Exposure of isolated perfused rabbit lungs (IPL) to ischemia-reperfusion causes a transient increase in pulmonary arterial (PA) pressure at the onset of reperfusion. Because thromboxane A2 (TxA2) is a potent vasoconstrictor, we hypothesized that it may contribute to the ischemia-reperfusion-induced pressor response. To evaluate this hypothesis, we exposed IPL perfused with a cell-free solution to 40 min of warm ischemia followed by reperfusion and measured perfusate immunoreactive thromboxane B2 (iTxB2) and 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha). We observed that ischemia-reperfusion IPL compared with controls had an increase in PA pressure (40.2 +/- 4.8 vs. 9.3 +/- 0.3 mmHg, P < 0.05), lung edema (29.3 +/- 6.3 vs. -0.2 +/- 0.2 g, P < 0.05), iTxB2 perfusate levels (155 +/- 22 vs. < 50 pg/ml, P < 0.05), and i6-keto-PGF1 alpha (436 +/- 33 vs. 61 +/- 16 pg/ml, P < 0.05). In ischemia-reperfusion IPL, infusion of SQ 29548 (10(-6) M), a specific TxA2/prostaglandin H2 receptor antagonist, attenuated the PA pressor response and the degree of edema. We conclude that pulmonary hypertension associated with ischemia-reperfusion results in part from pulmonary release of TxA2. Furthermore, TxA2 directly through membrane effects or indirectly through hydrostatic mechanisms increases the severity of ischemia-reperfusion-induced lung edema.

Serum cholecystokinin (CCK) levels were measured in 10 patients with chronic duodenal ulcers, fasting and at intervals after two standard tests meals (300 ml of 40 mmol/1 phenylalanine solution), one given before and one during H2-receptor blockade with metiamide (200 mg four times a day). Fasting serum CCK levels were lower in all patients during treatment with metiamide (the mean level falling from 306-0 +/- 102-0 (SEM) to 82-1 +/- 23-6 pg/ml after treatment (p less than 0-01)). In contrast, peak serum CCK levels after the meal were not significantly different (7400 +/- 1141 pg/ml before treatment and 7569 +/- 1293 pg/ml on metiamide). We conclude that in duodenal ulcer patients CCK secretion under basal condtions may be in part dependent on stimulation of the small intestinal mucosa by gastric acid, but that, after an amino acid meal, gastric acid secretion is less important in determining the amount of CCK released.

This study examines the pathophysiology of stroke secondary to focal cerebral ischemia. The interaction of arachidonic acid metabolites and polyamines, a class of ubiquitous ornithine-derived molecules with important membrane effects on edema, Ca++-dependent endocytosis, platelet function, and prostaglandin (PG) formation, are correlated with regional changes in H2 clearance, cerebral blood flow (rCBF), ischemic edema, and somatosensory evoked responses (SSERs) after middle cerebral artery (MCA) occlusion. Thirty cats were studied up to 3 hours before and 6 hours after right MCA occlusion. Four areas of brain showing different levels of perfusion after MCA occlusion were sampled for tissue levels of PGs: 6-keto-PGF1 alpha, PGE2, and as well as thromboxane B2 (TXB2), ornithine decarboxylase activity (ODC) (a measure of polyamine activity) and gravimetric determination of cerebral edema. After right MCA occlusion, right hemisphere SSER amplitude decreased and interpeak latency increased markedly. rCBF was distributed into zones of dense, partial, and no ischemia ranging from 12.6 to 59.4 ml/100 g/minute. Ischemic edema was distributed inversely to rCBF and was increased in areas of dense ischemia (85.2 +/- 0.5%) and ischemia (82.7 +/- 0.7%), but not in partially ischemic or control areas. 6-Keto-PGF1 alpha (1257.3 pg/mg), PGE2 (1628.5 pg/mg), and TXB2 (1572.8 pg/mg) were all significantly (P less than 0.05) increased in areas of partial ischemia that had not yet developed edema. ODC levels were significantly elevated (3812 pmole/g/hour, P less than 0.05) and increased with time in areas of slightly denser ischemia that showed an intermediate increase in edema, but not the presence of infarction. This is the first demonstration that ODC, the rate-limiting enzyme for polyamine synthesis, is stimulated by cerebral ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

