet aggregation in PRP diluted with autologous PPP was enhanced in castrated rats as compared with sham-operated rats, and DHT (2 nM) replacement suppressed platelet aggregation in castrated PRP to the level similar to that of sham-operated rats. However, presence of flutamide (3 microM) significantly increased platelet aggregation in PRP diluted with autologous PPP or Tyrode's buffer. DHT (2 nM) replacement significantly inhibited the ADP-induced platelet adhesion. However, presence of flutamide (3 microM) increased ADP-induced platelet adhesion again. DHT replacement obviously reduced the ratio of TXB(2) to 6-keto-PGF(1alpha) in castrated rats. However, administration of flutamide and DHT to castrated rats caused an increase in the ratio of TxB(2) to 6-keto-PGF1alpha. CONCLUSION: Inhibition of experimental arterial thrombosis by androgen at physiological doses and its receptor is mediated via modulation of platelet activation.

Screening method for nonsteroidal antiinflammatory drugs based on the cyclooxygenase 2 pathway activated by serum-free stimulation in A549 cells.

Cyclooxygenase 2 (COX-2) pathway inhibitors were regarded as promising nonsteroidal antiinflammatory drugs (NSAIDs). We discovered that the COX-2 pathway in A549 cells, a human lung cancer cell line, was activated by serum-free stimulation, and a drug screening model for NSAIDs was established based on this principle with simple performance and sufficient reliability. The COX-2 pathway was activated by treating with serum-free medium for 12 h. The activated cells were incubated with NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), acetyl salicylic acid (ASA) (nonselective COX inhibitor) at 37 degrees C for 15 min. Then the cells were incubated with 10 microM of arachidonic acid (AA) for another 30 min prostaglandin E2 and 6-keto-prostaglandin F(1alpha) were assayed in an enzyme immunoassay (EIA). The results showed that the COX-2 pathway was dominant in A549 cells whether activated by serum-free medium or not, and the COX-1 pathway could be ignored. The model accepted the positive inhibition threshold as NS398 2 microM; if a compound (10 microM) inhibited COX-2 pathway more than NS398 (2 microM), it was regarded as a hit. The COX-2 pathway inhibition experiment showed that the Z;-factor of the screening model was 0.62, which suggests that the model is suitable for COX-2 pathway inhibitor screening.

Gastric mucosal levels of prostaglandins and leukotrienes in patients with gastric ulcer after treatment with rabeprazole in comparison to treatment with ranitidine.

AIM: Prostaglandins (PGs) and leukotrienes (LTs) are major factors involved in the defense of the gastric mucosa against ulcer formation. However, little is still known about the gastromucosa-protecting action of proton pump inhibitors (PPIs) and histamine H(2) receptor antagonists (H(2) blockers) in patients with gastric ulcer. We therefore examined the effectiveness of a PPI in protecting the gastric mucosa. METHODS: We compared the PGE(2) and LTB(4) levels and the expression levels of cyclooxygenase (COX)-1 and COX-2 mRNA in the gastric mucosa in gastric ulcer patients between the group treated for 8 weeks with a PPI, rabeprazole (PPI group; n=5), and the group treated for 8 weeks with an H(2) blocker, ranitidine (H(2) blocker group; n=6), as well as in nonulcer subjects (control group; n=5). RESULTS: The mucosal levels of PGE(2) and COX-2 mRNA expression were significantly lower in the ulcer patients than those in the nonulcer patients, whereas the LTB(4) level was significantly higher in the ulcer patients than that in the nonulcer patients, and it was also significantly lower in the ulcerated mucosa than that in the nonulcerated mucosa. The PPI group had a significantly increased PGE(2) and decreased LTB(4) levels in comparison to the H(2) blocker group during the ulcer-healing stage. The COX-1 mRNA expression showed no difference among the PPI and H(2) blocker groups or between before and after the treatment. However, the COX-2 mRNA expression increased in the PPI group more than that in the H(2) blocker group during the ulcer-healing stage. CONCLUSION: These findings demonstrated the significant gastric-mucosa-protecting effect of PPI by increasing the PGE(2) production and reducing the LTB(4) production.

Gastro-protective action of lafutidine mediated by capsaicin-sensitive afferent neurons without interaction with TRPV1 and involvement of endogenous prostaglandins.

