Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH.

Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion (O(2)(-))] and the mechanisms underlying O(2)(-) production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O(2)(-) and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O(2)(-) generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9-11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P < 0.05). O(2)(-) levels were elevated (70-90%; P < 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91(ds-tat) (50 microM) or by the mitochondrial respiratory chain inhibitors antimycin (10 microM) and rotenone (50 microM). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 microM) also reduced (P < 0.05) O(2)(-) production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P < 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O(2)(-) generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O(2)(-) production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed.

Characterization of beta3-adrenoceptors in human internal mammary artery and putative involvement in coronary artery bypass management.

OBJECTIVES: The aim of the present study was to analyze whether beta3-adrenoceptors (beta3-ARs) were effectively present and functional in the human internal mammary artery (IMA). BACKGROUND: The beta1- and beta2-adrenoceptors classically mediate the relaxant effects of catecholamines in the vessels. In vitro and in vivo studies performed in various animal species described vasodilating effects due to activation of a third beta-ARs subtype (beta3). METHODS: Reverse transcription-polymerase chain reaction analysis, Western blot experiments, and pharmacological studies were carried out in human IMA samples harvested from 27 patients undergoing coronary bypass surgery. RESULTS: The beta3-ARs messenger ribonucleic acid and protein were detected in intact IMA, but were absent in endothelium-free samples. This finding was confirmed by immunohistochemical experiments. In organ baths, a beta3-AR agonist, SR 58611A, induced an endothelium-dependent relaxation of phenylephrine-precontracted IMA rings. This vasodilation was not modified by beta1/beta2-AR antagonists, but was greatly altered in the presence of L-748,337, a selective human beta3-AR antagonist. Moreover, the inhibition of nitric oxide (NO) synthases abolished the beta3-adrenergic vasodilation, suggesting the involvement of a NO-signaling pathway. CONCLUSIONS: Those results demonstrated the presence of beta3-ARs in the endothelial layer of human IMA. The present work highlights the role of beta3-ARs in vasomotor control of IMA and opens new fields of investigation in coronary bypass graft management, heart failure, and hypertension.

Impairment of the low-affinity state beta1-adrenoceptor-induced relaxation in spontaneously hypertensive rats.

1 In hypertension, a decrease of the vascular beta-adrenergic relaxation has been described. However, the specific involvement of each beta-adrenoceptor (beta-AR) subtype, in particular the low-affinity state of beta1-AR, has not yet been evaluated. We investigated whether the low-affinity state of beta1-AR-induced relaxation was impaired in Spontaneously Hypertensive Rats (SHR). 2 The relaxant responses to CGP 12177 and cyanopindolol, low-affinity state beta1-AR agonists (with beta1-/beta2-AR antagonistic and partial beta3-AR agonistic properties) were evaluated on thoracic aortic rings isolated from 12-weeks-old Wistar Kyoto rats (WKY) and SHR. 3 In WKY, CGP 12177 and cyanopindolol produced an endothelium and nitric oxide (NO)-independent relaxation. CGP 12177-induced endothelium-independent relaxation was not modified either by beta1-, beta2-AR (nadolol) or beta3-AR (L-748337 or SR 59230A) antagonists but was significantly reduced by high concentrations of CGP 20712A (P<0.05). This relaxation was also reduced by adenylyl cyclase inhibitors, SQ 22536 or MDL 12330A. 4 In SHR, CGP 12177 produced mainly an endothelium and NO-dependent relaxation. This effect was not modified by nadolol, but was strongly reduced by beta3-AR blockade. Endothelium-independent relaxation to CGP 12177 was not altered by adenylyl cyclase inhibition, but was amplified in preparations from pertussis toxin-pretreated SHR. 5 The immunohistochemical analysis revealed an upregulation of beta3-AR in the endothelial layer of SHR aorta, whereas the beta3-AR-induced relaxation was not modified. 6 In conclusion, we demonstrated an impaired low-affinity state of the beta1-AR-induced relaxation and an upregulation of the beta3-AR in hypertension. Some clinical implications of those findings are discussed.

Beta 3-adrenoceptor in rat aorta: molecular and biochemical characterization and signalling pathway.

1. We have previously demonstrated that beta(3)-adrenoceptor (beta(3)-AR) stimulation induces endothelium-dependent vasorelaxation in rat aorta through the activation of an endothelial NO synthase associated with an increase in intracellular cGMP. The aim of the present study was to localise beta(3)-AR to confirm our functional study and to complete the signalling pathway of beta(3)-AR in rat aorta. 2. By RT-PCR, we have detected beta(3)-AR transcripts both in aorta and in freshly isolated endothelial cells. The absence of markers for adipsin or hormone-sensitive lipase in endothelial cells excluded the presence of beta(3)-AR from adipocytes. The localization of beta(3)-AR in aortic endothelial cells was confirmed by immunohistochemistry using a rat beta(3)-AR antibody. 3. To identify the G protein linked to beta(3)-AR, experiments were performed in rat pre-treated with PTX (10 microg kg(-1)), a G(i/0) protein inhibitor. The blockage of G(i/0) protein by PTX was confirmed by the reduction of vasorelaxation induced by UK 14304, a selective alpha(2)-AR agonist. The cumulative concentration-response curve for SR 58611A, a beta(3)-AR agonist, was not significantly modified on aorta rings from PTX pre-treated rats. 4. At the same level of contraction, the relaxations induced by 10 microM SR 58611A were significantly reduced in 30 mM-KCl pre-constricted rings (E(max)=16.7+/-8.4%, n=5), in comparison to phenylephrine (0.3 microM) pre-constricted rings (E(max)=49.11+/-11.0%, n=5, P<0.05). In addition, iberotoxin (0.1 microM), glibenclamide (1 microM) and 4-aminopyridine (1 mM), selective potassium channels blockers of K(Ca), K(ATP), and K(v) respectively, decreased the SR 58611A-mediated relaxation. 5. We conclude that beta(3)-AR is preferentially expressed in rat aortic endothelial cells. Beta(3)-AR-mediated aortic relaxation is independent of G(i/0) proteins stimulation, but results from the activation of several potassium channels, K(Ca), K(ATP), and K(v).

Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity.

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.