Protection of salvianolic acid A on rat brain from ischemic damage via soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH) have been associated with ischemic stroke. Salvianolic acid A (SAA) is proved to display potent cerebroprotection. However, little information is available about the link between them. This study aimed to investigate whether SAA exhibits its protective effects in rats subjected to middle cerebral artery occlusion (MCAO) through sEH and EETs. The results showed that SAA treatment ameliorated neurological deficits and reduced infarct volume. Notably, the beneficial effects of SAA were attenuated by co-administration of (14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE)), a putative selective EETs antagonist. Furthermore, SAA increased the 14,15-EET levels in the blood and brain of sham and MCAO rats. Assay for hydrolase activity showed that 1 and 3 mg/kg of SAA significantly diminished brain sEH activity of MCAO rats. A fluorescent assay in vitro indicated that SAA could inhibit recombinant human sEH activity in a concentration-dependent manner (IC₅₀ = 1.62 μmol/l). Immunohistochemical analysis showed that SAA at the doses of 1 and 3 mg/kg significantly decreased sEH protein expression in hippocampus CA1 region of MCAO rats. In conclusion, cerebral protection of SAA is mediated, at least in part, via inhibiting sEH to increase EETs levels.     

Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs.     

Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP ω-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.     

Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.     

Novel selective and metabolically stable inhibitors of anandamide cellular uptake

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested on: (i) cannabinoid CB(1) and CB(2) receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid amide hydrolase (FAAH). The (R)- and, particularly, the (S)-1'-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA (OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells (K(i) ranging between 2.4 and 17.7 micro M), the oleoyl analogues (OMDM-1 and OMDM-2, K(i) 2.4 and 3.0 micro M, respectively) being 6- to 7-fold more potent than the arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB(1) (K(i)> or = 5 micro M) or CB(2) (K(i)>10 micro M) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular calcium assay carried out with intact cells over-expressing the human VR1 (EC(50)> or = 10 micro M); and (iii) no activity as inhibitors of FAAH in N18TG2 cell membranes (K(i)>50 micro M). The oleoyl- and arachidonoyl-N'-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and OMDM-6), inhibited ACU (K(i) 4.8 and 7.0 micro M, respectively), and were more potent as VR1 agonists (EC(50) 75 and 50nM, respectively), weakly active as CB(1) receptor ligands (K(i) 4.9 and 3.2 micro M, respectively), and inactive as CB(2) ligands (K(i)>5 micro M) as well as on FAAH (K(i)> or = 40 micro M). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one "hybrid" agonist of CB(1) and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very stable to enzymatic hydrolysis by rat brain homogenates.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

Nitroreductive metabolic activation of some carcinogenic nitro heterocyclic food contaminants in rat mammary tissue cellular fractions

Several nitrofurans and nitroimidazoles have been widely used in veterinary medicine. Some of these compounds are breast carcinogens in rodents and their mechanism of action is hypothesized to be related to reactive metabolites generated by nitroreduction and/or via oxygen-dependent redox cycling. The present work describes the nitroreductive metabolism of nitrofurazone, nitrofurantoin, furazolidone, and metronidazole by the cytosolic and microsomal fractions of mammary tissue from female Sprague–Dawley rats. The data obtained were compared with those obtained with nifurtimox and benznidazole, two well-known rodent carcinogen/mutagens nitroheterocycles. The nitroreductase activity of pure milk xanthine-oxidoreductase (XOR) was evaluated for screening purposes. All the nitrofurans were nitroreduced either by the pure XOR or the cytosolic fraction in the presence of hypoxanthine, and these activities were inhibited by allopurinol. Furthermore, they were nitroreduced by the microsomal fraction in the presence of NADPH, except for the nitrofurazone, suggesting the participation of cytochrome P450 reductase. Nitrofurans metabolism was significantly more intense than that of NFX. No equivalent nitroreductase activity was observed in either subcellular fraction using nitroimidazolic compounds as substrates. These results suggest that the nitroreductive metabolism of nitrofurans and the subsequent redox cycling might be involved in the associated mammary tissue carcinogenic effects.     

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol,and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.     