AIM: Lafutidine, a histamine H2 receptor antagonist, exhibits gastro-protective action mediated by capsaicin-sensitive afferent neurons (CSN). We compared the effect between lafutidine and capsaicin, with respect to the interaction with endogenous prostaglandins (PG), nitric oxide (NO) and the afferent neurons, including transient receptor potential vanilloid subtype 1 (TRPV1). METHODS: Male SD rats and C57BL/6 mice, both wild-type and prostacyclin IP receptor knockout animals, were used after 18 h of fasting. Gastric lesions were induced by the po administration of HCl/ethanol (60% in 150 mmol/L HCl) in a volume of 1 mL for rats or 0.3 mL for mice. RESULTS: Both lafutidine and capsaicin (1-10 mg/kg, po) afforded dose-dependent protection against HCl/ethanol in rats and mice. The effects were attenuated by both the ablation of CSN and pretreatment with N(G)-nitro-L-arginine methyl ester, yet only the effect of capsaicin was mitigated by prior administration of capsazepine, the TRPV1 antagonist, as well as indomethacin. Lafutidine protected the stomach against HCl/ethanol in IP receptor knockout mice, similar to wild-type animals, while capsaicin failed to afford protection in the animals lacking IP receptors. Neither of these agents affected the mucosal PGE2 or 6-keto PGF(1alpha) contents in rat stomachs. Capsaicin evoked an increase in [Ca2+]i in rat TRPV1-transfected HEK293 cells while lafutidine did not. CONCLUSION: These results suggest that although both lafutidine and capsaicin exhibit gastro-protective action mediated by CSN, the mode of their effects differs regarding the dependency on endogenous PGs/IP receptors and TRPV1. It is assumed that lafutidine interacts with CSN at yet unidentified sites other than TRPV1.

Oxygen-radical regulation of renal blood flow following suprarenal aortic clamping.

OBJECTIVE: Renal insufficiency continues to be complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. One proposed mechanism of renal injury after suprarenal aortic clamping (above the superior mesenteric artery) and reperfusion (SMA-SRACR) is the loss of microvascular renal blood flow with subsequent loss of renal function. This study examines the hypothesis that the loss of medullary and cortical microvascular blood flow following SMA-SRACR is due to oxygen-derived free radical down-regulation of endogenous medullary and cortical nitric oxide synthesis. METHODS: Anesthetized male Sprague-Dawley rats (about 350 g) either had microdialysis probes or laser Doppler fibers inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored. The microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer's solution. The animals were subjected to SMA-SRACR (or sham) for 30 minutes, followed by 60 minutes of reperfusion. Laser Doppler blood flow after the 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion was compared with the time zero (basal) and with the corresponding sham group and reported as percent change compared with the time zero baseline. The microdialysis fluid was collected at time zero (basal) and compared with the dialysis fluid collected after 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion as well as the corresponding sham group. The microdialysis dialysate was analyzed for total nitric oxide (microM) and prostaglandin E2 (PGE2), 6-keto-PGF(1alpha) (PGI2 metabolite), and thromboxane B2 synthesis. The data are reported as percent change compared with the baseline time zero. The laser Doppler blood flow and microdialysis groups were treated with either saline carrier, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (30 mg/kg, nitric oxide synthesis inhibitor), L-arginine (400 mg/kg, nitric oxide precursor), superoxide dismutase (SOD, 10,000 U/kg, oxygen-derived free radical scavenger), L-NAME + SOD, or L-arginine + SOD. SOD was given 30 minutes before the reperfusion, and the other drugs were given 15 minutes before reperfusion. The renal cortex and medulla were separated and analyzed for inducible nitric oxide synthase (iNOS), cyclooxygenase-2, prostacyclin synthase, and PGE2 synthase content by Western blot. RESULTS: Superior mesenteric artery-SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical nitric oxide synthesis. These changes were further accentuated by L-NAME treatment but restored toward sham levels by L-arginine treatment after SMA-SRACR. The kidney appeared to compensate for these changes by increasing cortical and medullary PGE2 synthesis and release. SOD treatment restored renal cortical and medullary nitric oxide synthesis and blood flow in the ischemia-reperfusion group and in the ischemia-reperfusion group treated with L-NAME. CONCLUSIONS: These data show that nitric oxide is important in maintaining renal cortical and medullary blood flow and nitric oxide synthesis. These data also support the hypothesis that the loss of medullary and cortical microvascular blood flow following SRACR is due in part to oxygen-derived free radical downregulation of endogenous medullary and cortical nitric oxide synthesis.

