To test the hypothesis that epoxyeicosatrienoic acids (EETs) facilitate pulmonary responses to hypoxia, male wild-type (WT) and soluble-epoxide hydrolase knockout (sEH-KO) mice, and WT mice chronically fed a sEH inhibitor (t-TUCB; 1 mg·kg-1·day-1) were used. Right ventricular systolic pressure (RVSP) was recorded under control and hypoxic conditions. The control RVSP was comparable among all groups. However, hypoxia elicited increases in RVSP in all groups with predominance in sEH-KO and t-TUCB-treated mice. 14,15-EEZE (an EET antagonist) attenuated the hypoxia-induced greater elevation of RVSP in sEH-deficient mice, suggesting an EET-mediated increment. Exogenous 5,6-; 8,9-, or 14,15-EET (0.05 ng/g body wt) did not change RVSP in any conditions, but 11,12-EET enhanced RVSP under hypoxia. Isometric tension was recorded from pulmonary arteries isolated from WT and sEH-KO mice, vessels that behaved identically in their responsiveness to vasoactive agents and vessel stretch. Hypoxic pulmonary vasoconstriction (HPV, expressed as increases in hypoxic force) was significantly greater in vessels of sEH-KO than WT vessels; the enhanced component was inhibited by EEZE. Treatment of WT vessels with 11,12-EET enhanced HPV to the same level as sEH-KO vessels, confirming EETs as primary players. Inhibition of cyclooxygenases (COXs) significantly enhanced HPV in WT vessels, but attenuated HPV in sEH-KO vessels. Blocking/inhibiting COX-1, prostaglandin H2 (PGH2)/thromboxane A2 (TXA2) receptors and TXA synthase prevented the enhanced HPV in sEH-KO vessels but had no effects on WT vessels. In conclusion, an EET-dependent alteration in PG metabolism that favors the action of vasoconstrictor PGH2 and TXA2 potentiates HPV and hypoxia-induced elevation of RVSP in sEH-deficient mice.

Histamine plays important physiological roles in the upper gastrointestinal tract and acts via the H2 receptor. The -1018 G>A (rs2067474) in an enhancer element of the promoter and non-synonymous rs79385261 (Asn46Thr) were identified in HRH2. We attempted to clarify the associations of these polymorphisms with gastric carcinogenesis. The study was performed in 321 patients with gastric cancer and 599 subjects with no evidence of gastric malignancies on upper gastroduodenal endoscopy. The genotypes were determined using a one-tube multiplex PCR-SSCP method. The degree of gastritis was assessed in 496 subjects and serum pepsinogen (PG) I/II levels were measured in 124 subjects without gastric cancer. The minor allele of Asn46Thr could not be detected. The frequencies of the -1018 A allele in the non-GC and GC groups were 13.5% and 8.26%, respectively (p=0.00077). Overall, -1018 GG homozygotes had an increased risk for developing gastric cancer (OR 1.68; 95% CI 1.17-2.42; p=0.0052), especially intestinal type cancer (OR 1.94; 95% CI 1.23-3.08; p=0.0047). In subjects aged >60 years, the adjusted risk for gastric cancer among individuals who were -1018 GG homozygotes was 1.87 (range 1.19-2.93; p=0.0065) compared with A carriers. In the gastric cancer cases located in the antrum and at comparative advanced stage, -1018 GG homozygosity was a significantly increased risk factor. In subjects >60 years, the metaplasia score was significantly higher in -1018 GG homozygotes than A carriers. Both atrophy and metaplasia scores were significantly increased with age only in -1018 GG homozygotes. The PG I/II ratio was significantly decreased in H. pylori positive GG homozygotes than negative GG homozygotes and positive A carriers. Our results suggest that -1018 GG homozygosity of HRH2 may be associated with the severity of gastric mucosal atrophy. This genotype has an increased risk for the subsequent development of gastric cancer, especially intestinal type, at advanced age.