Reciprocal regulation of human soluble and particulate guanylate cyclases in vivo

BACKGROUND & PURPOSE: We demonstrated previously that reciprocal regulation of soluble (sGC) and particulate (pGC) guanylate cyclases by NO and natriuretic peptides coordinates cyclic cGMP-mediated vasodilatation in vitro. Herein, we investigated whether such an interaction contributes to vascular homeostasis in mice and humans in vivo. EXPERIMENTAL APPROACH: Mean arterial blood pressure (MABP) changes in anaesthetized mice were monitored in response to i.v. administration of cGMP- and cAMP-dependent vasodilators in wild-type (WT), endothelial NO synthase (eNOS) and natriuretic peptide receptor (NPR)-A knockout mice. Forearm blood flow (FBF) in response to intra-brachial infusion of ANP (25, 50, 100, 200 pmol min(-1)) in the absence and presence of the NOS inhibitor NG-methyl-L-arginine (L-NMA; 4 micromol min(-1)) and the control constrictor noradrenaline (240 pmol min(-1)) was assessed in healthy volunteers. KEY RESULTS: Sodium nitroprusside (SNP; NO-donor) and atrial natriuretic peptide (ANP) produced dose-dependent reductions in MABP in WT animals that were significantly enhanced in eNOS KO mice. In NPR-A K mice, SNP produced a dose-dependent reduction in MABP that was significantly greater than that in WT mice. Responsiveness to the cAMP-dependent vasodilator epoprostenol was similar in WT, eNOS KO and NPR-A KO animals. ANP caused vasodilatation of the forearm resistance vasculature that was significantly greater in individuals lacking endothelium-derived NO (i.e. L-NMA treated). CONCLUSIONS & IMPLICATIONS: These data demonstrate that crosstalk occurs between the NO-sGC and ANP-pGC pathways to regulate cGMP-dependent vasodilatation in vivo in both mice and humans. These findings have implications for understanding the link between natriuretic peptide activity and cardiovascular risk.     

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.     

Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6

S-(-)-Nicotine and 13 of the most prevalent nicotine-related alkaloids and metabolites (i.e., S-(-)-nornicotine, myosmine, beta-nicotyrine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-anabasine, S-(-)-N-methylanabasine, anabaseine, S-(-)-anatabine, nicotelline, and 2,3'-bipyridyl) were evaluated as inhibitors of human cDNA-expressed cytochrome P-450 2A6 (CYP2A6) mediated coumarin 7-hydroxylation. Tobacco alkaloids myosmine, S-(-)-nornicotine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-N-methylanabasine, anabaseine, and nicotelline had Ki values for inhibition of coumarin 7-hydroxylation ranging from 20 microM to more than 300 microM whereas nicotine and S-(-)-anatabine were much more potent (i.e. 4.4 and 3.8 microM, respectively). The tobacco alkaloids 2,3'-bipyridyl (7.7 microM) and S-(-)-anabasine (5.4 microM), were somewhat less potent compared with S-(-)-nicotine or S-(-)-anatabine in inhibition of human CYP2A6. beta-Nicotyrine, in which the N-methylpyrrolidino moiety of nicotine was replaced by the aromatic N-methylpyrrole ring, was shown to inhibit human CYP2A6 with much greater potency (Ki=0.37 microM) compared with S-(-)-nicotine. Among the compounds examined, only nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6. The potency of the mechanism-based CYP2A6 inhibitors suggests that, for smokers, modulation of CYP2A6 may be greater than that predicted on the basis of serum concentration of these alkaloids. Our results indicate that the prominent nicotine-related alkaloid beta-nicotyrine present after smoking potently inhibits human CYP2A6.     

The effect of haem biosynthesis inhibitors and inducers on intestinal iron absorption and liver haem biosynthetic enzyme activities

The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p<0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p<0.005, p<0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p<0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p<0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p<0.05, p<0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p<0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p<0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p<0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption.     

Differential binding to the alpha/beta-tubulin dimer of vinorelbine and vinflunine revealed by nuclear magnetic resonance analyses

The binding of two antitumour alkaloids, vinorelbine and vinflunine, to the alpha/beta-tubulin dimer has been investigated at equilibrium by nuclear magnetic resonance (NMR) spectroscopy. Tubulin stability and assembly induced by these drugs has been checked under NMR experimental conditions, and tubulin spirals were found in majority. Then, using increasing ligand concentrations, the alkaloids were titrated against tubulin. A non-specific binding of both compounds to tubulin (K(d)>10(-5)M) was characterised by broad NMR ligand signal at 4 and 30 degrees. The tubulin dimer exhibited also 2.7 (sigma: 0.3) and 2.6 (sigma: 0.6) binding sites with a K(d)<10(-5)M for vinorelbine at 4 and 30 degrees, respectively. In contrast, if the tubulin dimer exhibited 2.7 (sigma: 0.2) binding sites for vinflunine at 4 degrees, these sites were not detected at 30 degrees. This NMR study revealed for the first time the presence of specific binding sites and a clear differential affinity of vinorelbine and vinflunine to the tubulin dimer at physiological temperatures which could possibly account for their differential cytotoxicity.     