Antithrombin reduces reperfusion-induced hepatic metastasis of colon cancer cells.

AIM: To examine whether antithrombin (AT) could prevent hepatic ischemia/reperfusion (I/R)-induced hepatic metastasis by inhibiting tumor necrosis factor (TNF)-alpha-induced expression of E-selectin in rats. METHODS: Hepatic I/R was induced in rats and mice by clamping the left branches of the portal vein and the hepatic artery. Cancer cells were injected intrasplenically. The number of metastatic nodules was counted on day 7 after I/R. TNF-alpha and E-selectin mRNA in hepatic tissue, serum fibrinogen degradation products and hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI2, were measured. RESULTS: AT inhibited increases in hepatic metastasis of tumor cells and hepatic tissue mRNA levels of TNF-alpha and E-selectin in animals subjected to hepatic I/R. Argatroban, a thrombin inhibitor, did not suppress any of these changes. Both AT and argatroban inhibited I/R-induced coagulation abnormalities. I/R-induced increases of hepatic tissue levels of 6-keto-PGF(1alpha) were significantly enhanced by AT. Pretreatment with indomethacin completely reversed the effects of AT. Administration of OP-2507, a stable PGI2 analog, showed effects similar to those of AT in this model. Hepatic metastasis in congenital AT-deficient mice subjected to hepatic I/R was significantly increased compared to that observed in wild-type mice. Administration of AT significantly reduced the number of hepatic metastases in congenital AT-deficient mice. CONCLUSION: AT might reduce I/R-induced hepatic metastasis of colon cancer cells by inhibiting TNF-alpha-induced expression of E-selectin through an increase in the endothelial production of PGI2. These findings also raise the possibility that AT might prevent hepatic metastasis of tumor cells if administered during the resection of liver tumors.

Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis.

The Gram-negative periodontal pathogen Porphyromonas gingivalis synthesizes several classes of novel phosphorylated complex lipids, including the recently characterized phosphorylated dihydroceramides. These sphingolipids promote the interleukin-1 (IL-1)-mediated secretion of inflammatory mediators from fibroblasts, including prostaglandin E2 and 6-keto prostaglandin F2alpha, and alter gingival fibroblast morphology in culture. This report demonstrates that one additional class of phosphorylated complex lipids of P. gingivalis promotes IL-1-mediated secretory responses and morphological changes in cultured fibroblasts. Structural characterization identified the new phospholipid class as 1,2-diacyl phosphatidylethanolamine, which substituted predominantly with isobranched C15:0 and C13:0 fatty acids. The isobranched fatty acids, rather than unbranched fatty acids, and the phosphoethanolamine head group were identified as the essential structural elements required for the promotion of IL-1-mediated secretory responses. These structural components are also observed in specific phosphorylated sphingolipids of P. gingivalis and likely contribute to the biological activity of these substances, in addition to the phosphatidylethanolamine lipids described in this report.

Chronic administration of ethyl docosahexaenoate reduces gerbil brain eicosanoid productions following ischemia and reperfusion.

Arachidonic acid (AA) and its vasoactive metabolites have been implicated in the pathogenesis of brain damage induced by cerebral ischemia. The membrane AA concentrations can be reduced by changes in dietary fatty acid intake. The purpose of the present study was to investigate the effects of chronic ethyl docosahexaenoate (E-DHA) administration on the generation of eicosanoids of AA metabolism during the period of reperfusion after ischemia in gerbils. Weanling male gerbils were orally pretreated with either E-DHA (100, 200 mg/kg) or vehicle, once a day, for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 10 min. E-DHA (200 mg/kg) pretreatment significantly decreased the content of brain lipid AA at the termination of treatment, prevented postischemic impaired regional cerebral blood flow (rCBF) and reduced the levels of brain prostaglandin (PG) PGF(2alpha) and 6-keto-PGF(1alpha), and thromboxane B(2) (TXB(2)), as well as leukotriene (LT) LTB(4) and LTC(4) at 30 and 60 min of reperfusion compared with the vehicle, which was well associated with the attenuated cerebral edema in the E-DHA-treated brain after 48 h of reperfusion. These data suggest that the E-DHA (200 mg/kg) pretreatment reduces the postischemic eicosanoid productions, which may be due to its reduction of the brain lipid AA content.