Type II diabetes increases mitochondrial DNA mutations in the left ventricle of the Goto-Kakizaki diabetic rat.

Mitochondrial dysfunction has a significant role in the development of diabetic cardiomyopathy. Mitochondrial oxidant stress has been accepted as the singular cause of mitochondrial DNA (mtDNA) damage as an underlying cause of mitochondrial dysfunction. However, separate from a direct effect on mtDNA integrity, diabetic-induced increases in oxidant stress alter mitochondrial topoisomerase function to propagate mtDNA mutations as a contributor to mitochondrial dysfunction. Both glucose-challenged neonatal cardiomyocytes and the diabetic Goto-Kakizaki (GK) rat were studied. In both the GK left ventricle (LV) and in cardiomyocytes, chronically elevated glucose presentation induced a significant increase in mtDNA damage that was accompanied by decreased mitochondrial function. TTGE analysis revealed a number of base pair substitutions in the 3' end of COX3 from GK LV mtDNA that significantly altered the protein sequence. Mitochondrial topoisomerase DNA cleavage activity in isolated mitochondria was significantly increased in the GK LV compared with Wistar controls. Both hydroxycamptothecin, a topoisomerase type 1 inhibitor, and doxorubicin, a topoisomerase type 2 inhibitor, significantly exacerbated the DNA cleavage activity of isolated mitochondrial extracts indicating the presence of multiple functional topoisomerases in the mitochondria. Mitochondrial topoisomerase function was significantly altered in the presence of H2O2 suggesting that separate from a direct effect on mtDNA, oxidant stress mediated type II diabetes-induced alterations of mitochondrial topoisomerase function. These findings are significant in that the activation/inhibition state of the mitochondrial topoisomerases will have important consequences for mtDNA integrity and the well being of the diabetic myocardium.

Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats.

Hyperglycemia represents the main cause of complication of diabetes mellitus and oxidative stress, resulting from increased generation of reactive oxygen species (ROS), and plays a crucial role in their pathogenesis. Impairment of vascular responses in diabetic rats, as a result of an increase in superoxide (O2-), formation is a major complication in diabetes. Since heme oxygenase (HO) expression regulates the level of ROS by increasing antioxidant, such as glutathione and bilirubin, we investigated whether upregulation of HO-1 modulates the levels of iNOS and eNOS and altered vascular responses to phenylephrine (PE) and acetylcholine (Ach) in aorta and femoral arteries of diabetic (streptozotocin (STZ)-induced) rats. Our results showed that iNOS expression was increased, but HO activity was reduced, in diabetic compared to nondiabetic rats (p<0.05). Upregulation of HO-1 expression by cobalt protoporphyrin (CoPP), an inducer of HO-1 protein and activity, conferred an increase in eNOS and differentially decreased iNOS protein levels (p<0.05). Isolated aortic and femoral arteries obtained from diabetic rats exhibited contraction to PE and relaxation to Ach, which were markedly increased and decreased, respectively. However, HO-1 induction in diabetic rats normalized relaxation compared to controls. Therefore, overexpression of HO-1 may mediate an increase in eNOS and a decrease in iNOS, potentially contributing to restoration of vascular responses in diabetic rats.

Prostaglandins and nitric oxide mediate superoxide-induced myocardial contractile dysfunction in isolated rat hearts.

Oxygen-derived free radicals have been implicated in the pathogenesis of myocardial injury. We therefore investigated the pathophysiology of myocardial injury induced in isolated rat hearts by perfusion with superoxide radical generated by reacting 2.5 mmol/l purine, 0.03 U/ml xanthine oxidase and 300 U/ml catalase. Perfusion with superoxide significantly (P<0.05) increased left ventricular end-diastolic pressure within 15 to 20 min. During the same time period, heart rate and left-ventricular developed pressure significantly declined to 44.6+/-8.2% and 31.0+/-4.9% of control, respectively. Superoxide perfusion also significantly increased production of prostaglandins, nitric oxide (detected as nitrites) and peroxynitrite (detected immunohistochemically as nitrotyrosine). N(G)-nitro-l-arginine (100 micromol/l), a nitric oxide synthase inhibitor, attenuated superoxide-induced generation of peroxynitrite, increased synthesis of prostacyclin, and partially blocked myocardial dysfunction, as did 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (30 micromol/l), a selective inhibitor of soluble guanylate cyclase, and ONO-3708 (10 micromol/l), a selective thromboxane A(2)receptor antagonist. In contrast, nitroglycerin (4 micromol/l) and sodium nitroprusside (1 micromol/l) each exacerbated the superoxide-induced myocardial dysfunction. These results suggest that nitric oxide and related reactive species contribute to myocardial injury induced by superoxide. Moreover, they suggest that oxidative stress can be delayed or inhibited by reducing levels of nitric oxide, by inhibiting soluble guanylate cyclase, and by blocking thromboxane/prostaglandin receptors.

