The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.

Studies of the hierarchies of agonist and antagonist affinity for the prostaglandin (PG)H2/thromboxane (Tx)A2 receptor have been performed to establish whether distinct receptor subtypes exist in platelets and vascular smooth muscle cells (VSMC). They have yielded conflicting results. The pattern of homologous desensitization of phospholipase C activation and [Ca++i] increase induced by the PGH2/TxA2 agonist U46619 in rat aortic SMC was similar to that previously observed in human platelets: rapid desensitization of both responses followed by a delayed loss of binding sites from the cell membrane. Recently, the pattern of receptor inactivation by the antagonist ligand, GR 32191, has identified two subtypes in platelets. GR 32191 binds reversibly (GRr) to a site that mediates platelet shape change and an increase [Ca++i] and irreversibly (GRirr) to a site linked to phospholipase C activation and aggregation. In contrast to platelets, studies of ligand dissociation only identified GRr sites in rat aortic SMC and GR 32191 failed to inactivate PGH2/TxA2 receptors as detected by the PGH2/TxA2 receptor antagonist, [3H]SQ 29548. Inhibition of U46619-induced contraction of both rat aortic and human saphenous vein was competitive, consistent with the absence of GRirr sites in VSMC. Platelet activating factor, which heterologously desensitizes U46619-evoked phospholipase C activation in platelets, had no such effect in VSMC. The biochemical events attendant to PGH2/TxA2 receptor desensitization are similar in SMC and platelets. However, both the pattern of receptor inactivation by GR 32191 and of heterologous desensitization by PAF, suggest that VSMC lack the receptor subtype that transduces aggregation of platelets.

Melatonin, a major hormone of pineal gland, was recently shown to attenuate acute gastric lesions induced by strong irritants because of the scavenging of free radicals but its role in ulcer healing has been little investigated. In this study we compared the effects of intragastric (i.g.) administration of melatonin and its precursor, L-tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on healing of chronic gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm2). The involvement of endogenous prostaglandins (PG), nitric oxide (NO) and sensory nerves in ulcer healing action of melatonin and L-tryptophan was studied in rats treated with indomethacin and NG-nitro-L-arginine (L-NNA) to suppress, respectively, cyclo-oxygenases (COX) and NO synthases or in those with functionally deactivated sensory nerves with capsaicin. The influence of melatonin on gastric secretion during ulcer healing was tested in separate group of rats with gastric ulcer equipped with gastric fistulas (GF). At day 8 and 15 upon the ulcer induction, the area of gastric ulcers was measured by planimetry, the mucosal blood flow (GBF) was determined by H2-gas clearance technique and gastric luminal NO2-/NO3- levels was assessed by Griess reaction. Plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for expression of constitutive NO-synthase (cNOS) and inducible NOS (iNOS) by reverse transcriptase-polymerase chain reaction (RT-PCR). Melatonin (2.5-20 mg/kg-d i.g.) and L-tryptophan (25-100 mg/kg-d i.g.) dose-dependently accelerated ulcer healing, the dose inhibiting by 50% (ED50) of ulcer area being 10 and 115 mg/kg, respectively. This inhibitory effect of melatonin (10 mg/kg-d i.g.) and L-tryptophan (100 mg/kg-d i.g.) on ulcer healing was accompanied by a significant rise in the GBF at ulcer margin and an increase of plasma melatonin. luminal NO2-/NO3- and plasma gastrin levels. Gastric acid and pepsin outputs were significantly inhibited during the ulcer healing in melatonin-treated gastric mucosa as compared with those in vehicle-treated animals. Luzindole abolished completely the healing effects of melatonin and L-tryptophan and attenuated significantly the rise in plasma gastrin evoked by the hormone and its precursor. Indomethacin (5 mg/kg-d i.p). that blocked PG biosynthesis by 90% or L-NAME (20 mg/kg i.v), inhibitor of NOS. that suppressed luminal NO release, attenuated significantly melatonin and L-tryptophan-induced acceleration of ulcer healing and accompanying rise in GBF at ulcer margin and luminal NO release. The melatonin-induced acceleration of ulcer healing, hyperemia at ulcer margin and increase in the release of NO were enhanced when L-arginine but not D-arginine was added to L-NAME. The ulcer healing and the GBF effects of melatonin and L-tryptophan were significantly impaired in rats with capsaicin-induced denervation of sensory nerves and both, ulcer healing and the hyperemia at ulcer margin were restored in these rats by addition of exogenous CGRP to melatonin and L-tryptophan. Expression of cNOS mRNA was detected by RT-PCR in the intact gastric mucosa as well as at the edge of gastric ulcers treated with both, vehicle and melatonin, while iNOS mRNA that was undetectable in the intact gastric mucosa, appeared during ulcer healing and especially this was strongly up-regulated in the melatonin-treated gastric mucosa. We conclude that (1) exogenous melatonin and that derived from its precursor, L-tryptophan, accelerate ulcer healing probably via interaction with MT2 receptors; (2) this ulcer healing action is caused by an enhancement by melatonin of the microcirculation at the ulcer margin possibly mediated by COX-derived PG and NO because of overexpression of iNOS and (3) gastrin, which exhibits trophic activity in the gastric mucosa and calcitonin gene related peptide (CGRP), released from sensory nerves, may also contribute to the ulcer healing action of melatonin.