Hydrogen sulfide (H2S) is a metabolite and signaling molecule in biological tissues that regulates many physiological processes. Reliable and sensitive methods for H2S analysis are necessary for a better understanding of H2S biology and for the pharmacological modulation of H2S levels in vivo. In this chapter, we describe the use of gas chromatography coupled to sulfur chemiluminescence detection to measure the rates of H2S production and degradation by tissue homogenates at physiologically relevant concentrations of substrates. This method allows separation of H2S from other sulfur compounds and provides sensitivity of detection to ~15 pg (or 0.5 pmol) of H2S per injected sample.

OBJECTIVE

Denudation and regeneration of the vascular endothelium are important in the pathogenesis of atherosclerosis. The aim of this study is to clarify the mechanisms of functional alterations in remodelled arteries following endothelial injury.

RESULTS

Non-mechanical endothelial injury was induced by 540-nm light irradiation of rose Bengal in femoral arteries of Wistar rats. Endothelium-dependent vasodilation was assessed by the response to acetylcholine (ACh) 1, 2, and 4 weeks after the injury. In control arteries, ACh-induced relaxation was mainly nitric oxide-dependent at all study time points. In injured arteries, this response was completely restored at 1 week, but was more dependent on KCl-sensitive endothelium-derived hyperpolarizing factor production during the first 2 weeks. Cyclooxygenase (COX) isoforms 1 and 2 were detected in the endothelium of injured arteries, and inhibition of prostanoids production with the non-specific COX inhibitor indomethacin substantially enhanced the ACh-induced vasorelaxation response in injured arteries, but did not affect control arteries. Similar effects were observed with the COX-1 inhibitor SC-560, the COX-2 inhibitor NS-398, the thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist SQ29548 and the PGF2alpha receptor antagonist AL-8810. However, the TX synthetase inhibitor OKY-046 had no effect on ACh-induced relaxation in injured arteries.

CONCLUSIONS

In remodelled arteries following photochemical endothelial injury, the vasoconstrictive prostanoids PGH2 and PGF2alpha, but not TXA2, contribute to changes in endothelium-dependent vascular response via COX-1- and 2-dependent pathways.

Lymphocytes have surface receptors for a variety of hormones that play an important part in modulating the immune response. Most previous studies, however, have examined the effects of hormone agonists on heterogeneous bulk populations of cells, making it difficult to precisely identify the responding target cells. We have therefore studied a set of well characterized T cell clones for a series of adenylate cyclase-linked hormone receptors and examined changes in receptor expression that occur after cell activation. All clones tested had receptors for histamine, isoproterenol, and PGE1, but not for several other cAMP-active hormone agonists. The apparent receptor affinities and their specificities were characteristic of typical histamine H2, beta 2-adrenergic, and PGE receptors. The cAMP response to PG was higher and longer lasting than that to histamine or isoproterenol, both of which appear to undergo receptor desensitization. After activation of quiescent cells in IL-2-containing media, the cAMP response to all three ligands increased, peaking 4 to 5 days after stimulation, and then returned to basal levels as the cells ceased proliferating. Inasmuch as this effect did not require Ag, it appears that the coordinate regulation of responsiveness to these ligands is a direct result of lymphocyte activation. This increase in hormone receptor activity is functionally analogous to the up-regulation of receptors for other ligands that occurs after lymphocyte activation and further demonstrates the important immunoregulatory role played by the changing repertoire of surface receptors that is associated with activation.

The cytoprotective action of roxatidine acetate HCl (roxatidine) was investigated. We also studied the involvement of endogenous prostaglandins (PGs) in the cytoprotective action of roxatidine and the effect of roxatidine on SRS content in pleurisy induced by A23187. Simultaneously, these effects of roxatidine were compared with those of other histamine H2-receptor antagonists at the same anti-secretory activity level. Roxatidine prevented formation of the gastric mucosal lesions induced by abs. ethanol, 0.6 N HCl and 0.2 N NaOH, but it failed to prevent 30% NaCl-induced gastric mucosal lesions. Cimetidine, ranitidine and famotidine failed to prevent formation of the gastric mucosal lesions induced by necrotizing agents. The cytoprotective action of roxatidine was not abolished by pretreatment with indomethacin. Roxatidine did not greatly influence SRS production. Consequently, it appears that roxatidine has a cytoprotective action and that this action is not associated with endogenous PGs and SRS.