A vasoconstrictor-induced prostacyclin (PGI2) production in a perfused rat heart was found, suggesting a mitigating role of PGI2 on coronary vasoconstriction. Treatment of the heart with cyclooxygenase inhibitors (aspirin or indomethacin) decreased PGI2 production by more than 90% and paradoxically reduced the vasoconstriction response. The attenuating effect of cyclooxygenase blockade suggested that endogenous prostanoids contribute to serotonin-, vasopressin- or U46619-induced vasoconstriction. Two prostaglandin (PG) H2/thromboxane A2 (TXA2) receptor antagonists, i.e., 13-azaprostanoic acid (13-APA) and SQ 29,548 were used to investigate putative endogenous vasoconstrictor prostanoids on the exogenously induced vasoconstriction. Retrogradely perfused (5-6 ml/min) rat hearts were rendered guiescent, yet responsive to stimuli, by local injection of lidocaine to the atrioventricular node. Changes in coronary vascular resistance (i.e., perfusion pressure at constant flow) were monitored and the cardiac effluent was collected for analysis of 6-keto PGF1 alpha (the stable metabolite of PGI2) as well as PGF2 alpha by radioimmunoassay. Three vasoconstrictors, i.e., serotonin, vasopressin and the TXA2/PGH2 analog U46619, as well as authentic PGD2, PGE2 and PGF2 alpha were infused. PGD2, PGE2 and PGF2 alpha exerted a dose-related coronary vasoconstriction, as did U46619, serotonin and vasopressin. Treatment with 13-APA (100 microM) or SQ 29,548 (100 nM) almost abolished U46619-induced vasoconstriction. The addition of PGH2/TXA2 receptor antagonists also significantly reduced the pressor effect of exogenously administered PGs, serotonin and vasopressin, with the exception that SQ 29,548 did not significantly antagonize PGE2-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

In dog mesenteric vein strips, contractions induced by histamine relative to those induced by 5 mM Ba++ were potentiated by removal of endothelium. The induced contractions were potentiated by AA861, a lipoxygenase inhibitor, and methylene blue, a guanylate cyclase inhibitor, to an appreciably greater extent in the strips with endothelium than in those with damaged endothelium. Indomethacin did not potentiate the contraction induced by histamine. Cimetidine potentiated the contraction in control strips and those without endothelium to a similar extent whereas chlorpheniramine suppressed the contraction. Contractile responses to acetylcholine, norepinephrine, serotonin and prostaglandin (PG) F2 alpha were not potentiated by removal of endothelium. It may be concluded that histamine activates histaminergic receptors, possibly H1 but not H2, in endothelial cells and results in a release of vasodilator substance produced by lipoxygenase, which accumulates cellular cyclic GMP and relaxes mesenteric veins. The H1 and H2 receptors in smooth muscle cells appear to be responsible for contractions and relaxations, respectively. Acetylcholine, norepinephrine, serotonin and PGF2 alpha do not seem to release vasodilator substances from endothelium in an amount sufficient to cause significant relaxations of venous smooth muscle.