We tested the hypothesis that the role of histamine in the control of intestinal secretion is mediated by prostaglandins (PGs). The effects of histamine on ion transport were examined in muscle-stripped sheets of mucosa/submucosa set up in flux chambers. Histamine evoked a transient concentration-dependent increase in short-circuit current (Isc) that was reduced by the Cl- transport inhibitor bumetanide. Histamine also caused the release of PGE2. The Isc response to histamine was reduced by indomethacin and piroxicam, which block PG formation, but not by nordihydroguaiaretic acid, which prevents production of lipoxygenase products. 2-Methylhistamine, but not dimaprit, evoked a concentration-dependent increase in Isc. The Isc response to histamine was reduced by the H1-blocker pyrilamine, but not by the H2-antagonist cimetidine. In addition to its direct effect, histamine augmented the responses of endogenously released neurotransmitters with and without indomethacin and hexamethonium. Tetrodotoxin (TTX) reduced the Isc response to 10(-3) M histamine. In the presence of TTX, exogenous histamine amplified the responses to PGs, vasoactive intestinal polypeptide, 2-chloroadenosine, bethanechol, and carbachol. These results suggest that histamine acts at H1-receptors on cells within the gut to mediate intestinal Cl- secretion in part by releasing PGs and by augmenting the actions of endogenously released neurotransmitters. Our results indicate that histamine has a role in the regulation of colonic transport function.

BACKGROUND

Fibroblast growth factor (FGF)-21 is a novel regulator of glucose and lipid metabolism. Recently, increased FGF-21 mRNA expression in muscle was found in patients with type 2 diabetes, but the role for FGF-21 in muscle is not well understood. Patients with HIV-infection and lipodystrophy are characterised by various degree of lipid-driven insulin resistance. We hypothesized that muscle FGF-21 mRNA would be altered in HIV patients with lipodystrophy.

METHODS

Twenty-five HIV-infected men with lipodystrophy (LD) and 15 age-matched healthy controls, received an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp (50 mU/m2/min) combined with 6,6-H2 glucose infusion. Muscle biopsies were obtained and FGF-21 mRNA and glycogen synthase (GS) activity were measured.

RESULTS

Subjects with HIV were insulin resistant compared with non-HIV subjects. Compared to controls, HIV subjects demonstrated a twofold increase of plasma FGF-21 from 70.4±56.8 pg/ml vs 109.1±71.8 pg/ml, respectively (p = 0.04) and an eight-fold increase in muscular FGF-21 mRNA expression (p = 0.001). Muscle FGF-21 mRNA correlated inversely with the rate of disappearance of glucose during insulin clamp (r = -0.54, p = 0.0009), and the GS fractional velocity in muscle (r = -0.39, p = 0.03), and directly with fasting insulin (r = 0.50, p = 0.0022), HOMA-IR (r = 0.47, p = 0.004), triglycerides (r = 0.60. P = 0.0001), waist-to-hip ratio (r = 0.51, p = 0.0001) and limb fat mass (-0.46, p = 0.004), but not to plasma FGF-21.