Lipopolysaccharide (LPS) is recognized as a key molecule in the pathogenesis of Gram negative sepsis and septic shock. In the present study, we demonstrate that LPS (1-1000 pg/ml) concentration dependently up-regulated the expression of intercellular adhesion molecule (ICAM)-1, B7.1, and B7.2 on human monocytes using fluorescence-activated cell sorting analysis, and that tumor necrosis factor (TNF)-alpha production induced by LPS in peripheral blood mononuclear cells (PBMCs) was inhibited by the addition of antibodies against these adhesion molecules, suggesting the dependence of TNF-alpha production on cell-cell interaction through these adhesion molecules. Moreover, we found that histamine (10(-7)-10(-4) M) concentration dependently inhibited the expression of ICAM-1 and B7.1, but not B7.2 on monocytes induced by LPS. Histamine also inhibited the responses of TNF-alpha production induced by LPS. The modulatory effects of histamine on ICAM-1 and B7.1 expression and TNF-alpha production were all concentration dependently antagonized by famotidine but not by d-chlorpheniramine and thioperamide, and were mimicked by selective H2-receptor agonists but not by H1-, H3-, and H4-receptor agonists, indicating the involvement of H2-receptors in the histamine action. Dibutyryl cAMP down-regulated ICAM-1 and B7.1 expression on monocytes stimulated by LPS, suggesting the mediation by the cyclic adenosine monophosphate-protein kinase A pathway of H2-receptor activation. These results as a whole indicated that histamine via H2-receptor inhibited the LPS-induced TNF-alpha production through the regulation of ICAM-1 and B7.1 expression, leading to the reduction of innate immune response stimulated by LPS.

Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour.

U-44069 is a stable prostaglandin (PG) H2 analogue and a potent vasoconstrictor. Its in vivo and in vitro actions mimic those of thromboxane A2. We have studied the effects of the calcium antagonist diltiazem upon the vasoconstriction induced by U-44069 using isolated rat aortic smooth muscle and isolated perfused rat kidney (IPRK). The administration of 10(-6)M U-44069 elicited maximally effective contractions in isolated aortic rings and increased 45Ca uptake from a control value of 285 +/- 6 mumol/kg to 344 +/- 8 mumol/kg. Diltiazem reduced U-44069-induced tension development and 45Ca uptake of isolated aortic smooth muscle 73 +/- 2 and 91 +/- 3%, respectively. The dose dependency of each of these effects of diltiazem was similar (EC50 = 369 nM and 334 nM for tension and 45Ca flux, respectively). When administered to the IPRK, 10(-6) M U-44069 caused a 82 +/- 3% decrease in glomerular filtration rate (GFR) and a 80 +/- 4% decrease in filtration fraction but reduced renal perfusate flow (RPF) only 13 +/- 8% (P less than 0.005). Diltiazem completely reversed the actions of U-44069 on the IPRK (EC50 = 288 nM and 323 nM for GFR and RPF, respectively). Diltiazem thus inhibited U-44069-induced tension development and 45Ca uptake by vascular smooth muscle and increased GFR within identical dose ranges. The contractile response of isolated rat glomeruli was also assessed. U-44069 reduced the volume of isolated glomeruli, but this action was neither prevented nor reversed by diltiazem. These results are consistent with the hypothesis that diltiazem increased GFR by inhibiting U-44069-induced Ca influx at preglomerular vessels.

