Intragastric administration of aspirin (ASA) plus 0.15 M HCl to fasted rats produced typical gastric ulcers accompanied by almost complete disappearance of mucosal prostaglandins (PGs). Pretreatment with various exogenous PGs that were biologically inactive (e.g. 6-keto-PGF1 alpha or PGF2 beta) or active (PGE2 and PGI2) but used in non-antisecretory doses prevented the formation of these gastric lesions ('cytoprotection'). Besides PGs, antisecretory compounds such as ranitidine, a new H2-receptor antagonist, and probanthine were also found to be cytoprotective, even when given in non-antisecretory doses. Mucosal generation of PGs in animals treated with ASA and HCl plus ranitidine or probanthine was very low and not significantly different from those receiving only ASA and HCl. Thus, the cytoprotection appears to be the property not only of PGs but also of conventional gastric antisecretory compounds such as H2-receptor antagonists or anticholinergics. This cytoprotection can be demonstrated under conditions excluding any role of gastric secretory inhibition and in the absence of endogenous PGs.

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.

Prostaglandin (PG) release is characteristic of most inflammatory diseases. The committed step in the formation of free arachidonic acid into PG products is catalyzed by cyclooxygenase (COX, prostaglandin H2 synthase, PGHS), which exists as two genetically distinct isoforms. COX-1 is constitutively expressed and produces PGs and thromboxane A2 during normal physiologic activities, while COX-2 is an inducible enzyme stimulated by growth factors, lipopolysaccharide, and cytokines during inflammation or cell injury. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) released into the amniotic fluid in the setting of infection have been proposed to signal amnion and decidual cells to produce PGs that may culminate in preterm labor. However, since the molecular control of this phenomenon has not been established, this study used amnion-derived WISH cells to determine if TNF-alpha promoted the formation of PGs through COX-2 activity. Treatment of WISH cells with TNF-alpha (0.1 ng/mL-100 ng/mL) caused a dose-dependent increase in COX-2 expression and the subsequent biosynthesis of PGE2 that persisted for at least 48 hrs. In contrast, COX-1 mRNA and protein levels were unaltered by TNF-alpha treatment as determined by RT-PCR and immunoblot analysis, respectively. TNF-alpha-stimulated COX-2 expression and the subsequent formation of PGE2 were inhibited by dexamethasone (0.1 microM). In addition, indomethacin (1 microM) and the novel COX-2-selective inhibitor, NS-398 (IC50 approximately 1.1 x 10(-9) M), attenuated TNF-alpha-elicited PGE2 production. Results presented here demonstrate that TNF-alpha elicits prolonged and regulatable induction of COX-2 in WISH cells, while COX-1 is constitutively expressed and unchanged in response to TNF-alpha stimulation.

We have reported previously that thromboxane A2/prostaglandin (PG)H2 and serotonin independently mediate the occurrence of cyclic flow variations (CFVs) in a canine preparation of severe coronary artery narrowing. This may be due to an effect of these substances on platelets and/or the vascular wall. We tested the hypothesis that there is a cooperative effect between thromboxane A2/PGH2 and serotonin receptor stimulation in the development of CFVs in this animal preparation. After placement of a hard plastic cylindrical constrictor around the left anterior descending coronary artery, CFVs develop and are characterized by repetitive cycles of declines in coronary blood flow and abrupt increases in flow. In a control group of dogs, CFV frequency (cycles/hour) and severity (lowest coronary blood flow just before its restoration) did not change significantly over a 3 hr interval. In a second group of dogs, CFVs were established after constrictor placement, abolished with the serotonin (5HT2) receptor antagonist ketanserin, and reestablished by the continuous infusion of serotonin into the left atrium. Serotonin-induced CFVs were then abolished with a thromboxane A2/PGH2 receptor antagonist, SQ29,548, or a thromboxane synthetase inhibitor, dazoxiben (UK37,248). The relative specificity of the respective antagonists, SQ29,548 and ketanserin, was determined in canine platelets and rat aortic vascular strips. No significant cross-reactivity between ketanserin and SQ29,548 was found. Thus, the data obtained in these studies demonstrate a cooperative interaction between thromboxane A2/PGH2 and serotonin S2 receptors that contributes to the development of CFVs in this experimental preparation.