Flavonois derived from sophoradine are known to exhibit gastroprotective and ulcer healing properties but the mechanism of these actions are not fully explained. In this study we determined the effect of novel flavonoid derivative of sophoradin, SU-840, on gastric secretion, acute gastric lesions induced by acid-independent (100% ethanol) or acid-dependent ulcerogens (acidified aspirin (ASA) and stress) and on the healing of chronic gastric ulcers in rats. The number and area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured using H2-gas clearance technique and the mucosal samples were excised for the measurement of PGE2 generation by radioimmunoassay. Exposure of rats to 100% ethanol or acidified ASA (100 mg/kg dissolved in 0.2 N HCl) or to water immersion and restraint stress (WRS) resulted in hemorrhagic gastric lesions accompanied by drastic fall in the GBF as compared to the values recorded in vehicle treated gastric mucosa. SU-840 (6.25-100 mg/kg i.g.) reduced dose-dependently gastric acid and pepsin secretion and gastric lesions induced by ethanol, acidified ASA and WRS, the dose inhibiting by 50% of these lesions (ID50) being 28, 17 and 95 mg/kg, respectively. This protection required much lower doses as compared to original sofalcone or sucralfate and was obtained when this sofalcone-like drug was administered via parenteral route. The protective effect of SU-840 given i.g. or i.p. was accompanied by a marked rise in the GBF and mucosal generation of PGE2. The protective activity of SU-840 showed longer duration of the action than that of sofalcone and occurred in the doses that failed to affect gastric secretion. Pretreatment with indomethacin to suppress endogenous PG reversed completely the protective and hyperemic effects of SU-840 against ethanol and stress induced damage whereas L-NNA, a potent inhibitor of NO-synthase, failed to affect protection but completely abolished the hyperemia evoked by this agent. NEM, an sulfhydryl alkylator, significantly attenuated the protective and hyperemic effects of SU-840 suggesting that endogenous sulfhydryls are involved in these effects. Seven day treatment with SU-840 accelerated significantly healing rate of chronic gastric ulcers and increased the GBF at the ulcer crater and ulcer margin. These effects were reversed by L-NNA and further restored by the addition to L-NNA of L-arginine, a substrate for NO-synthase. We conclude that SU-840 exhibits gastroprotective and hyperemic activity against acid-independent and acid-dependent irritants involving endogenous PG, sulfhydryls and hyperemia mediated by NO and 2) enhancement in gastric blood flow in the ulcer area mediated by NO appears to be essential for the acceleration of the ulcer healing by SU-840.

Hypertension is associated with an endothelial dysfunction characterized by an increased endothelium-dependent contraction and a decreased endothelium-dependent relaxation. Angiotensin converting enzyme (ACE) inhibition with cilazapril or captopril can remarkably improve the endothelial function in spontaneously hypertensive rats (SHRs). The goal of the present study was to investigate whether ACE inhibitors were acting by decreasing endothelium-dependent contraction or by increasing endothelium-dependent relaxation. Endothelial function was estimated by calculating the ratio of maximal contraction to serotonin on isolated aortic rings with endothelium to maximal contraction on paired rings without endothelium, termed the serotonin ratio. The serotonin ratio was greater than 1 in SHRs, indicating the release of a vasoconstrictor substance by the endothelium. This substance was identified as prostaglandin (PG) H2, because the serotonin ratio was significantly decreased by thromboxane (TX) A2/PGH2 receptor antagonists but not by TXA2 synthetase inhibitors. Two weeks of treatment of SHRs with cilazapril led to a marked decrease in the serotonin ratio, although acute administration of cilazaprilat was without any effect. However, after 2 weeks of treatment, the serotonin ratio still could be lowered further by TXA2/PGH2 receptor antagonists, indicating that cilazapril did not act by inhibition of PGH2 synthesis. In contrast, the effect of a 4-week treatment with cilazapril could be completely reversed by inhibiting the action of endothelium-derived relaxing factor with methylene blue. The same result was found after treatment with captopril. We speculate that ACE inhibitors improve endothelial function in SHRs not by inhibiting the synthesis of PGH2 but by increasing the release or the action of endothelium-derived relaxing factor.

We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 +/- 2.9 to 51.6 +/- 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H1,H2) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB2) and 6-keto-prostaglandin (PG)F1 alpha from basal values of 0.96 +/- 0.09, 2.7 +/- 0.7 and 3.4 +/- 0.28 ng/5 min respectively, to 5.9 +/- 0.9, 48.4 +/- 3.4 and 6.9 +/- 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 +/- 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB2 and 6-keto-PGF1 alpha. Simultaneous blockade of H1 and H2 receptors abolished release of 6-keto-PGF1 alpha, while release of TXB2 and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA2.