Studies were designed to test the role of the endothelium and endogenous release of thromboxane (Tx) A2 in the contractile response of rat thoracic aortic rings to the TxA2/prostaglandin (PG) H2 mimetic, U-46,619. U-46,619 caused a dose-dependent contraction of rings with endothelium (mean ED50 = 6.54 +/- 3.02 x 10(-9) M; n = 13) which was abolished by the TxA2/PGH2 receptor antagonist, SQ-29,548. Removal of endothelium greatly potentiated (P less than .05) the contractile response to U-46,619 (ED50 = 4.78 +/- 2.14 x 10(-10) M; n = 14). On addition to the organ bath, oxyhemoglobin (10(-6) M), an inhibitor of endothelium-derived relaxing factor, increased vascular smooth muscle contraction in response to U-46,619 and abolished the difference in response between rings with endothelium (ED50 = 6.63 +/- 0.38 x 10(-11) M) and those without (ED50 = 5.13 +/- 0.18 x 10(-11) M). Vascular contraction with U-46,619 (10(-7] was associated with release of immunoreactive TxB2 and 6-keto PGF1 alpha as well as increased conversion of [14C]arachidonate to [14C]TxB2 and 6-keto-[14C]PGF1 alpha. To test the role of endogenous TxA2 in response to U-46,619, the TxA2 synthetase inhibitor UK-38,485 (10(-6) M) was added directly to the organ bath; this diminished (P less than .05) the contractile responses to U-46,619 of rings with and without endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen sulfide (H2S) is a mediator with demonstrated protective effects for the cardiovascular system. On the other hand, prostaglandin (PG)E2 is involved in vascular wall remodeling by regulating matrix metalloproteinase (MMP) activities. We tested the hypothesis that endogenous H2S may modulate PGE2, MMP-1 activity and endogenous tissue inhibitors of MMPs (TIMP-1/-2). This regulatory pathway could be involved in thinning of abdominal aortic aneurysm (AAA) and thickening of saphenous vein (SV) varicosities. The expression of the enzyme responsible for H2S synthesis, cystathionine-γ-lyase (CSE) and its activity, were significantly higher in varicose vein as compared to SV. On the contrary, the endogenous H2S level and CSE expression were lower in AAA as compared to healthy aorta (HA). Endogenous H2S was responsible for inhibition of PGE2 synthesis mostly in varicose veins and HA. A similar effect was observed with exogenous H2S and consequently decreasing active MMP-1/TIMP ratios in SV and varicose veins. In contrast, in AAA, higher levels of PGE2 and active MMP-1/TIMP ratios were found versus HA. These findings suggest that differences in H2S content in AAA and varicose veins modulate endogenous PGE2 production and consequently the MMP/TIMP ratio. This mechanism may be crucial in vascular wall remodeling observed in different vascular pathologies (aneurysm, varicosities, atherosclerosis and pulmonary hypertension).

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.

Metformin confers vascular benefits beyond glycemia control, possibly via pleiotropic effects on endothelial function. In type-1-diabetes-mellitus (T1DM-)patients metformin improved flow-mediated dilation but also increased prostaglandin(PG)-F2α, a known endothelial-contracting factor. To explain this paradoxical finding we hypothesized that metformin increased endothelial-vasodilator mediators (e.g. NO and EDHF) to an even larger extent. Spontaneously-hypertensive-rats (SHR) display impaired endothelium-dependent relaxation (EDR) involving contractile PGs. EDR was studied in isolated SHR aortas and the involvement of PGs, NO and EDHF assessed. 12-week metformin 300 mg/kg/day improved EDR by up-regulation of NO and particularly EDHF; it also reduced blood pressure and increased plasma sulphide levels (a proxy for H2S, a possible mediator of EDHF). These effects persisted in SHR with streptozotocin (STZ)-induced T1DM. Vildagliptin (10 mg/kg/day), targeting the incretin axis by increasing GLP-1, also reduced blood pressure and improved EDR in SHR aortas, mainly via the inhibition of contractile PGs, but not in STZ-SHR. Neither metformin nor vildagliptin altered blood glucose or HbA1c. In conclusion, metformin reduced blood pressure and improved EDR in SHR aorta via up-regulation of NO and particularly EDHF, an effect that was independent from glycemia control and maintained during T1DM. A comparison to vildagliptin did not support effects of metformin mediated by GLP-1.

Among the potential novel therapeutics to treat bacterial infections, antimicrobial peptides (AMPs) are a very promising substitute due to their broad-spectrum activity and rapid bactericidal action. AMPs strongly interact with the bacterial membrane, and the need to have a correct understanding of the interaction between AMPs and lipid bilayers at a molecular level prompted a wealth of experimental and theoretical studies exploiting a variety of AMPs. Here, we studied the effects of magainin H2 (Mag H2), an analog of the well-known magainin 2 (wt Mag 2) AMP endowed with a higher degree of hydrophobicity, on giant unilamellar vesicles (GUVs) concentrating on its permeabilization activity and the effect on the lipid bilayer mechanical properties. We demonstrated that the increased hydrophobicity of Mag H2 affects its selectivity conferring a strong permeabilization activity also on zwitterionic lipid bilayers. Moreover, when lipid mixtures including PG lipids are considered, PG has a protective effect, at variance from wt Mag 2, suggesting that for Mag H2 the monolayer curvature could prevail over the peptide-membrane electrostatic interaction. We then mechanically characterized GUVs by measuring the effect of Mag H2 on the bending constant of lipid bilayers by flickering spectroscopy and, by using micropipette aspiration technique, we followed the steps leading to vesicle permeabilization. We found that Mag H2, notwithstanding its enhanced hydrophobicity, has a pore formation mechanism compatible with the toroidal pore model similar to that of wt Mag 2.