CONCLUSIONS

FGF-21 mRNA is increased in skeletal muscle in HIV patients and correlates to whole-body (primarily reflecting muscle) insulin resistance, but not to plasma FGF-21. Those findings add to the evidence that FGF-21 is a myokine and may suggest that muscle FGF-21 is working in a local manner.

The secretion of HCO3(-) in the duodenum is increased by mucosal acidification, and this process is modulated by gas mediators such as nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO), in addition to prostaglandins (PGs). The secretion is increased by NOR3 (NO donor), NaHS (H2S donor), and CORM-2 (CO donor). The HCO3(-) responses to NOR3 and CORM-2 are attenuated by indomethacin, while that to NaHS is mitigated by indomethacin and L-NAME as well as sensory deafferentation. NOR3 and CORM-2 increase mucosal PGE2 production, while H2S increases mucosal PGE2 content and luminal NO release. The HCO3(-) response to mucosal acidification is attenuated by indomethacin, propargylglycine, and SnPP, each inhibiting PG, H2S and CO production, respectively. The acid-induced duodenal damage is worsened when either PG, H2S or CO is lacking. These findings suggest that 1) NO, H2S, and CO, generated endogenously or exogenously, stimulate HCO3(-) secretion in the duodenum; 2) the stimulatory action of NO and CO is mediated, at least partly, by endogenous PGs, while that of H2S is mediated by PGs and NO as well as sensory neurons; 3) these gas mediators are involved in the local regulation of acid-induced HCO3(-) secretion, in addition to endogenous PGs; 4) the acid-induced duodenal damage is worsened by agents inhibiting the endogenous production of NO, H2S or CO. It is assumed that these gas mediators play a role in maintaining the integrity of the duodenal mucosa by modulating the secretion of HCO3(-).

Histamine has been reported to attenuate adrenergic responses in cardiovascular tissues. In guinea pig atria preloaded with [3H]norepinephrine, histamine diminishes the field stimulation-induced efflux of radioactivity; this effect has been attributed to an inhibition of norepinephrine release from nerve endings. To assess the possible physiological relevance of these findings, we have reinvestigated the effects of histamine on cardiac sympathetic responses and on the release of endogenous norepinephrine in the guinea pig heart isolated with its intact sympathetic innervation. Heart rate, left ventricular contractile force, and perfusion pressure all increased with increasing frequencies of sympathetic nerve stimulation (2-8 Hz). Histamine (3 X 10(-8) to 3 X 10(-7) M) caused dose-dependent attenuation of the responses to sympathetic stimulation. The ability of histamine to modulate nerve stimulation-induced norepinephrine overflow into the coronary effluent was dependent on whether the heart had been preloaded with norepinephrine. Whereas histamine did not cause a significant reduction in nerve stimulation-induced norepinephrine overflow in hearts from untreated animals, histamine significantly reduced stimulation-induced norepinephrine overflow in hearts from guinea pigs that had been pretreated with norepinephrine before sacrifice. Histamine also attenuated the increases in left ventricular contractile force, perfusion pressure, and heart rate, which result from the intracardiac administration of norepinephrine (0.16-microgram bolus injection). In this respect, histamine was as effective as it was in inhibiting the responses elicited by nerve stimulation. Thus, in normal animals, the negative modulatory effect of histamine on adrenergic responses can be attributed largely, if not totally, to a postjunctional mechanism. In contrast, a prejunctional action of histamine may contribute significantly to the negative modulation observed in norepinephrine-preloaded hearts. Since we have observed a large increase in the amount of endogenous histamine present in the coronary effluent after sympathetic stimulation (930 pg during the 30 seconds poststimulation vs. 240 pg during 30 seconds prestimulation), as well as a prolongation of nerve stimulation-induced cardiac responses in the presence of the H2 receptor antagonist tiotidine, we postulate that histamine plays a physiological role as a modulator of sympathetic responses in the heart.

These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, levuglandin (LG) E2 and D2. We previously have demonstrated that LGE2 reacts with the epsilon-amine of lysine to form both the lysyl-levuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam adducts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolation of the adducted amino acid residues, these adducts were identified by liquid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be formed from PGH2 can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formation of adducts of other proteins present in the reaction solution. The reactivity of the PGH-synthase adducts themselves is demonstrated by the formation of intermolecular cross-links.