Synthetic cyclic somatostatin-14 (SRIF-14) was conjugated with carbodiimide onto hemocyanin to immunize rabbits, and anti-sera with high titers (1:5,000-100,000) and good sensitivity and specificity were obtained. 125I-Tyr1-SRIF was made and purified on CM-52 cation exchange column. The immunoreactive specific activity (SP.AC.) of the tracer was around 1013 mCi/mg, which was much higher than the commercial products and other reports. The sensitivity of the assay was around 16 pg/ml, and the detectable range 20-2000 pg/ml. Human plasma was freshly prepared in the presence of aprotinin. Rat hypothalamus was homogenized and extracted with HC1 followed by heating at 95 degrees C then centrifuged, and the supernatant assayed. The standard curves of the acetate buffer (ph 5.2), plasma doses, plasma recovery doses, hypothalamic extract doses, and hypothalamic extract recovery doses were all in good parallelism with each other. The recovery of SRIF from human plasma and from rat hypothalamus were 98.8 +/- 6.3% and 86.9 +/- 6.8%, respectively. The specificity of the antisera were very high, and they only cross-react with H2-SRIF and Tyr1-SRIF at 15% and 170%, respectively, of the immunoreactivity to cyclic SRIF-14. The Scatchard plot of the binding data showed a straight line with a Kd of 3.52 X 10(-12)M and binding capacity of 4.06 X 10(-10)M. The intra- and inter-assay coefficient of variation were 4.5% and 12%, respectively. When the synthetic cyclic SRIF (Stilamin) was infused intravenously into normal volunteers, there were reproducible plasma time-dose curves of SRIF-LI (SRIF Like Immunoreactivity), which revealed its plasma half life around 1.5 minutes, and metabolic clearance rate about 50 ml/kg/min.

Up to now, no relevant tumor antigen has been identified in medullary thyroid carcinoma (MTC). The aim of the present study was to prove the concept of an immunization with an amino acid-modified calcitonin (CT) for the treatment of MTC in a transgenic mouse model. Amino acid-modified (human) CT has been chosen for vaccination because of its higher binding affinity to the murine H2-Kb-MHC molecule. Mice were immunized over 6 months with monthly injections of amino acid-modified CT-pulsed dendritic cells. For enumeration of tumor epitope-specific CD8+ cytotoxic T cells, tetramer analyses were performed. CT peptide-treated mice revealed a mean 0.73 +/- 0.45 and 0.91 +/- 0.59% positive cells, depending on the two tetramers tested, whereas no increase was seen in control protein-immunized mice (0.08-0.12% tetramer-positive cells). Importantly, the subset of CT-specific CD8+ T cells also showed a high expression of interferon-gamma. In line with these results, CT-immunized mice also showed an intratumor infiltration with CD8+ T lymphocytes. Importantly, we also found a diminished tumor outgrowth of -57% and a decrease of the serum CT levels (2.0 +/- 0.1 pg/ml) compared with control protein-immunized Ret/Cal mice (3.0 +/- 0.4 pg/ml). In summary, we show that amino acid-modified CT is recognized from the immune system leading to a specific antitumor immune response and a diminished tumor outgrowth in transgenic MTC mice. The results are of potential importance because they might be applicable to patients with metastatic spread of a MTC.

It is still controversial whether gastrin stimulates acid secretion by interacting with specific gastrin receptors on parietal cells or via endogenous mediators, e.g., histamine. Therefore, it was our aim to determine in healthy human volunteers (n = 14; 3 females, 11 males; age 23-28 years) the degree by which the specific histamine H2-receptor antagonist famotidine or the muscarinergic antagonist atropine block acid secretion in response to synthetic human gastrin (hG) (1-17). Famotidine was deliberately administered at a supramaximal dose (40 mg i.v. bolus) to reliably block any and all effects of endogenous histamine on the parietal cells. After an overnight fast famotidine or saline were injected i.v., and gastric secretions were collected via a nasogastric tube for the ensuing 60 min to assess basal secretion. Thereafter, hG (1-17) was infused for 60 min in randomized order at two different rates: 0.75 ng/kg/min resulting in postprandial plasma gastrin levels (55-66 pg/ml), and 1.5 ng/kg/min yielding supraphysiologic levels (110-136 pg/ml). Both rates increased basal acid secretion (meq/10 min) from 0.5 +/- 0.2 to 3.8 +/- 0.6 and 4.7 +/- 0.5, respectively. Famotidine abolished basal acid secretion and completely blocked acid and volume secretion in response to both hG (1-17) doses. After injection of famotidine both hG (1-17) doses resulted in plasma levels exceeding those in controls by 18-27 pg/ml. A similar increase (14-16 pg/ml) was observed after famotidine injection without simultaneous hG (1-17) infusion indicating that this increase was due to the release of endogenous gastrin when the acid feedback inhibition was blocked by famotidine. To study a potential additional role of cholinergic mechanisms the effect of atropine (7 micrograms/kg i.m.) on hG (1-17)-induced acid secretion was examined. Atropine reduced basal acid secretion from 0.8 +/- 0.1 to 0.1 +/- 0.08 meq/15 min. Similarly, the response to 0.75 ng/kg/min hG (1-17) was reduced by 72.9%. Basal gastrin release was not altered by atropine which, however, tended to increase serum gastrin levels during infusion of hG (1-17) by 16-24 pg/ml. We conclude that in man histamine and muscarinic mechanisms are essential mediators of gastrin-stimulated acid secretion. The present data argue against a significant direct effect of gastrin alone on human parietal cells but rather support potentiating interaction with histamine and cholinergic mechanisms.