Although in most patients with duodenal ulcer disease the ulcer heals after 8 weeks of treatment with standard doses of H2 blockers or other agents, in about 10% the ulcer does not heal. These patients are considered 'refractory' to treatment. Reasons often cited for non-healing include poor patient compliance, cigarette smoking, and non-steroidal anti-inflammatory drug (NSAID) use. Gastric acid hypersecretion also appears to be an important factor in non-healing with standard doses of antisecretory agents. We have defined idiopathic gastric acid hypersecretion as a basal acid output of greater than 10 mmol/h in the absence of an elevated fasting serum gastrin level (or a negative secretin test if gastrin level greater than 100 pg/ml) to exclude persons with Zollinger-Ellison syndrome. Among the acid/peptic-related disorders in which idiopathic gastric acid hypersecretion should be considered are refractory duodenal ulcer, refractory gastro-oesophageal reflux disease (especially patients with oesophagitis), postbleeding duodenal ulcer, and certain rare disorders such as hereditary angioedema. Some children with atypical abdominal pain may also be hypersecretors of gastric acid. Once identified, patients with refractory duodenal ulcer or gastro-oesophageal reflux disease are treated with incremental doses of ranitidine titrated against the level of gastric acid secretion that remains during therapy. Ranitidine was selected to avoid the dose-related antiandrogenic effects and potential hepatic cytochrome P450 system-related drug interactions that may occur with cimetidine. In most cases of refractory duodenal ulcer, doubling the standard dose of ranitidine (to 300 mg b.d.) is sufficient to achieve symptomatic relief and mucosal healing. Higher doses appear to be necessary for refractory oesophagitis. To date, no side effects have been associated with high doses of ranitidine (up to 1800 mg/day) for periods of longer than 6 months. Idiopathic gastric acid hypersecretion is an important factor in explaining why not all patients respond to a 'standard' ulcer-healing dose of H2 blocker, and it provides a rationale for use of higher-dose therapy as a safe and effective alternative to omeprazole or to combination drug therapy in refractory acid/peptic disease.

Harpagophytum procumbens has a long story of use for the treatment of inflammatory diseases. Considering both the antiinflammatory effects of H. procumbens in multiple tissues and the stability of harpagoside in artificial intestinal fluid, the aim of the present study was to explore the possible protective role of a microwave-assisted aqueous Harpagophytum extract (1-1000 μg/mL) on mouse myoblast C2C12 and human colorectal adenocarcinoma HCT116 cell lines, and isolated rat colon specimens challenged with lipopolysaccharide (LPS), a validated ex vivo model of acute ulcerative colitis. In this context, we evaluated the effects on C2C12 and HCT116 viability, and on LPS-induced production of serotonin (5-HT), tumor necrosis factor (TNF)-α, prostaglandin (PG)E2 and 8-iso-prostaglandin (8-iso-PG)F2α . Harpagophytum extract was well tolerated by C2C12 cells, while reduced HCT116 colon cancer cell viability. On the other hand, Harpagophytum extract reduced H2 O2 -induced (1 mM) reactive oxygen species (ROS) production, in both cell lines, and inhibited LPS-induced colon production of PGE2 , 8-iso-PGF2α , 5-HT and TNFα. Concluding, we demonstrated the efficacy of a microwave-assisted Harpagophytum aqueous extract in modulating the inflammatory, oxidative stress and immune response in an experimental model of inflammatory bowel diseases (IBD), thus suggesting a rational use of Harpagophytum in the management and prevention of ulcerative colitis in humans. Copyright © 2017 John Wiley & Sons, Ltd.