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

Hydrogen sulfide (H2S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H2S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H2S precursor L-cysteine, H2S-donor NaHS, the H2S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H2S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H2S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H2S and or L-cysteine, the H2S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H2S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H2S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

Prostaglandin E synthase (PGES) is a recently identified terminal enzyme that acts downstream of cyclooxygenase and catalyzes the conversion of prostaglandin (PG) H2 to PGE2. At least three isozymes have been cloned so far, which are called membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES. Among them, mPGES-1 is induced by various inflammatory stimuli in some cells and tissues. Induction of mPGES-1 in the component of articular tissues of patients with rheumatoid arthritis and osteoarthritis has been demonstrated in vitro. Recent studies using adjuvant induced arthritis model have shown the increase of mPGES-1 expression resulted in the increase of PGE2 production at the sites of inflammation. In addition, reports of mPGES-1-deficient mice clearly suggest the role of mPGES-1 in the process of chronic inflammation such as collagen-induced arthritis and collagen antibody induced arthritis in vivo. Thus, recent in vitro and in vivo findings suggest that mPGES-1 may be a novel therapeutic target for arthritis. This paper introduces recent advances in research about the role of PGES in the pathophysiology of arthritis.

Prostaglandin (PG) D2 has been postulated to be an endogenous sleep-promoting factor in rats, and SeCl4 and Na2SeO3 recently have been shown to inhibit the PGD synthase (prostaglandin-H2 D-isomerase, EC 5.3.99.2) activity of rat brain. The effect of these selenium compounds on sleep-wake activities was examined in freely moving rats along with their effects on brain temperature, food and water intake, and behavior. Test substances were administered for 6 hr into the third ventricle of rats, using a microdialysis technique. SeCl4, time- and dose-dependently, inhibited sleep at perfusion rates of 60 pmol/0.2 microliter per min and higher, and the inhibition was almost complete at rates greater than 200 pmol/0.2 microliter per min. The effect was reversible and was followed by a rebound. Na2SeO3 exhibited similar effects, but Na2SO3 did not show any effect on sleep. Simultaneous administration of dithiothreitol eliminated the sleep-inhibiting effects of these selenium compounds. These findings indicate that the decrease in sleep is due to inhibition of the PGD synthase activity in the brain by SeCl4 as well as Na2SeO3. During the inhibition of sleep, the rats in general showed an activation of behavior with moderate elevation of brain temperature and a detectable increase in food and water intake, suggesting that the sleep-inhibited state of the rats was similar to the physiological state of wakefulness and that the inhibitory effect was not due to the general toxicity of selenium.

Prostaglandin endoperoxide synthases (PTGS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze the key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H2 (PGH2). Although COX and prostaglandins have been implicated in a wide variety of physiologic processes, an evaluation of the role of prostaglandins in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus: COX null mouse embryos develop normally during embryogenesis. Here, we verify that inhibition of COX-1 results in zebrafish gastrulation arrest and shows that COX-1 expression becomes restricted to the posterior mesoderm during somitogenesis and to posterior mesoderm organs at pharyngula stage. Inhibition of COX-1 signaling after gastrulation results in defective vascular tube formation and shortened intersomitic vessels in the posterior body region. These defects are rescued completely by PGE(2) treatment or, to a lesser extent, by PGF(2alpha), but not by other prostaglandins, such as PGI(2), TxB(2), or PGD(2). Functional knockdown of COX-1 using antisense morpholino oligonucleotide translation interference also results in posterior vessel defect in addition to enlarged posterior nephric duct, phenocopying the defects caused by inhibition of COX-1 activity. Together, we provide the first evidence that COX-1 signaling is required for development of posterior mesoderm organs, specifically in the vascular tube formation and posterior nephric duct development.