Interactions of ONO3708, a thromboxane (TX) A2 analog, and epithio-methano-TXA2 (sTXA2) or prostaglandins (PGs) were investigated in helical strips of dog cerebral, coronary, renal, and mesenteric arteries. In these arterial strips sTXA2 (10(-10) to 10(-7) M) produced a dose-dependent contraction, whereas ONO3708 up to 10(-6) M failed to contract the arteries but antagonized the contractile response to sTXA2. The inhibition tended to be greater in renal arteries than in the other arteries. Contractions induced by PGF2 alpha, PGE2, and PGD2 were also suppressed by treatment with low concentrations (3 X 10(-9) and 10(-8) M) of ONO3708. The attenuations of the response to sTXA2, PGF2 alpha, PGE2, and PGD2 did not appreciably differ. Norepinephrine-induced contractions were not influenced by ONO3708 up to 2 X 10(-7) M. On the other hand, relaxant responses to PGI2 of cerebral and renal arteries were not reduced by ONO3708. Prostaglandin H2 produced a transient contraction followed by a relaxation in cerebral and renal arteries. The contractile response was abolished by 10(-7) M ONO3708, and the relaxation was potentiated. It may be concluded that ONO3708 selectively antagonizes the vasoconstrictor action of TXA2, its analogs, and PGs but does not alter the action of vasodilator PGs. At least in part, sTXA2, PGF2 alpha, PGE2, and PGD2 appear to share the same receptive site responsible for vascular contraction.

The metabolism of [1-14C]arachidonic acid by microsomal and purified prostaglandin (PG) H synthase was investigated. HPLC analysis confirmed that arachidonic acid (20:4) was extensively converted into prostaglandin G2 (PGG2) and/or prostaglandin H2 (PGH2) but several minor labelled products were formed in addition. Their formation, mediated by PGH synthase was established by inhibition with aspirin and indomethacin [Hecker, M., Hatzelmann, A. & Ullrich, V. (1987) Biochem. Pharmacol. 36, 851-855]. Upon comparison with authentic reference material these unknown PGH synthase metabolites were identified with respect to chromatographic properties, ultraviolet spectroscopy and mass spectrometry as 11 (R)-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-OOH-20:4), 12(S)-hydroperoxy-5Z,8E,10E-heptadecatrienoic acid (OOH-17:3), 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (OH-17:3), 15(RS)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoi c acid (15-OOH-20:4), 15(RS)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-OH-20:4), 13-hydroxy-5Z,14Z-prostaglandin H2, 15(S)-hydroxy-8-iso-5Z,13E-prostaglandin H2 and 15-oxo-prostaglandin H2. Unlike PGG2 and PGH2, 8-iso-PGH2, 13-hydroxy-PGH2 and 15-oxo-PGH2 failed to induce aggregation of washed human platelets and to form thromboxane upon incubation with homogeneous human platelet thromboxane synthase. In contrast to the formation of OOH-17:3, 15-oxo-PGH2 and OH-17:3 which can be attributed to the heme-catalyzed decomposition of PGG2 and PGH2, 11-OOH-20:4,15-(O)OH-20:4-,8 iso-PGH2 and 13-hydroxy-PGH2 represent potential side products of arachidonic acid conversion into PG endoperoxides. Their formation allows to conclude on PGH synthase mechanism and its intermediates for which an extended reaction scheme is proposed.

BACKGROUND

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1.

METHODS

Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured.

RESULTS

Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation.

CONCLUSIONS

mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.