Proton pump inhibitors (PPIs) block gastric acid secretion and may increase serum gastrin concentration. The aim of this study was to determine whether fasting serum gastrin concentration predicts gastric acid suppression in patients on PPI therapy. Ambulatory pH monitoring with one pH probe in the distal esophagus and a second probe in the stomach was performed in patients with persistent symptoms of GERD despite PPI treatment. Upon completion of pH monitoring, blood was drawn for measurement of fasting serum gastrin concentration. In all, 51 patients were studied: 26 on PPIs, 1 on H2-receptor antagonists, and 24 off acid suppression. Fasting serum gastrin correlated inversely with percent time of gastric pH < 4 for all patients (r = -0.553; P<0.001) and for the subgroup of 26 patients on PPIs (r = -0.435; P = 0.027). In patients on PPIs, an elevated gastrin >> or =100 pg/ml) was associated with gastric pH < 4 for 25+/-7% of the time compared to 54+/-5% when the gastrin was normal (P = 0.004). Therapeutic gastric acid suppression (gastric pH < 4 for <50% of time) was present in 6 of 7 (86%) patients with an elevated fasting serum gastrin, compared with only 8 of 19 (42%) patients with a normal serum gastrin (P<0.05). In conclusion, there is a significant inverse correlation between the fasting serum gastrin concentration and gastric acid profile in patients with GERD. An elevated fasting serum gastrin concentration while on PPI therapy suggests that gastric acid secretion is adequately suppressed.

Serum gastrin levels were determined in 120 consecutive patients (43 females, 77 males) with peptic disease 24 h post dose after 4 weeks continuous omeprazole (40 mg daily) treatment. Serum gastrin levels were elevated in 33 (28%) but exceeded the twice normal range in only 6 patients (5%). Age and sex did not influence the magnitude of gastrin levels. Gastrin increments induced by omeprazole compared to pretreatment with H2-blockers in 60 cases were similar in both males (42 +/- 10 pg/ml) and females (44 +/- 9 pg/ml). It is concluded that the magnitude of gastrin increases observed during omeprazole therapy are small and independent of age and sex.

1. The present study was aimed to assess the interaction between nitric oxide (NO) and thromboxane (Tx) A2-prostaglandin (PG) H2 in single-pass perfused isolated kidneys of the rat. 2. Noradrenaline (NA, 63 and 110 nM) dose-dependently elevated the renal vascular resistance (RVR), the glomerular filtration rate (GFR) and the urinary excretion of sodium (UNa V). Infusion of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), an inhibitor of NO synthesis, enhanced the effects of NA on RVR and on UNa V, but decreased those on GFR. The TxA2-PGH2 (TP) receptor blockade by GR32191B (10 microM) attenuated this potentiating effect of L-NAME. 3. When renal perfusion pressure was stepwise increased from 90 to 150 mmHg, L-NAME similarly decreased renal perfusion flow rate and GFR. 4. The venous excretion of TxB2 and 6-keto-PGF1 alpha was increased by L-NAME in baseline conditions as well as after NA or increasing renal perfusion pressure (RPP). 5. These results suggest that: (1) TxA2 and PGH2 play an important role in the overall effect of the renal prostanoids, (2) NO strongly interacts with the cyclo-oxygenase pathway and reduces the prostanoid synthesis in the kidney, and (3) the pressor effect of L-NAME partly relies upon the vasoconstrictor effect of TxA2 and PGH2.

Using [125I]I-S-145-OH, a radiolabeled derivative of a thromboxane (TX) A2 receptor antagonist, we have studied the expression of the TXA2 receptor in several lines of cultured leukemia cells. Specific binding of the ligand was observed in cells of two human erythroleukemia cell lines, K562 and HEL. However, only negligible binding was seen in HL-60 human promyelocytic leukemia cells and L-1210 murine leukemia cells. Scatchard analyses revealed a curvilinear plot which indicated the existence of two classes of binding sites in these cells. The Kd and Bmax values of the high and low affinity bindings in HEL cells were 2.4 nM and 24 fmol/10(6) cells, and 58 nM and 360 fmol/10(6) cells, respectively. These values in K562 cells were 2.8 nM and 16 fmol/10(6) cells, and 18 nM and 46 fmol/10(6) cells, respectively. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the cultures of K562 and HEL cells caused a concentration- and time-dependent increase in the binding activity. TPA at 10(-8) M increased the Bmax values of both high and low affinity bindings approximately 3 times without significant change in their Kd values and these increases were inhibited by the addition of actinomycin D. Both classes of the binding in cells of each cell line were specifically displaced by several TXA2/prostaglandin (PG) H2 analogues, although the relative specificities to the analogues were different in the two classes. These results suggest that both HEL and K562 cells express the TXA2 receptor on their cell surface and TPA strongly induces this expression in these cells.