Microsomal prostaglandin (PG) E synthase (mPGES)-1 is an inducible enzyme that acts downstream of cyclooxygenase (COX) and specifically catalyzes the conversion of prostaglandin (PG)H2 to PGE2, most prominently in inflammatory conditions. Specific inhibitors of mPGES-1 are not yet available, however, mice with genetic deletion of mPGES-1 have been generated that have given insight into the specific role of mPGES-1 in eicosanoid biosynthesis in vivo and in peritoneal macrophages. We created mouse embryo fibroblast (MEF) cell lines that would facilitate investigation of the effect of mPGES-1 genetic deletion on prostanoid biosynthesis in fibroblast lineage cells and its subsequent effect on the expression of inducible NOS (iNOS) and nitrite biosynthesis using cells derived from mPGES-1 wild-type (WT), heterozygous (Het), and null mice. The results show that genetic deletion of mPGES-1 results in a dramatic decrease in PGE2 production in Het and null MEFs under basal conditions and after stimulation with interleukin (IL)-1beta, suggesting that mPGES-1 is critically important for PGE2 production. Furthermore, we show that mPGES-1 gene deletion results in diversion of prostanoid production from PGE2 to 6-keto PGF1alpha (the stable metabolic product of PGI2; prostacyclin) in a gene dose-dependent manner in Het and null MEFs compared with their WT counterparts, suggesting a shunting phenomenon within the arachidonic acid (AA) metabolic pathway. In addition, we show that mPGES-1 gene deletion and subsequent decrease in PGE2 levels results in a differential induction profile of iNOS and nitrite levels (the stable breakdown product of nitric oxide (NO) in mPGES-1 WT MEFs compared with null MEFs. These results provide important information regarding the therapeutic potential for pharmacologic inhibition of mPGES-1 in inflammatory conditions.

1. Injection or infusion of histamine intraarterially into the isolated perfused rabbit ear dose-dependently stimulated the release of prostaglandins (PGs) as measured by radioimmunoassay (PGE), bovine coronary artery strips (PGI2) and by the prelabeling technic with [1-14C]-arachidonic acid (PGI2, PGE2, PGF2 alpha, PGD2). 2. PG release was abolished by indometacin (1-3 microgram/ml) and reduced by the phospholipase A2 inhibitor quinacrine (10 microgram/ml) as well as by perfusing with calcium-free, 1 mM EGTA containing solution. 3. The histamine H2-receptor antagonists burimamide (5 microgram/ml) and cimetidine (2 microgram/ml) did not influence histamine-induced PG release. The H1-receptor antagonist mepyramine (0.1-1 microgram/ml) abolished histamine-induced mepyramine (0.1-1 microgram/ml) abolished histamine-induced PG release. 4. In the presence, but not in the absence, of bovine serum albumin there was a basal release of high amounts of arachidonic acid. Histamine tended to increase the released amount of radioactive arachidonic acid. In contrast to indometacin which only blocked PG release, mepyramine significantly reduced the histamine-stimulated release of arachidonic acid, too. 5. The results show that in the peripheral vascular bed, histamine, via H1-receptors, activates a phospholipase A2 mainly by increasing a transfer of extracellular calcium into the cell. Activation of a phospholipase A2 results in the release of arachidonic acid possibly from a rather small endogenous pool which specifically provides substrate for the PG synthetase system.

Anaphylaxis of human lung is accompanied by the synthesis of prostaglandins (PG), including PGF2 alpha and PGE. In an analysis of the tissue source of these prostaglandins, parenchymal preparations of both human and guinea pig (GP) lungs were compared. Peripheral, relatively airway-free preparations of human lung generate PGF2 alpha and PGE in response to histamine and 2-methylhistamine, on H1 agonist, but not to dimaprit, an H2 agonist. GP parenchymal preparations respond in a similar fashion. Stimulation of these same preparations with KCl or carbachol caused no increase in the synthesis of either PG. In human airway preparations all three agonists (histamine, KCl, and carbachol) caused the selective generation of PGE. However, stimulation of GP airway preparations with the agonists caused the production of both PGE and PGF2 alpha. These data indicate that (1) human and GP peripheral lung tissues respond to H1, but not H2, stimulation with the generation of PGF2 alpha and PGE; (2) these parenchymal responses are specific and may not be attributed to muscle contraction; and (3) stimulation of muscle contraction in human airway preparations results in the selective generation of PGE while GP airways produce both PGE and PGF2 alpha.