The growth hormone release-inhibiting hormone or somatostatin is a potent inhibitor of gastric acid secretion. In the present paper these inhibiting properties were tested on isolated gastric glands from rabbit fundic mucosae, prepared as according to Berglindh & Obrink (1976). Parietal cell activity was measured as [14C]aminopyrine (AP) accumulation and O2-consumption. Glandular histamine release was determined after condensation with o-phthalaldehyde and measured fluorometrically. In the gastric glands there are two possible main processes that can be inhibited, namely (1) the release of histamine from some endocrine cells and (2) the activity of the parietal cell itself. It was found that somatostatin acted on both mechanisms. Basal histamine release was, however, not affected by somatostatin while the release induced by pentagastrin (Pg) at a concentration of 3 X 10(-9) M, or acetylcholine (10(-5) M) was dose-dependently (10(-12) to 10(-6) M) inhibited by this peptide. Maximal inhibition, which was about 70%, occurred at a dose of 10(-8) M somatostatin. Somatostatin also depressed parietal cell activity induced by histamine (10(-6) to 10(-4) M), isobutyl-methyl-xanthine (IMX, 10(-5) to 10(-4) M) or the combination of IMX (10(-5) M) and Pg (3 X 10(-9) M) Basal parietal cell activity was, however, unaffected. The IMX (10(-4) M) induced parietal cell activity in cimetidine-treated (10(-4) M) glands was also depressed by somatostatin, which indicates an action directly on the parietal cell not mediated via H2-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Because previous studies suggest an antinociceptive role for the neuromodulator histamine (HA) in the periaqueductal grey or the nearby dorsal raphe (PAG/DR), a detailed pharmacological investigation of the effects of intracerebral HA on the hot-plate nociceptive test was performed in rats. Intracerebral microinjections of HA (1 microgram) into the PAG/DR or into the median raphe evoked a mild, reversible antinociceptive response; injections into lateral or dorsal midbrain evoked either a delayed response or no response, respectively. In the PAG/DR, the HA dose-response curve had an inverted U-shape, showing that HA can induce both antinociceptive (0.3-3 micrograms) and pro-nociceptive (10-30 micrograms) responses. Larger doses of HA (e.g., 100 micrograms) produced irreversible and highly variable antinociceptive responses that were accompanied by behavioral and histopathological changes; such effects, indicative of toxicity, were not observed after 0.3 microgram of HA, the peak antinociceptive dose. HA (0.3 microgram) antinociception was completely inhibited by intracerebral co-administration of the opiate antagonist naloxone (1 ng), the H1-receptor antagonist temelastine (20 pg), and the H2-receptor antagonist tiotidine (1 ng); none of these drugs altered nociceptive scores in the absence of HA. These results show that: (1) HA, a neurotransmitter in the PAG, can evoke antinociception in the absence of other behavioral or toxic effects; and (2) HA antinociception depends on the activation of both opiate and HA receptors in the PAG/DR.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.

When prostaglandin H2 (PGH2) was incubated with a mixture of glutathione S-transferases (GSTs) obtained from S-hexylglutathione affinity chromatography, as much as 40% of it was transformed into a prostanoid whose Rf value corresponded to that of the standard PGF2 alpha. The reaction product was identified as PGF2 alpha by cochromatography with a standard on TLC and HPLC. The stereochemistry of the hydroxyl groups on C-9 and C-11 of the cyclopentane ring was confirmed by mass-spectral analysis of the butylboronate derivative of the reaction product. Neither PGE2 nor PGD2 could substitute for PGH2 in the reaction mixture, indicating that the mechanism of formation of PGF2 alpha is a direct two-electron reduction of the endoperoxide moiety and not through a reduction of the keto group on PGE2 or PGD2. Individual GST isozymes exhibited distinct differences in their catalytic rates of formation of PGF2 alpha from PGH2. Among various GSTs, isozyme IV, a homodimer of Ya size subunit showed the highest activity with a Vmax value of approximately 6000 nmol.min-1.mg-1. In general, the isozymes containing Ya and Yc subunits exhibited relatively high activity toward PGH2, indicating that it is the non-selenium-dependent glutathione peroxidase activity associated with the GSTs that might be responsible for the reduction of PGH2 to PGF2 alpha. Interestingly, isozyme IV also exhibited the highest PGE2 forming activity with a Vmax value of approximately 3000 nmol.min-1.mg-1 followed by isozyme I, a homodimer of Yb subunit, which had a Vmax value of 420 nmol.min-1.mg-1. Based on these results, it appears that the GSTs play an important role in the biosynthesis of classical PGs. Therefore, it is conceivable that the tissue-specific formation of PGF2 alpha and PGE2 might, in part, be due to the relative distribution of these enzyme activities in a given tissue. Our results have not only confirmed the previously published reports (E. Christ-Hazelhof et al. (1976) Biochim. Biophys. Acta 450, 450-461), but also have characterized the specificity of GST isozymes in the formation of PGF2 alpha.