We tested the hypothesis that the production of prostaglandin E2 (PGE2) is impaired in duodenal ulcer disease and affected by treatment and healing. This was investigated by a study of maximal PGE2 synthesis rates in duodenal and antral mucosal biopsies obtained at endoscopy. The patients were divided into three groups. Group (a): endoscopically normal controls (n = 56); group (b): treatment controls (non-DU disease: gastric ulcer or oesophagitis treated by histamine H2 receptor antagonists) (n = 41); and group (c): patients with DU disease (n = 183) further subdivided into group (c1) active ulcer not on treatment (n = 47), (c2) treated active ulcer (n = 35), (c3) healed ulcer on treatment (n = 86), and (c4) healed ulcer not on treatment (n = 15). Group (a) synthesised (mean (SD] 106.6 (39.0) pg PGE2/mg wt of tissue from the duodenal bulb and 129.9 (56.9) from the second part of the duodenum. No difference was found between group (a) and (b) at either site. Group (c1) ulcer rim made 49.8 (22.7) and at all stages ulcer rim and scar made less than the control duodenal bulb (p less than 0.02). Uninvolved duodenal bulb form groups (c1) (63.4 (31.0], (c2) (83.6 (38.5], and (c3) (81.5 (31.1], however, also made significantly less than controls (p less than 0.02) and a similar though non-significant trend was seen in group (c4). Biopsies from the second part of the duodenum did not synthesise significantly less than the control group but a similar trend was noticed at each stage of ulcer treatment. Biopsies of control antrum synthesised 124.5 (32.2) but only 93.7 (44.2) in group (cl) (p < 0.005). All stages of duodenal ulcer healing were associated with a decreased capacity to synthesise the major prostaglandin PGE2 at the ulcer site and the uninvolved duodenal bulb and, in acute untreated duodenal ulcer, the uninvolved antrum. This decreased capacity may be the consequence of the disease process itself and not secondary to the treatment, indicating a basic pathophysiological abnormality which may explain the characteristic tendency of the disease to relapse.

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.