We compared the effects of modulating the postsynaptic histamine receptor subtype 2 (H2) and inhibitory presynaptic autoreceptor subtype 3 (H3) on memory processing in the septum. Mice were partially trained on footshock avoidance in a T-maze. Immediately after training, saline or a drug solution was infused into the septum. One week later, retention was tested by continuing training until the mice made five avoidance responses in six consecutive trials. The results indicate that dimaprit, an H2 agonist, facilitated retention (25 and 50 pg) with a U-shaped dose-response curve typical of drugs acting at postsynaptic receptors. Cimetidine, an H2 antagonist, impaired retention (15-50 ng). The H3 agonist. imetit, impaired retention (25-200 ng), while the H3 antagonist, thioperamide, facilitated retention (10-400 ng). An unusual feature of the dose-response curve for thioperamide was that it did not appeal to yield a U-shaped curve as occurs with drugs acting postsynaptically, but facilitated retention to approximately the same degree from 50 to 400 ng. As histamine neurons project to various limbic system structures involved in memory processing, it may play an important role in regulating the activity of structures such as the septum, hippocampus and amygdala.

Results of several experiments have suggested that histamine-2 receptors play an inhibitory role in regulating gastrin release. We evaluated this prospectively in healthy human beings by infusing intravenously either histamine (0.33 micrograms/kg/min) or cimetidine (3.33 mg/min) during a continuous 3-h intragastric infusion of a 3% mixed amino acid meal, a potent stimulus of gastrin release. In order to be certain that effects of histamine or cimetidine on gastrin release were independent of their known effects on gastric acid secretion, intragastric pH was maintained at 5.0 by in vivo intragastric titration with sodium bicarbonate or hydrochloric acid. Although histamine and cimetidine had significant effects on gastric acid secretion, neither significantly affected the rises in serum gastrin concentrations during intragastric amino acid infusion. For example, mean gastrin rises above basal concentrations were 39 +/- 9 pg/ml on the control day, 39 +/- 9 pg/ml on the histamine day and 44 +/- 11 pg/ml on the cimetidine day (P greater than 0.05). Thus, blockade or stimulation of H2-receptors at the doses tested had no effect on gastrin release in response to an amino acid meal in humans when intragastric pH was maintained at 5.0.

Ferret trachea and bronchus contract to carbachol greater than 5-HT greater than bradykinin (BK) greater than PGF2alpha greater than histamine greater than 2-methylhistamine (2-MeH: a specific H1-receptor agonist). Rat trachea is insensitive to the spasmogenic actions of histamine, 2-MeH, PGF2alpha, BK and 5-HT. Ferret trachea and bronchus and rat trachea (which were partially contracted to carbachol) relax to isoproterenol greater than PGE1 greater than E2 greater than 4-methyl-histamine (4-MeH: a specific H2-histamine receptor agonist). High doses of histamine also relax rat trachea. The contractions of ferret airways to 2-MeH coupled with the antagonism of histamine-induced contractions by mepyramine showed the presence of H1-histamine receptors in the trachea and bronchus of ferret. After H1-blockade, the relaxant responses to histamine on ferret bronchus and trachea, and rat trachea were not affecte by "specific" doses of burimamide, metiamide, and cimetidine (H2-receptor antagonists), indomethacin (PG-synthetase inhibitor) and propranolol (a beta-adrenoceptor blocker). These results appear to suggest the existence of an atypical histamine receptor subtype (i.e. not of the classical H1 or H2 type) in the airways of rat and ferret.