Term and preterm parturition is associated with elevated intrauterine PG production. Although an increase of PG synthesis by the fetal membranes during term labor is well documented, there is little data available regarding the prostanoid production of these tissues at term, before the spontaneous onset of labor. In the present study, we determined the expression of PG H2 synthase (PGHS), the committing and rate-limiting enzyme of prostanoid biosynthesis, in the chorion laeve during gestation. Tissues were collected from 18 patients at term (37-41 weeks of gestation) and from 13 patients between 17 and 35 weeks of pregnancy. None of the patients were in labor. PGHS-specific activity and the abundance of messenger RNAs (mRNAs) encoding the two PGHS isoenzymes (the constitutive PGHS-1 and the inducible PGHS-2) were measured by a cell-free enzyme assay and specific ribonuclease protection assays, respectively. PGHS-specific activity as well as PGHS-1 and -2 mRNA levels were significantly (P < 0.01) higher at term before labor than earlier during gestation. Furthermore, PGHS activity at term exhibited significant positive correlation with PGHS-2 mRNA levels, but not with PGHS-1 mRNA levels. In situ hybridization indicated that the expression of both PGHS mRNAs increased in the epithelial and the mesenchymal cells of the amnion and the chorion laeve at term. Additionally, PGHS activity and mRNA levels were determined in the chorion laeve of a group of patients who gave birth spontaneously before term (30.6 +/- 1 weeks, mean +/- SEM, n = 5), and the values were compared with a group who delivered by cesarean section before labor at a similar gestational age (31.9 +/- 1.4 weeks, n = 5, P>> 0.05 vs. the preterm labor group). None of the patients exhibited signs of genital tract infection. PGHS-specific activity and PGHS-1 and -2 mRNA levels were significantly higher in the preterm labor group than in the group who delivered preterm without labor. In situ hybridization suggested that the enhanced PGHS-1 and -2 mRNA expression occurred predominantly in the mesenchymal cells of the fetal membranes at preterm labor. Thus, PGHS-1 and -2 expression increases in the chorion laeve at term before labor, with PGHS-2 as the functionally prevalent isoform. This supports the possibility that PGs originating in the fetal membranes promote the onset of normal labor. Furthermore, preterm labor is associated with the elevated expression of the two PGHS isoenzymes in the chorion laeve. The maturation of the fetal membranes in preparation for term labor involves both the epithelial and the mesenchymal cells, whereas preterm labor is accompanied by the maturation of the mesenchymal tissue components, as reflected by PGHS expression. This difference may have implications in the early recognition of preterm labor.

BACKGROUND

Hydrogen (H2) has been reported to be effective in the treatment of oxidative injury, which plays an important role in the process of decompression sickness (DCS). This study was designed to test whether H2-rich saline (saline saturated with molecular hydrogen) protected rats against DCS.

METHODS

Models of DCS were induced in male Sprague-Dawley rats weighing 300-310 g. H2-rich (0.86 mmol x L(-1)) saline was administered intraperitoneally (10 ml x kg(-1)) at 24 h, 12 h, immediately before compression, and right after fast decompression.

RESULTS

H2-rich saline significantly decreased the incidence of DCS from 67.57 to 35.14% and partially counteracted the increases in the total concentration of protein in the bronchoalveolar lavage from 0.33 +/- 0.05 to 0.14 +/- 0.01 mg x ml(-1) (mean +/- SD; P < 0.05), myeloperoxidase activity from 0.86 +/- 0.16 to 0.44 +/- 0.13 U/g, levels of malondialdehyde (MDA) from 0.80 +/- 0.10 to 0.48 +/- 0.05 nmol x mg(-1), 8-hydroxydeoxyguanosine from 253.7 +/- 9.3 to 191.2 +/- 4.8 pg x mg(-1) in the lungs, and MDA level from 1.77 +/- 0.20 to 0.87 +/- 0.23 nmol x mg(-1) in the spinal cord in rat DCS models. The histopathology results also showed that H2-rich saline ameliorated DCS injuries.