Activation of TxA2/PGH2 receptors and protein kinase C contribute to coronary dysfunction in superoxide treated rat hearts.

We have previously shown that superoxide anion (O2-) stimulates the release of vasoconstrictor prostanoids and induces a prolonged rise in coronary perfusion pressure (CPP) that persists even after removal of O2-. In this study, we tested the hypothesis that the increased CPP is mediated by activation of TxA2/ PGH2 (TP) receptors and protein kinase C (PKC)-dependent mechanisms. In Langendorff perfused rat hearts, O2- was applied for 15 min and then washed out over a period of 20 min. Application of O2- increased the release of vasoconstrictive (TxA2 and PGF2alpha) and decreased vasodilating (PGI2 and PGE2) prostanoids. Although indomethacin (10 microM), a cyclooxygenase inhibitor, attenuated the rise in CPP during O2- perfusion, the increase was not completely blocked. OKY 046Na (10 microM), a thromboxane synthase inhibitor, had no effect on O2--induced increases in CPP, whereas ONO 3708 (10 microM), a TP receptor antagonist, suppressed this effect. PKC activity was also elevated by more than 50% by O2- perfusion. CPP typically increased throughout the O2- wash-out. This post-O2- vasoconstriction was not inhibited by indomethacin, nitroglycerin or nitrendipine. In contrast, ONO 3708 (10 microM) and two PKC inhibitors, staurosporine (10 nM) and calphostin C (100 nM), completely blocked the rise in CPP, and even elicited vasodilation. PDBu enhanced the post-O2- vasoconstriction. We conclude that O2--induced coronary vasoconstriction is initially mediated by TP receptors, but activation of PKC sustains the response.

NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO.

The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from Fe(2+) to Fe(3+), which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 microM FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 microM S-nitroso-N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 microM NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 microM diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 microM 6-aminonicotinamide (6-AN) and 100 microM epiandrosterone (Epi)], or 1 microM DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited (P < 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 microM 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its Fe(3+)-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its Fe(2+) oxidation state.

A flavoprotein mechanism appears to prevent an oxygen-dependent inhibition of cGMP-associated nitric oxide-elicited relaxation of bovine coronary arteries.

The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 micromol/L DPI under an atmosphere of 21% O(2) (5% CO(2)). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N(2) (5% CO(2)). If BCAs are treated with DPI under 21% O(2) and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N(2), there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O(2) but not under N(2). Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 micromol/L DPI under 21% O(2), but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O(2)-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.

Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and H2O2.

The inhibitor of soluble guanylate cyclase (sGC) stimulation by nitric oxide (NO), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), was examined for its effects on the prolonged relaxation of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to peroxynitrite (ONOO-) and on H2O2-elicited relaxation and sGC stimulation. Our previous studies suggest that ONOO- causes a prolonged relaxation of BPA by regenerating NO and that a 2-min exposure of BCA or BPA to 50 nM NO causes an ONOO--elicited relaxation. The relaxation of K+-precontracted BCA to 50 nM NO or 100 microM ONOO- was essentially eliminated by 10 microM ODQ. ODQ also eliminated relaxation to 0.1 nM-10 microM of NO donor S-nitroso-N-acetyl-penicillamine (SNAP), but it did not alter relaxation to 1-300 microM H2O2. Similar responses were also observed in BPA. ODQ did not increase lucigenin-detectable superoxide production in BCA, and it did not alter luminol-detectable endogenous ONOO- formation observed during a 2-min exposure of BCA to 50 nM NO. In addition, ODQ did not affect tissue release of NO after 2 min exposure of BCA to 50 nM NO. The activity of sGC in BPA homogenate that is stimulated by endogenous H2O2 was not altered by ODQ, whereas sGC activity in the presence of 10 microM SNAP (+fungal catalase) was reduced by ODQ. Thus relaxation of K+-precontracted BCA and BPA to ONOO- appears to be completely mediated by NO stimulation of sGC, whereas the actions of ODQ suggest that NO is not involved in H2O2-elicited relaxation and sGC stimulation. This study did not detect evidence for the participation of additional mechanisms potentially activated by ONOO- in the responses studied.

Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD.