The role of T helper-17 (Th17) lymphocytes in the regulation of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma is unknown. This study examined the effect of Th17 cytokines (IL-17 and IL-22) on granulocyte recruitment and functions during SEA-induced granuloma formation in vitro in Schistosoma-infected and noninfected individuals. Granulocytes were isolated from 27 Schistosoma-infected patients and 13 controls and were used for granuloma induction using SEA-conjugated polyacrylamide beads in the presence of Th17 cytokines. Granuloma index was assessed, and granulocyte mediators such as tumour necrosis factor (TNF-α), hydrogen peroxide (H2 O2 ) and nitric oxide (NO) were measured in the culture supernatant at the 7th day using enzyme-linked immunosorbent assay (ELISA). Schistosoma-infected patients had significant larger SEA-induced granuloma than controls. IL-17 (125 pg/mL) induced the optimum size for granuloma within 3-7 days. However, IL-22 at different concentrations up to 300 pg/mL had no effect on granuloma formation. Using both cytokines simultaneously, IL-22 suppressed the effect of IL-17 and prevented granuloma formation. IL-17 significantly decreased TNF-α, H2 O2 and NO levels in Schistosoma-infected individuals. In contrast, IL-22 increased TNF-α and H2 O2 levels. In conclusion, IL-17 accelerates SEA-induced granuloma formation and inhibits granulocytes functions in Schistosoma-infected patients, while IL-22 inhibited the granuloma formation, but enhanced granulocyte functions.

1 The prostaglandin endoperoxide analogue U-46619 potently contracted human isolated myometrium, suggesting that prostaglandin H2 (PGH2) may be a major stimulant of myometrial contractions. 2 Sodium mefenamate, flufenamate or meclofenamate 2 microgram/ml greatly reduced contractions of the myometrium induced by the PGH2 analogue. 3 Flufenamate, but not the other two drugs, also significantly inhibited contractions to acetylcholine. 4 Sodium meclofenamate 2 microgram/ml did not consistently antagonize contractions to PGF2alpha. 5 The relief of dysmenorrhoea by fenamates may be explained both by inhibition of PG synthesis, and by antagonism of contractions to PGH2 produced by incompletely blocked PG synthesis.

Polymorphonuclear leukocytes (PMNs) produce arachidonic acid (AA) metabolites including thromboxane A2 (TXA2). These cells are the first line of defense against bacterial invasion, which often causes endotoxin shock. TXA2 which plays an important role in the pathogenesis of endotoxin shock is synthesized by three consecutive enzyme activation, cytosolic phospholipase A2 (cPLA2), prostaglandin H2 synthase (PHS type 1 and type 2) and TXA2 synthase. Among them, cPLA2- and PHS-2 activity is known to be transcriptionally and/or posttranscriptionally up-regulated by various bioactive substances including lipopolysaccharide (LPS), a bacterial endotoxin, in many cell types. We investigated the action of LPS on TXA2 synthesis in human PMNs. A23187-stimulated production of thromboxane B2 (TXB2, a stable metabolite of TXA2), assayed by specific radioimmunoassay (RIA), was significantly increased from 566.7+/-44.1 pg/10(6) cells to 966.7+/-44.1 pg/10(6) cells (p<0.05) after 6 h-exposure to LPS at the concentration of 100 ng/ml. Messenger RNA for PHS-2, PHS-1, TXA2 synthase and cPLA2, which was assessed by reverse transcription-polymerase chain reaction (RT-PCR), was expressed in PMNs without LPS stimulation. Although PHS-2 was putatively an inducible enzyme, abundance of mRNA for PHS-2 in PMNs without LPS stimulation was detectable. Messenger RNA abundance for PHS-2 and cPLA2, but not for PHS-1 and TXA2 synthase, was enhanced by LPS-treatment, indicating that the increased production of TXB2 was attributable to the up-regulation of cPLA2 and PHS-2. We conclude that (1) PHS-2 plays a more important role than PHS-1 in the production of TXA2 in human PMNs and (2) TXA2 synthesis in human PMNs is transcriptionally up-regulated by new induction of cPLA2 as well as PHS-2, when the cells encounter endotoxin producing bacteria.