CONCLUSIONS

It is concluded that H2-rich saline may have a protective effect against DCS, possibly due to its antioxidant action.

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35 ± 9.8 mm(2)); increased levels of TNF-α, IL-1β, and MDA (2311 ± 302.3 pg/mL, 901.9 ± 106.2 pg/mL, 121.1 ± 4.3 nmol/g, respectively); increased MPO activity (26.1 ± 3.8 U/mg); and reduced GSH levels (180.3 ± 21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77 ± 5.3 mm(2)); reduced TNF-α, IL-1β, and MDA formation (1502 ± 150.2 pg/mL, 632.3 ± 43.4 pg/mL, 78.4 ± 7.6 nmol/g, respectively); lowered MPO activity (11.7 ± 2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9 ± 40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive gamma-ketoaldehydes. Isoprostanes (IsoPs) are PG-like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2-like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4-hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the epsilon-amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.

BACKGROUND

In view of reports that CD8+ T cells may produce T(H2)-type cytokines and our own finding that levels of intracellular IL-4 are higher in CD8+ than CD4+ T cells in healthy nonatopic subjects, we have hypothesized that the capacity of CD8+ T cells to produce IL-4 may be increased in atopic asthma, a disease characterized by high production of T(H2) cytokines.

METHODS

Levels of IL-4 and interferon-gamma were measured by ELISA in cell lysates and in 20- and 48-hour cultures of concanavalin A-stimulated purified peripheral blood CD8+ T cells in seven patients with mild atopic asthma and seven healthy nonatopic subjects.

RESULTS

Resting CD8+ T cells in patients with asthma contained significantly more IL-4 than those of healthy nonatopic subjects (median, 26 pg/10(6) cells; range, 17 to 84 pg/10(6) cells vs 16 pg/10(6) cells; 10 to 28 pg/10(6) cells), with no difference in intracellular interferon-gamma levels. In the healthy control subjects, but not in the patients with asthma, levels of intracellular IL-4 correlated negatively with levels of interferon-gamma in resting CD8+ T cells (r[s] = -0.9411, p = 0.005). Stimulation with concanavalin A produced a consistent and significant increase in secretion of interferon-gamma, but not IL-4, with no difference between the two groups of subjects.

CONCLUSIONS

The results of this study suggest that CD8+ T cells from patients with asthma may be an important source of the T(H2)-type cytokine IL-4. This capacity appears to be acquired in vivo, possibly by conditioning by IL-4 produced in the inflamed airways.

In this work, a new multi-residue method using ultra-performance liquid chromatography (UPLC) quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was developed for screening and confirmation of 29 pharmaceutical compounds belonging to different therapeutical classes: analgesics and antiinflammatories, lipid regulating agents cholesterol lowering statin agents, psychiatric drugs, anti ulcer agents, histamine H2 receptor antagonist, antibiotics and beta-blockers. UPLC uses columns packed with 1.7 microm particles and enables elution of sample components in much narrower, more concentrated bands, resulting in better chromatographic resolution and increased peak height. The typical peak width was 5-10s at base, permitting very good separation of all compounds in 10 min, which represented an approximate three-fold reduction in the analysis time in comparison to conventional high-performance liquid chromatography (HPLC). Unequivocal identification of target pharmaceutical compounds was based on accurate mass measurement of the molecular ions in the TOF mode and by performing collision induced dissociation (CID) in the Q-TOF mode in order to generate accurate mass measurement of the product ions. Using lock mass correction the accurate masses calculated for the product ions deviated from the theoretical masses by 0.2 to 1.3 mDa (root mean square (RMS) value=0.67) and 0.7-6.4 ppm (RMS=3.53), respectively. Quantitation was carried out working in the TOF mode using the narrow window extracted ion chromatograms (nwXICs) of each compound (extracted using a 20 mDa window) yielding relative standard deviation (RSD) from 0.5 to 5.3% (run-to-run) and from 2.1 to 9.1% (day-to-day) and instrumental detection limits (IDLs) from 1 to 200 pg. Analysis of wastewater treatment plant (WWTP) samples gave method detection limits (MDLs) ranging from 10 to 500 ng/L. The UPLC-Q-TOF method was successfully applied to analyze pharmaceutical residues in WWTP samples.