We have previously reported that inhibition of Cu/Zn superoxide dismutase (SOD) in endothelium-removed bovine pulmonary arteries (BPA) attenuates nitrovasodilator-elicited relaxation and that a NADH oxidase linked to the redox status of cytosolic NADH is the major detectable source of superoxide (O-2) production in this tissue. In the present study, we investigated whether NADH oxidase-derived O-2 participated in inhibition of nitrovasodilator-elicited relaxation and soluble guanylate cyclase (sGC) stimulation. Lactate (10 mM) and pyruvate (10 mM) were employed to increase and decrease, respectively, NADH-dependent O-2 production in the BPA presumably by modulating cytosolic NAD(H) through the lactate dehydrogenase reaction. A 30-min pretreatment with 10 mM diethyldithiocarbamate (DETCA) was used to inhibit Cu/Zn SOD, and S-nitroso-N-acetylpenicillamine (SNAP) was employed as a source of nitric oxide (NO). Lactate or pyruvate did not alter relaxation to NO. However, when the response to NO was inhibited by DETCA, lactate potentiated and pyruvate reduced the inhibitory effects of DETCA. SOD attenuated the inhibitory effects of DETCA plus lactate. In the presence of 10 microM SNAP, the activity of sGC in a BPA homogenate preparation (which was reconcentrated to approximate tissue conditions) was not altered by SOD. However, NADH (0.1 mM) decreased sGC activity by 70%, and this effect of NADH was attenuated in the presence of SOD. Thus cytosolic NADH redox and Cu/Zn SOD activity have important roles in controlling the inhibitory effects of O-2 derived from NADH oxidase on sGC activity and cGMP-mediated relaxation to nitrovasodilators in BPA.

Superoxide and nitroglycerin stimulate release of PGF2 alpha and TxA2 in isolated rat heart.

The aim of this study was to investigate the effects of nitroglycerin (NTG), a nitric oxide (NO) donor used as a vasodilating agent, on prostanoid [e.g., prostaglandin (PG)] release in the O2(-)-pretreated rat heart. Perfusion of O2-, generated by a xanthine oxidase-purine coupling, caused elevation (P < 0.05) of the coronary perfusion pressure (CPP) after 20 min (from 57.1 +/- 3.9 during the control period to 72.2 +/- 3.9 mmHg, P < 0.05). O2- caused increased release of PGF2 alpha from 3.6 +/- 0.7 to 20.6 +/- 4.4 pmol.min-1.g-1 and of thromboxane A2 (TxA2) from 2.4 +/- 0.4 to 9.6 +/- 1.6 pmol.min-1.g-1 (P < 0.001) with no significant changes in PGE2 and PGI2 release. During the 20-min washout of O2- from the heart with normal Krebs solution, release of PGF2 alpha and TxA2 decreased to 8.7 +/- 1.4 and 6.3 +/- 1.7 pmol.min-1.g-1, respectively, and the release of PGE2 and PGI2 markedly increased from 11.1 +/- 2.9 to 25.4 +/- 3.6 and 157.2 +/- 16.4 to 413.2 +/- 41.4 pmol.min-1.g-1, respectively (P < 0.05), without lowering the elevated CPP. Administration of 4 microM NTG during the washout period paradoxically augmented the elevated CPP to 133.3 +/- 0.6% and was associated with a doubling (P < 0.05) of PGF2 alpha and TxA2 release with no significant changes in PGE2 and PGI2 release. The NTG-induced CPP elevation was inhibited (P < 0.05) by indomethacin, a cyclooxygenase inhibitor, or ONO-3708, a TxA2 receptor blocker, whereas arachidonic acid, a substrate for PG synthesis, augmented the CPP elevation. These results indicate that NTG stimulates the synthesis of vasoconstrictive PG in the O2(-)-pretreated rat heart, inducing a paradoxical elevation in CPP.

Single-dose treatment of trichomonal vaginitis: a comparison of tinidazole and metronidazole.

A randomized, comparative trial was carried out in 100 patients with trichomonal vaginitis to compare the efficacy and toleration of a single 2 g dose of tinidazole and metronidazole. Tinidazole produced parasitological cure in 94% of patients (47/50) and a satisfactory clinical response in 96% of patients (48/50); for metronidazole the figures were 64% (32/50) and 72% (36/50) respectively. Side-effects were reported by 52% of patients (25/50) on tinidazole and 82% of patients (41/50) on metronidazole. Severity of side-effects and their frequency per patient were significantly less (p less than 0-045 to 0-01) with tinidazole than with metronidazole. Statistical evaluation of the results showed that tinidazole was significantly better than metronidzole with respect parasitological cure (p less 0-01) and clinical improvement (p less than 0-01) and also with regard to the incidence and intensity of side-effects (p less than 0-01).