This study was designed to characterize the endothelium-dependent contracting factor (EDCF) released by arachidonic acid (AA) and methacholine (MeCH) in the rabbit pulmonary artery. AA and MeCH contract the rabbit pulmonary artery; however, the effects of both are blocked by denuding the vessels and by administration of indomethacin (a cyclooxygenase inhibitor), dazoxiben (a thromboxane [TX] synthase inhibitor), and SQ29548 (a TXA2/prostaglandin [PG] H2 receptor antagonist). When segments of rabbit pulmonary artery were incubated with [14C]AA and the [14C] metabolites were resolved by reverse-phase high-performance liquid chromatography (HPLC), radioactive products were observed that comigrated with 6-keto-PGF1 alpha and TXB2, the stable metabolites of prostacyclin and TXA2. The TXB2 radioactive peak was rechromatographed on normal-phase HPLC and again migrated with TXB2. Finally, the structures of derivatized [14C]6-keto-PGF1 alpha and [14C]TXB2 peaks were confirmed by gas chromatography/mass spectrometry. The synthesis of [14C]6-keto-PGF1 alpha and [14C]TXB2 was inhibited by removal of the endothelium and by indomethacin. Dazoxiben inhibited the synthesis of [14C]TXB2 but not [14C]6-keto-PGF1 alpha. Using specific radioimmunoassays, AA and MeCH stimulated 6-keto-PGF1 alpha and TXB2 release. Indomethacin blocked the production of both 6-keto-PGF1 alpha and TXB2, whereas dazoxiben only blocked TXB2. In a superfusion/bioassay system, AA stimulated an endothelium-intact donor vessel to release a labile substance that contracted an indomethacin-treated endothelium-denuded recipient vessel. The EDCF released by AA had an approximate half-life of 30 seconds. Cultured rabbit pulmonary arterial endothelial cells synthesized 6-keto-PGF1 alpha but not TXB2. Immunohistochemical studies indicated the presence of cyclooxygenase, but not TX synthase, in pulmonary artery endothelial cells. TXA2 appears to be the EDCF released by AA and MeCH in rabbit pulmonary artery; however, TXA2 is not produced by endothelial cells but may arise from cells that adhere to the luminal surfaces, such as platelets or macrophages.

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure-function relationships. Using an approach of the combined structure prediction, molecular docking, site-directed mutagenesis, and enzymatic activity assay, we have developed the first three-dimensional (3D) model of the substrate-binding domain (SBD) of mPGES-1 and its binding with substrates prostaglandin H2 (PGH2) and glutathione (GSH). In light of the 3D model, key amino acid residues have been identified for the substrate binding and the obtained experimental activity data have confirmed the computationally determined substrate-enzyme binding mode. Both the computational and experimental results show that Y130 plays a vital role in the binding with PGH2 and, probably, in the catalytic reaction process. R110 and T114 interact intensively with the carboxyl tail of PGH2, whereas Q36 and Q134 only enhance the PGH2-binding affinity. The modeled binding structure indicates that substrate PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the -SH group of GSH. The -SH group of GSH is expected to attack the peroxy group of PGH2, initializing the catalytic reaction transforming PGH2 to prostaglandin E2 (PGE2). The overall agreement between the calculated and experimental results demonstrates that the predicted 3D model could be valuable in future rational design of potent inhibitors of mPGES-1 as the next-generation inflammation-related therapeutic.

Malondialdehyde (MDA), a product of lipid peroxidation, causes mutations in bacterial and mammalian cells and cancer in rats. MDA reacts with deoxynucleosides in vitro and the monomeric adduct of MDA with deoxyguanosine (M1G-dR) is the major adduct formed. We have developed a sensitive analytical method to characterize and quantify M1G-dR from biological matrices using gas chromatography/electron capture negative chemical ionization mass spectrometry (GC/ECNCI MS). Reduction of M1G-dR with sodium borohydride produced a dihydro derivative (H2-M1G-dR). This more stable analog had improved high-performance liquid chromatographic characteristics which facilitated its isolation from biological fluids. H2-M1G-dR was converted to a monopentafluorobenzyl derivative with simultaneous depurination; it was then converted to the corresponding t-butyldimethylsilyl derivative and analyzed by GC/ECNCI MS. (2H2)H2-M1G was used as internal standard. Quantitative analysis was carried out using selected ion monitoring of m/z 302 and m/z 304 where the limit of detection was 10 pg (30 fmol) injected on-column. The level of M1G-dR in normal rat liver was 5.2 +/- 0.2 modified bases per 10(7) bases (n = 6 rats).

OBJECTIVE

Grapefruit-seed extract (GSE) containing flavonoids, possesses antibacterial and antioxidative properties but whether it influences the gastric defense mechanism and gastroprotection against ethanol- and stress-induced gastric lesions remains unknown.