Relative importance of maternal and embryonic microsomal epoxide hydrolase in 7,12-dimethylbenz[a]anthracene-induced developmental toxicity

Microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of epoxide intermediates derived from drugs and environmental chemicals. The response of in vivo (embryo) and in vitro (embryo fibroblast) tests were analyzed using mEH-null and wild-type mice to determine the relative role of maternal and embryonic mEH in the developmental toxicity induced by 7,12-dimethylbenz[a]anthracene (DMBA). Embryos derived from DMBA-treated [50mg/kg, daily from gestational day (GD) 11 to GD 15] dams were analyzed. Although weight (P=0.0009) and crown-rump length (P=0.0003) of wild-type fetuses on GD 18 were significantly lower than those of mEH-null fetuses, respectively, no significant difference was found between mEH-null and heterozygous fetuses of mEH-null dams. Cell viability was decreased to 50% in wild-type mouse embryo fibroblasts (MEFs) treated with 3 microM DMBA, but no significant decrease was found in mEH-null MEFs. DMBA-3,4-diol produced a significant decrease in cell viability and suppressed the proliferation of wild-type MEFs at a 10-fold lower concentration than did DMBA. Although mEH protein was expressed in liver microsomes from wild-type embryos (GD 15), DMBA-3,4-diol was not detected among the DMBA metabolites. However, it was detected in the serum of wild-type pregnant mice treated with DMBA, but not in that of mEH-null mice. These results suggest that maternal mEH plays a major role in DMBA-induced developmental toxicity, and embryonic mEH is less involved in the toxicity.     

Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling

Nitric oxide (NO) produced by the endothelium is an important protective molecule in the vasculature. It is generated by the enzyme endothelial NO synthase (eNOS). Similar to all NOS isoforms, functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via the flavins flavin adenine dinucleotide and flavin mononucleotide in the carboxy-terminal reductase domain, to the heme in the amino-terminal oxygenase domain. Here, the substrate L-arginine is oxidized to L-citrulline and NO. Cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia or cigarette smoking reduce bioactive NO. These risk factors lead to an enhanced production of reactive oxygen species (ROS) in the vessel wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in the presence of cardiovascular risk factors. NADPH-oxidase-derived superoxide avidly reacts with eNOS-derived NO to form peroxynitrite (ONOO(-)). The essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH(4) ) is highly sensitive to oxidation by this ONOO(-). In BH(4) deficiency, oxygen reduction uncouples from NO synthesis, thereby converting NOS to a superoxide-producing enzyme. Among conventional drugs, compounds interfering with the renin-angiotensin-aldosterone system and statins can reduce vascular oxidative stress and increase bioactive NO. In recent years, we have identified a number of small molecules that have the potential to prevent eNOS uncoupling and, at the same time, enhance eNOS expression. These include the protein kinase C inhibitor midostaurin, the pentacyclic triterpenoids ursolic acid and betulinic acid, the eNOS enhancing compounds AVE9488 and AVE3085, and the polyphenolic phytoalexin trans-resveratrol. Such compounds enhance NO production from eNOS also under pathophysiological conditions and may thus have therapeutic potential.     

Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators

Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.     

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.     

Reduction of high glucose and phorbol-myristate-acetate-induced endothelial cell permeability by protein kinase C inhibitors LY379196 and hypocrellin A

Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. Although recent studies have established a link between protein kinase C (PKC) pathway and hyperglycaemic-induced vascular permeability, it is unclear which PKC isoforms involve increased endothelial cell permeability. In the present study, we investigated whether high glucose induced endothelial hyperpermeability via distinct PKC isoforms in human umbilical vein endothelial cells (HUVECs) and whether increased endothelial permeability could be substantially reversed by PKC inhibitors LY379196 and hypocrellin A (HA). High glucose (20 mM) and phorbol-myristate-acetate (PMA)-induced endothelial hyperpermeability was almost abolished by 150 nM HA and partially reduced by 30 nM PKC beta inhibitor (LY379196). LY379196 and HA inhibited the membrane fraction of PKC activity in a dose-dependent manner. Western blot analysis revealed high-glucose-induced overexpression of PKC alpha and PKC beta2 in the membrane fraction of HUVECs. LY379196 (30 and 150 nM) selectively inhibited PKC beta2 with no significant effect on PKC alpha expression. HA (150 nM) significantly reduced PKC alpha expression with no inhibitory effect on PKC beta2. At higher concentrations (300 nM), both LY379196 and HA were no longer selective for PKC beta or alpha, respectively. This study showed that both PKC alpha and beta2 contributed to endothelial hyperpermeability. Since reduction of endothelial hyperpermeability was greater with inhibition of PKC alpha rather than PKC beta2, we conclude that PKC alpha may be a major isoform involved in endothelial permeability in HUVECs, and that PKC alpha-mediated endothelial permeability was significantly reversed by the PKC inhibitor HA.