A high-affinity thromboxane (TX)A2/prostaglandin (PG) H2 receptor antagonist, I-SAP [7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid] and its radiolabeled analog [125I]SAP (Mais et al., 1991) are characterized in the present study. I-SAP antagonized I-BOP ([1S-(1 alpha, 2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2y l]-5'heptenoic acid) and U46619 [15S-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5Z,13E-dienoic acid)], two different TXA2/PGH2 mimetics, induced aggregation of washed human platelets in a similar manner (pA2 of 8.11 +/- 0.09, Kd = 7.8 nM, n = 3; pA2 = 8.01 +/- 0.05, Kd = 9.7 nM, n = 8, respectively). I-SAP also had agonistic activity, producing platelet shape change (EC50 = 9.7 nM +/- 0.6 nM at pH 7.4, n = 3) which was blocked by pretreatment of platelets with SQ29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept- 2-yl]-5-heptenoic acid), a TXA2/PGH2 receptor antagonist. Radioligand binding studies were performed with [125I]SAP using washed human platelets. Competition of three agonists and four antagonists for binding with [125I]SAP was determined. The compounds showed the appropriate rank order potencies, including stereoselective competition by a pair of stereoisomeric antagonists. In washed human platelets, the Kd for I-SAP was 468 +/- 49 pM and the maximum binding (Bmax) was 2057 +/- 156 sites/platelet at pH 7.4 (n = 6). The Bmax was significantly increased 49% to 3072 +/- 205 sites/platelet at pH 6.5 (P less than .01 but the Kd was unchanged (490 +/- 18 pM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, 80-85]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PG) H2 to TXA2 and 12-hydroxy-heptadecatrienoic acid (HHT). Both conversions of PGH2 to TXA2 and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY-046 (5 microM).

For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has been known to be a product of thromboxanesynthase (TX-Syn) when synthesized with thromboxane A2 (TXA2). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much information about its physiological role. This review presents a summary of the physicochemical properties of HHT, its chemical synthesis, the impact of various biological systems on its enzymatic and non-enzymatic production and its physiological function and metabolization, as well as a survey of the most important methods for analyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT, no immunological system for HHT quantification has been developed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enhanced enzymatic method using platelet-rich plasma (PRP). With an effective and modified liquid-liquid and solid-phase extraction method we were able to produce highly purified HHT (97% purity by GC/MS) in sub-milligram ranges. These fractions were used for the synthesis of BSA-antigen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized using prostaglandin H synthase for the production of prostaglandin H2 (PGH2) followed by an aqueous reaction with Fe(2+)-solution to rear-range PGH2 to HHT. The dynamic range of the assay was from 30-400 pg/tube, with a sensitivity of approximately 40 pg/tube. The evaluation of the assay was performed by a HPLC-RIA method as well as by correlation with a quantitative HPLC method and correlation with TXB2 concentrations in a blood coagulation study. The assay may be useful for the quantification of HHT in several tissues and body fluids under various physiological conditions and may also help to understand the possible physiological role of HHT in biological processes.