METHODS

We compared the effects of GSE on gastric mucosal lesions induced in rats by topical application of 100% ethanol or 3.5 h of water immersion and restraint stress (WRS) with or without (A) inhibition of cyclooxygenase (COX)-1 activity by indomethacin and rofecoxib, the selective COX-2 inhibitor, (B) suppression of NO-synthase with L-NNA (20 mg/kg ip), and (C) inactivation by capsaicin (125 mg/kg sc) of sensory nerves with or without intragastric (ig) pretreatment with GSE applied 30 min prior to ethanol or WRS. One hour after ethanol and 3.5 h after the end of WRS, the number and area of gastric lesions were measured by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance technique and plasma gastrin levels and the gastric mucosal generation of PGE2, superoxide dismutase (SOD) activity and malonyldialdehyde (MDA) concentration, as an index of lipid peroxidation were determined.

RESULTS

Ethanol and WRS caused gastric lesions accompanied by the significant fall in the GBF and SOD activity and the rise in the mucosal MDA content. Pretreatment with GSE (8-64 mg/kg i g) dose-dependently attenuated gastric lesions induced by 100% ethanol and WRS; the dose reducing these lesions by 50% (ID50) was 25 and 36 mg/kg, respectively, and this protective effect was similar to that obtained with methyl PGE2 analog (5 microg/kg i g). GSE significantly raised the GBF, mucosal generation of PGE2, SOD activity and plasma gastrin levels while attenuating MDA content. Inhibition of PGE2 generation with indomethacin or rofecoxib and suppression of NO synthase by L-NNA or capsaicin denervation reversed the GSE-induced protection and the accompanying hyperemia. Co-treatment of exogenous calcitonine gene-related peptide (CGRP) with GSE restored the protection and accompanying hyperemic effects of GSE in rats with capsaicin denervation.

CONCLUSIONS

GSE exerts a potent gastroprotective activity against ethanol and WRS-induced gastric lesions via an increase in endogenous PG generation, suppression of lipid peroxidation and hyperemia possibly mediated by NO and CGRP released from sensory nerves.

We hypothesized that endothelin in addition to prostaglandin (PG)H2 may also contribute to the enhanced myogenic tone of skeletal muscle arterioles of spontaneously hypertensive (SH) rats. Changes in the diameter of isolated, cannulated arterioles (approximately 60 microm) from cremaster muscles of 30-week-old normotensive Wistar Kyoto (WKY) and SH rats were measured as a function of perfusion pressure (20 to 140 mm Hg). Pressure-induced constrictions were significantly enhanced between 60 to 140 mm Hg in arterioles of SH rats compared with those of WKY rats; at 80 and 140 mm Hg the normalized diameter of arterioles (expressed as a percentage of corresponding passive diameter) of SH rats was 11.0% and 15.4% less (P<.05) than that of WKY rats. After inhibition of thromboxane A2-PGH2 receptors by SQ 29,548 (10[-6] mol/L), the still enhanced myogenic response of SH arterioles was eliminated by the removal of endothelium or the administration of BQ-123 (10[-7] mol/L), an endothelin A (ET-A) receptor blocker, which also inhibited constrictions to exogenous ET-1 (10[-11] to 5x10[-10] mol/L). ET-1 elicited comparable responses in arterioles of SH and WKY rats. Thus, in SH rats the enhanced arteriolar constriction to increases in intravascular pressure seems to be due to the production of endothelium-derived constrictor factors PGH2 and endothelin.

Because endogenous thromboxane A2 (TXA2) potentiates the tubuloglomerular feedback response (TGF), we studied the mechanism of action of TXA2 by using a stable TXA2/prostaglandin (PG) H2 mimetic, U-46,619. Intravenous infusion of U-46,619 at 100 ng.kg-1.min-1 reduced the GFR and the single-nephron (SN)GFR measured from the distal tubule (macula densa function intact), whereas the SNGFR measured from the proximal tubule (macula densa function interrupted) was not changed consistently. 10-100-fold higher rates of infusion of U-46,619 were required to raise blood pressure or femoral vascular resistance. The regulation of glomerular capillary pressure (PGC) by TGF was assessed in anesthetized rats from changes in proximal stop flow pressure (PSF) and/or SNGFR during perfusion of the loop of Henle (LH) with artificial tubular fluid (ATF). Orthograde loop perfusion and retrograde perfusion of U-46,619 into the macula densa segment reduced PSF. Responses to luminal U-46,619 were blunted by a TXA2-PGH2 receptor antagonist. Orthograde loop perfusions with luminal U-46,619 increased net Cl absorption, whereas coperfusion with furosemide (10(-4) M) blunted the response to U-46,619 by 68%. These data indicated that the luminal U-46,619 might increase the signal for TGF activation by increasing Cl reabsorption in macula densa cells. However, since 80 +/- 4% of [3H]U-46,619 perfused via the LH was reabsorbed peritubular capillaries (PTC) were perfused with U-46,619 to test additional extra-luminal actions. PTC perfusion with U-46,619 again increased TGF by reducing PSF selectively only while macula densa function was intact during perfusion of the LH with ATF.