To determine the prostaglandin (PG) H2 synthase (generally referred to as cyclooxygenase [COX]) isozyme responsible for producing uterotonic PGs during parturition, we used PGF2alpha receptor-deficient mice, which exhibit parturition failure due to impaired withdrawal of serum progesterone at term. On ovariectomy-induced parturition in these mice, uterine COX-2 mRNA expression was drastically induced in the myometrium, whereas COX-1 mRNA expression in the endometrial epithelium decreased. The concomitant administration of progesterone with ovariectomy resulted in a delay in parturition and the disappearance of both the increase in COX-2 mRNA and the decrease in COX-1 mRNA. Thus, the expression of myometrial COX-2 and the occurrence of parturition are closely associated in this model. Furthermore, administration of the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522, effectively delayed ovariectomy-induced parturition in these mice. These findings suggest that COX-2-derived PGs contribute to the onset of parturition after the decrease in serum progesterone level.

BACKGROUND

Increased dietary intake of polyunsaturated fatty acids (PUFAs) is known to be associated with a decrease in the incidence of peptic ulcer disease possibly due to increase in the synthesis of prostaglandins. But, it is also likely that conversion of PUFAs to PGs may not always be required for gastric mucosal protection. Present study was designed to study the role of PUFAs in pathobiology of steroid induce gastric damage in rats.

METHODS

Wistar rats were treated with 5 mg/kg bodyweight of dexamethasone to induce gastric mucosal ulcers. Effects of PUFAs was studied by supplementation of Fish oil (rich in n-3 EPA and DHA) and AA rich oil. Famotidine was used as a positive control. Generation of lipid peroxides, nitric oxide and the activity of anti-oxidant enzymes were also studied.

RESULTS

Dexamethasone induced ulceration was associated with changes in the phospholipid fatty acid profile, levels of lipid peroxidation products, nitric oxide and activity of anti-oxidant enzymes. The fatty acid profile showed an increase in LA and a decrease in other PUFAs like GLA, AA, EPA and DHA. When PUFAs were supplemented in the form of Fish oil and AA rich oil or when the animals were treated with H2-blocker, famotidine, there was a decrease in the incidence of ulceration in the animals associated with near normalization of changes in the phospholipid fatty acid profile. The levels of lipid peroxides, nitric oxide, and anti-oxidant activity also reverted to control values.

CONCLUSIONS

Dexamethasone induced gastric ulceration was prevented by PUFAs. This is supported by the results of our earlier study where in it was noted that in patients with DU plasma lipid peroxides, nitric oxide and phospholipid fatty acid pattern and red cell antioxidant activity were altered similar to those seen in dexamethasone treated group of the present study. These abnormalities, similar to the PUFA treated groups of the present study, reverted to normalcy following treatment of the patients with lansoprazole, a proton pump inhibitor. Further, PUFAs are known to inhibit the growth of Helicobacter pylori in vitro. Hence, it is concluded that PUFAs, free radicals, nitric oxide and anti-oxidants play a significant role in the pathobiology of peptic ulcer.

Prostaglandins (PG) are members of a large group of hormonally active fatty acids derived from free fatty acids. They are formed from arachidonic acid-the major PG precursor. Cyclooxygenase (COX)-1 and -2 are the rate-limiting steps in PG synthesis. COX-2 is overexpressed in many human non-endocrine and endocrine tumors including colon, breast, prostate, brain, thyroid, and pituitary. COX-2 has an important role in angiogenesis and tumor growth. Thromboxane synthase (TS) catalyzes the synthesis of thromboxane A2 (TXA2), which is derived from arachidonic acid and prostaglandin H2 and is a vasoconstrictor and inducer of platelet aggregation. TXA2 stimulates tumor growth and spread of some tumors and TS appears to have a critical role in tumorigenesis in some organ systems. In this review, we examine the role of COX-2 and TS in various non-endocrine tumors, especially colon, breast, prostate, and brain as well as in endocrine tumors. The accumulating evidence points to an increasingly important role of COX-2 and TS in tumor progression and metastasis.