The mechanisms underlying the biphasic response (BR) of the circular muscle of the guinea pig ileum (CMGPI) to bradykinin (BK) have been examined. Both BK and lysyl-BK (1 nM to 1 microM) caused graded contractions followed by relaxations of the CMGPI, yielding EC50 of 21 and 92 nM for contraction and of 10 and 27 nM for relaxation, respectively. The selective B1 receptor agonist Des-Arg9-BK was without effect up to 3 microM. The potencies of BK and lysyl-BK to evoke BR were markedly increased by enalapril (3 microM) and decreased by raising the preparation tone with the thromboxane A2/prostaglandin H2-mimetic U46619 (30 ng/ml). The BR of CMGPI to BK was unaffected by atropine, yohimbine, pyrilamine, propranolol, prazosin, phorbol ester, des-Arg9-[leu8]-BK (1 microM, each), tetrodotoxin (0.3 microM), [3,4,5-trimethoxybenzoic acid-8-(diethylamino) octyl ester (10 microM), [N-6-(aminohexyl)-5-chloro-1-naphthalenosulfonamide (10 microM), glibenclamide (0.3 microM), nordihydroguaiaretic acid (50 microM), phenidone (30 microM) or dexamethasone (0.1 microM). However, indomethacin (3 microM), ibuprofen (30 microM) and 3-amino, 1-(m-[trifluoromethyl] phenyl)2-pyrazoline (10 microM) each abolished the relaxant and increased the contractile response to BK, suggesting that a cyclo-oxygenase-derived eicosanoid mediates relaxation and limits contraction. Prostaglandin (PG) E2 (up to 100 nM) caused only graded relaxations, PGF2 alpha (up to 3 microM) and 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy prostaglandin F2 alpha (up to 300 ng/ml) caused only contractions and the PGI2 analog iloprost was without effect up to 1 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

First-principle density functional theory (DFT) calculation and molecular dynamic (MD) simulation are employed to investigate the hydrogen purification performance of two-dimensional porous graphene material (PG-ESX). First, the pore size of PG-ES1 (3.2775 Å) is expected to show high selectivity of H2 by DFT calculation. Then MD simulations demonstrate the hydrogen purification process of the PG-ESX membrane. The results indicate that the selectivity of H2 over several other gas molecules that often accompany H2 in industrial steam methane reforming or dehydrogenation of alkanes (such as N2, CO, and CH4) is sensitive to the pore size of the membrane. PG-ES and PG-ES1 membranes both exhibit high selectivity for H2 over other gases, but the permeability of the PG-ES membrane is much lower than the PG-ES1 membrane because of the smaller pore size. The PG-ES2 membrane with bigger pores demonstrates low selectivity for H2 over other gases. Energy barrier and electron density have been used to explain the difference of selectivity and permeability of PG-ESX membranes by DFT calculations. The energy barrier for gas molecules passing through the membrane generally increase with the decreasing of pore sizes or increasing of molecule kinetic diameter, due to the different electron overlap between gas and a membrane. The PG-ES1 membrane is far superior to other carbon membranes and has great potential applications in hydrogen purification, energy clean combustion, and making new concept membrane for gas separation.

The effects of methylprednisolone and hydrocortisone on platelet aggregation induced by arachidonic acid (AA), collagen, adenosine diphosphate (ADP), prostaglandin (PG) H2, and a stable PGH2 analog, were studied in platelet-rich plasma (PRP) from the rabbit. Incubation of either steroid in PRP inhibited AA-, collagen- and ADP-induced platelet aggregation in a concentration-related manner. The dose of methylprednisolone required to inhibit 0.02 mM AA-induced aggregation was lower than that required to inhibit either 0.08 microgram/ml collagen or 0.2 microM ADP-induced aggregation. Methylprednisolone produced a dose-dependent inhibition of platelet aggregation induced by PGH2 and the stable PGH2 analog. In washed platelets methylprednisolone was more effective in inhibiting AA-induced aggregation than ADP- or collagen-induced aggregation; however, the difference in effect was less than in PRP. Platelet responses to AA in PRP from rabbits treated with hydrocortisone or methylprednisolone, 100 mg/kg i.v., were inhibited in a transient manner, whereas aggregation induced by ADP under similar conditions was unchanged. Since inhibition of aggregation elicited by AA occurred at concentrations which do not influence PGH2-, PGH2 analog-, collagen- or ADP-induced aggregation, the present data suggest that the steroids may inhibit the incorporation, the release, or the metabolism of arachidonic acid in platelets. The actual mechanism of this relatively specific inhibition of AA-induced aggregation by anti-inflammatory steroids is uncertain but may be related to the membrane "stabilizing" properties of methylprednisolone and hydrocortisone.

An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.