CONCLUSIONS

(a) TGF is potentiated by U-46,619 given systematically, via the lumen of the LH by orthograde or retrograde perfusions or via the PTC; (b) at the lower doses tested, reduction of PGC and SNGFR by U-46,619 depends on tubular fluid delivery and reabsorption by the macula densa; (c) potentiation of TGF by U-46,619 entails preglomerular vasoconstriction which may be elicited in part by an increased signal due to increased net chloride reabsorption in the LH and presumably macula densa cells and by an increased sensitivity of the arteriole to macula densa-derived signals; (d) activation of TGF may contribute to the selective vasoconstriction of the renal vascular bed by low doses of U-46,619.

Stable synthetic mimetics of thromboxane (TX) A2 and prostaglandin (PG) H2 have been synthesized and reported to stimulate platelets and vascular smooth muscle. The synthetic agonists induce aggregation of isolated platelets and contraction of vascular tissue. The tritiated agonists [3H]U46619 and [3H]U44069 have been used in radioligand binding studies to characterize platelet and vascular smooth muscle TXA2/PGH2 receptors, but have limited usefulness due to their low specific activities and variable specific binding. In an attempt to overcome these problems, we have synthesized a stable, high affinity, 125I-radiolabeled TXA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP). I-BOP induced shape change, increased intracellular free calcium concentrations and aggregated isolated human platelets (EC50 = 0.21 +/- 0.05 nM, n = 3; 4.1 +/- 1.1 nM, n = 4; 10.8 +/- 3 nM, n = 9, respectively). The kinetically determined Kd was 1.02 +/- 0.33 nM (kobs = 0.19 +/- 0.05 min-1, k-1 = 0.097 +/- 0.02 min-1, k1 = 0.119 +/- 0.03 min-1 M, n = 4). Equilibrium binding studies of [125I]BOP to isolated human platelets indicated one class of high affinity sites, Kd = 2.2 +/- 0.3 nM and a maximum binding of 0.028 +/- 0.002 x 10(-12) mol/10(7) platelets (1699 +/- 162 sites/platelet, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

A model of early osteoarthritis (OA) induced in ovine joints by medial meniscectomy was used to study the effects of two hyaluronan (HA) preparations (AHA and DHA) on cartilage composition and proteoglycan (PG) metabolism. DHA was an HA preparation with an average molecular weight (MW) of approximately 2.0 x 10(6) d, and AHA had an MW of approximately 8.0 x 10(5) d. Both preparations were administered intraarticularly once a week for 5 weeks starting 16 weeks after meniscectomy, and animals (n = 5) were killed 5 weeks after the last injection. Meniscectomized, saline-injected (n = 5) and nonoperated (n = 5) animals were used for controls. At necropsy, 3-mm-diameter full-depth cartilage plugs were sampled under sterile conditions from specific locations on the medial and lateral femoral condyles, tibial plateaus, patella, and trochlear groove. The cartilage plugs were cultured in Hams-F12 medium supplemented with 10% fetal calf serum for 24 hours, then for a further 48 hours in the presence of H2(35)SO4 to determine the biosynthesis of PGs. The percentage of 35S-PGs and sulfated glycosaminoglycans released into the media was also ascertained. The cartilage adjacent to the plugs was analyzed for collagen and proteoglycan content and differential extractability with guanidine hydrochloride (GuHCl) solutions. The extractability of PGs with 0.4 mol/L GuHCl (nondissociative conditions) was lower from the medial femoral cartilages of the DHA-treated group than from the corresponding saline-treated group. In contrast, the release of 35S-PGs from the tibial cartilages of the DHA-treated animals was higher than in the saline-treated group. The biosynthesis of 35S-PGs, determined in vitro, for cartilage derived from the medial compartment was generally lower than for the lateral regions of the meniscectomized joints. The biosynthetic activity was further reduced in joints injected with the two HA preparations, but DHA reduced 35SO4 incorporation into PGs more than AHA. It was concluded that reduced biosynthesis of 35S-PGs and secretion into media was a consequence of increased loading of joints in the HA-treated animals rather than a direct effect of these preparations on chondrocyte metabolism.