Melatonin attenuates acute gastric lesions induced by topical strong irritants because of scavenging of free radicals, but its role in the pathogenesis of stress-induced gastric lesions has been sparingly investigated. In this study we compared the effects of intragastric (i.g.) or intracerebroventricular (i.c.v.) administration of melatonin and its precursor, L-tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on gastric lesions induced by water immersion and restraint stress (WRS). The involvement of pineal gland, endogenous prostaglandins (PG) and sensory nerves in gastroprotective action of melatonin and L-tryptophan against WRS was studied in intact or pinealectomized rats or those treated with indomethacin or rofecoxib to suppress cyclooxygenase (COX)-1 and COX-2, respectively, and with capsaicin to induce functional ablation of the sensory nerves. In addition, the influence of i.c.v. and i.g. melatonin on gastric secretion was tested in a separate group of rats equipped with gastric fistulas. At 3.5 hr after the end of WRS, the number of gastric lesions was counted, the gastric blood flow (GBF) was determined by H2-gas clearance technique and plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for determination of expression of mRNA for COX-1 and COX-2 by reverse transcriptase-polymerase chain reaction (RT-PCR) and of the mucosal generation of prostaglandin E2 (PGE2) by RIA. Melatonin applied i.g. (1.25-10 mg/kg) or i.c.v. (1.25-10 microg/kg) dose-dependently inhibited gastric acid secretion and significantly attenuated the WRS-induced gastric damage. This protective effect of melatonin was accompanied by a significant rise in the GBF and plasma melatonin and gastrin levels and in mucosal generation of PGE2. Pinealectomy, which suppressed plasma melatonin levels, aggravated the gastric lesions induced by WRS and these effects were counteracted by i.g. or i.c.v. application of melatonin. Luzindole abolished completely the gastroprotective effects of melatonin and L-tryptophan and attenuated significantly the rise in GBF evoked by the indoleamine and its precursor. Indomethacin and rofecoxib, which diminished PGE2 biosynthesis by c. 90 and 75% or capsaicin denervation, attenuated significantly melatonin- and L-tryptophan-induced protection and the rise in the GBF. Both the protection and the hyperemia were restored by addition of exogenous CGRP to capsaicin-denervated animals. COX-1 mRNA was detected by RT-PCR in the intact and melatonin-treated gastric mucosa, while COX-2 mRNA, which was undetectable in the intact gastric mucosa, appeared in WRS-exposed mucosa, especially in the melatonin-treated animals and this was accompanied by increased generation of PGE2 in gastric mucosa. Pinealectomy downregulated COX-2 mRNA and this effect was reversed by supplementation of pinealectomized animals with melatonin. We conclude that, (a) exogenous melatonin and its precursor, L-tryptophan, attenuates WRS-induced gastric lesions via interaction with MT2 receptors, (b) this protective action of melatonin is because of an enhancement of gastric microcirculation, probably mediated by PGE2 derived from COX-2 overexpression and activity, the activation of brain-gut axis involving CGRP released from sensory nerves, and the release of gastrin and (c) the pineal plays an important role in the limitation of WRS-induced gastric lesions via releasing melatonin, which exerts gastroprotective and hyperemic activities against stress ulcerogenesis.

Lipopolysaccharides (LPS) derived from gram-negative bacteria were reported to impair gastrointestinal mucosal integrity, but the results obtained are controversial. This study was undertaken to determine the effects of short-term administration of LPS on gastric secretion and gastric damage induced by 100% ethanol and to assess the role of the gene expression of two isoforms of cyclooxygenase (COX), constitutive (COX-1) and inducible (COX-2), and endogenous prostaglandins (PG) on these effects of LPS. Fasted rats received vehicle (control) or LPS (0.1-40 mg/kg i.g. or i.p.) without or with pretreatment with nonselective inhibitors of COX activity, indomethacin (5 mg/kg i.p.) and meloxicam (2 mg/kg i.g.), or the selective COX-2 inhibitor NS-398 (10 mg/kg i.g.), followed by intragastric application of 100% ethanol. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured by the H2-gas clearance technique, mucosal PGE2 generation was measured by radioimmunoassay, and expression of COX-1 and COX-1 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR with [32P]dCTP and immunohistochemistry. LPS applied intraperitoneally in various doses (0.1-10 mg/kg), dose dependently inhibited gastric acid and pepsin secretion and significantly reduced the area of gastric lesions induced by ethanol, and this was accompanied by an attenuation of the ethanol-induced fall in GBF and increased mucosal generation of PGE2. LPS applied in higher doses, such as 20 or 40 mg/kg, that caused systemic hypotension failed to protect the mucosa against 100% ethanol. Suppression of mucosal PGE2 generation by indomethacin or meloxicam, significantly reduced the inhibitory action of LPS on gastric secretion and abolished LPS-induced gastroprotection and elevation of GBF. NS-398 did not influence PGE2 generation, but significantly attenuated the protection and hyperemia induced by LPS suggesting that COX-2-derived products play an important role in gastroprotection. The expression of COX-1 mRNA, as determined by RT-PCR, quantitative RT-PCR and immunohistochemistry was found in intact gastric mucosa and after LPS administration. In contrast, the expression of COX-2 mRNA was undetectable in intact gastric mucosa but appeared in this mucosa 2, 4 and 8 h after LPS administration. COX-2 mRNA was not detected in rats treated with ethanol but, when LPS was applied before ethanol, the enhanced expression of COX-2 was detected without affecting COX-1 mRNA expression. We conclude that acute parenteral LPS affords gastroprotection against ethanol-damage through an increase in gastric microcirculation and overexpression of COX-2 and enhanced endogenous PG release.