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Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical ([??]OH) formation and the function of ROS/[??]OH in obesity is not known. In the present study, formation and function of ROS, including [??]OH, were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of [alpha]-tocopherol and ascorbic acid. Generation of either [??]OH, [??][O.sup.-.sub.2], and [H.sub.2][O.sub.2] was strongly inhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular [??]OH formation and ROS activity were reduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced [??]OH formation in lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-induced contractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs. untreated). Exogenous ROS, including [??]OH caused stronger vasodilation in obese animals (P < 0.05 vs. lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular [??]OH generation and provide evidence that ET-1 regulates vascular [??]OH formation. The data indicate that in obesity, vascular formation of ROS, including [??]OH is lower, whereas the sensitivity to ROS is increased, suggesting a novel and important role of ROS, including [??]OH in the regulation of vascular tone in disease status associated with increased body weight. oxidative stress; acetylcholine; vasoconstriction; murine; ob/ob; terephthalic acid; vitamin E; vitamin C

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. NixA was identified as a specific nickel uptake system in this organism. Addition of small amounts of nickel to media strongly stimulates urea hydrolysis. On the other hand, high nickel concentrations are deleterious to cell growth. As a possible protective reaction, nickel uptake seems to be reduced in H. pylori grown in nickel-rich media. These observations led to investigations of regulation of the expression of the nickel permease NixA. We found that increasing the nickel concentration in media reduced the amount of NixA. In order to address the question of whether this phenomenon was subject to transcriptional or translational regulation, we quantified nixA mRNA from H. pylori by real-time PCR. The amount of nixA mRNA was gradually reduced five- to sevenfold in a time- and concentration-dependent manner. Repression could be measured as soon as 5 min after nickel addition, and the maximum repression occurred after 20 to 30 min. The maximum repression was obtained with an external nickel concentration of 100 [micro]M. The observed nickel repression of NixA was dependent on nikR encoding the nickel-responsive regulatory protein NikR. In conclusion, we demonstrated that synthesis of the NixA nickel permease of H. pylori shows nickel-responsive regulation mediated by NikR to maintain the balance between effective nickel acquisition and a toxic overload.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Angiotensin II (Ang II), endothelin-1 (ET-1) and reactive oxygen species (ROS) have been implicated in the development of pathologic changes associated with obesity including hypertension and atherosclerosis. The aim of this study was to investigate the effects of dietary fat content on vasoreactivity and receptor expression at the level of gene and protein expression. Methods: C57BL/6 mice were fed diets of normal (Control, 12.3% kcal from fat), high (HF, 41% kcal from fat) and very high (VHF, 58% kcal from fat) fat content for 15 weeks. Glucose tolerance tests were performed, and aortic rings were exposed to ET-1 (0.01–300 nM) and Ang II (100 nM) in the presence of l-nitro-arginine-methyl ester (l-NAME; 300 μM). Gene and protein expressions of angiotensin and endothelin receptors were examined by real-time PCR and immunoblotting, respectively. The effects of diet on responses to acetylcholine (ACh 0.1–300 μM), in the absence or presence of l-NAME, and to exogenous ROS/lso investigated. Results: Both high fat diets similarly impaired glucose tolerance (P <0.05). Increasing dietary fat augmented contractions to Ang II in a step-wise manner (P <0.05). Conversely, increasing dietary fat had no effect on contractions to ET-1. Exposure to ROS/ed in a rapid vasodilation that was markedly augmented in a step-wise manner with increasing dietary fat (P <0.05). Endothelium-dependent relaxation to ACh was unaffected whereas vasoconstriction to high concentrations of ACh was enhanced in VHF animals (P <0.05 vs. control). Gene expression of the AT1B receptor was increased in the aorta of VHF mice, and aortic ETA receptor protein expression was increased after both high fat diets. Conclusions: These findings demonstrate that changes in dietary fat intake modulate vascular reactivity in response to Ang II and ROS, as well as expression of vascular angiotensin and endothelin receptors. Dietary fat intake may thereby directly affect cardiovascular risk. [Copyright &y& Elsevier]

Protein kinase Cμ (PKCμ) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino-terminal hydrophobic region. In order to analyse the potential role of PKCμ in signal-transduction pathways, stable PKCμ transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor-necrosis-factor (TNF)-induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF-κB-driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF-receptor-associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCμ transfectants. In support of a PKCμ action on NF-κB, we show enhancement and downregulation of TNF-induced expression of a NF-κB-dependent reporter gene by transient overexpression of wild-type and kinase-negative mutants of PKCμ, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCμ action downstream of IκB degradation, probably by modulation of the transactivation capacity of NF-κB. The dominant negative action of the kinase-negative mutant further suggest a regulatory role of PKCμ for NF-κB-dependent gene expression. [ABSTRACT FROM AUTHOR]

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17beta-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17Beta-estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17Beta-estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor beta was 10-fold higher than that of estrogen receptor alpha or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17beta-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17beta-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17beta-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.