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.

Pioglitazone, a specific ligand for peroxisome proliferator-activated receptor gamma (PPAR-gamma), was recently implicated in the control of inflammatory processes and in the modulation of the expression of various cytokines such as tumor necrosis factor alpha (TNF-alpha), but its role in the mechanism of gastric mucosal integrity has not been studied extensively. This study was designed to determine the effect of pioglitazone on gastric mucosal lesions induced in rats by topical application of 100% ethanol and by 3.5 h of water immersion and restraint stress (WRS) with or without pretreatment with indomethacin (5 mg/kg i.p.) to inhibit cyclooxygenase-1 (COX-1) and COX-2 enzyme activities and L-NNA (20 mg/kg i.p.) to suppress nitric oxide (NO)-synthase. In addition, the effect of pioglitazone on ulcer healing in rats with chronic acetic acid ulcers (ulcer area 28 mm2) was determined. Rats were killed 1 h and 3.5 h after ethanol administration or WRS exposure or at day 9 upon ulcer induction, and the number and area of gastric lesions were measured by planimetry, the gastric blood flow (GBF) was determined by H2-gas clearance technique and the mucosal PGE2 generation and gene expression and plasma concentration of TNF-alpha and IL-1beta were also evaluated. Pre-treatment with pioglitazone dose-dependently attenuated gastric lesions induced by 100% ethanol and WRS; the dose reducing these lesions by 50% (ID50) being 10 mg/kg and 7 mg/kg, respectively. The protective effect of pioglitazone was accompanied by the significant rise in the GBF, an increase in PGE2 generation and the significant fall in the plasma TNF-alpha and IL-1beta levels. Strong signals for IL-1beta- and TNF-alpha mRNA were recorded in gastric mucosa exposed to ethanol or WRS, and these effects were significantly decreased by pioglitazone. Indomethacin which suppressed PG generation by about 90%, while augmenting WRS damage, and L-NNA, that suppressed NO-synthase activity, significantly attenuated the protective and hyperaemic activity of this PPAR-gamma ligand. In the chronic study, pioglitazone significantly reduced the area of gastric ulcers on day 9 and significantly raised the GBF at the ulcer margin. The acceleration of ulcer healing by PPAR-gamma ligand was accompanied by a significant increase in the expression of PECAM-1 protein, a marker of angiogenesis. We conclude that (1) pioglitazone exerts a potent gastroprotective and hyperaemic actions on the stomach involving endogenous PG and NO and attenuation of the expression and release of proinflammatory cytokines TNF-alpha and IL-1beta, and (2) PPAR-gamma ligand accelerates ulcer healing, possibly due to the enhancement in angiogenesis at ulcer margin.

We tested the hypothesis that a prostanoid-mediated mechanism of vascular contraction is expressed in rats with aortic coarctation-induced hypertension. Rings of descending thoracic aorta taken from normotensive and hypertensive rats were contrasted in terms of constrictor responsiveness to arachidonic acid (AA), AA-induced release of eicosanoids, and ability to convert exogenous prostaglandin (PG) H2 to PGI2. AA (10(-8) to 10(-5) mol/L) increased isometric tension in aortic rings (bathed in Krebs' bicarbonate buffer) of hypertensive but not normotensive rats. AA (10(-5) mol/L) also elicited the release of PGI2, PGE2, thromboxane (TX) A2, and monohydroxyeicosatetraenoic acids (HETEs); this release from the aortic rings of hypertensive rats exceeded the corresponding release from the aortic rings of normotensive rats. However, the rate of conversion of exogenous PGH2 to PGI2 by aortic rings of hypertensive rats was < 50% the rate of conversion by aortic rings of normotensive rats. The constrictor effect of AA in aortic rings of hypertensive rats was abolished by an inhibitor of cyclooxygenase (indomethacin, 10 mumol/L) and a blocker of TXA2-PGH2 receptors (SQ29548, 1 mumol/L) but was not affected by an inhibitor of TXA2 synthesis (CGS13080, 10 mumol/L), suggesting mediation by PGH2. The lipoxygenase inhibitor baicalein (75 mumol/L) also attenuated the constrictor effect of AA in aortic rings of hypertensive rats while decreasing the associated release of HETEs and correcting the impairment in the conversion of PGH2 to PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)