Citations

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50of 32 μM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.

The purpose of this study was to test the hypothesis that the efficacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 h of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 min before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Confirmation of the effects of these serum glucose manipulations on brain intracellular pH (pHi) was confirmed in a group of acute experiments utilizing umbelliferone fluorescence. The animals were euthanized at 72 h for histology. 7-NI significantly ( P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach significance because moderate hypoglycemia in itself dramatically reduced infarction volume. We hypothesize that a mechanism to explain the published discrepancies on the effects of neuronal NOS inhibitors in vivo may be due to the effects by differences in ischemic brain acidosis on the production of NO.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37°C) to an adenoviral vector [107, 108, or 109plaque-forming units (pfu)/ml] encoding either the iNOS gene or the β-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (109pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10−9–10−3M) and diethylaminodiazen-1-ium-1,2-dioate (10−10–10−5M) were significantly shifted to the right in iNOS gene (109pfu/ml)-transduced rings compared with control and β-galactosidase-transduced rings ( P < 0.05, n = 5–6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings ( P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O[Formula: see text]·) production were elevated in iNOS-transduced rings ( P < 0.05, n = 7 for cGMP; P < 0.01, n = 6–9 for O[Formula: see text]· production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

Although hydrogen peroxide (H2O2) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding β-galactosidase (Adβgal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by β-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adβgal-transduced cells (536.8 ± 31 vs. 1,875.1 ± 132.9 vs. 1,347.5 ± 93.7 dpm/well, respectively; P < 0.05 vs. control and Adβgal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 ± 1.8% of cell counts in control, P < 0.05; and 45 ± 2% of cell count in Adβgal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H2O2(50 μM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H2O2is an important modulator of endothelial cell proliferation.

Our experiments were designed to determine the acute effects of 17β-estradiol on femoral veins from intact and ovariectomized female pigs. Rings of femoral veins with or without endothelium were suspended in organ chambers for measurement of isometric force. Concentration-response curves to 17β-estradiol (10−9to 10−5M) were obtained in veins contracted with prostaglandin F2αin the absence and presence of inhibitors of either estrogen receptors (ICI-182780; 10−5M), nitric oxide synthase [ NG-monomethyl-l-arginine (l-NMMA); 10−4M], soluble guanylate cyclase (1- H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10−5M), or potassium channels (tetraethylammonium; 10−2M). Estrogen receptors were identified with the use of Western blotting and immunostaining in veins of both groups. 17β-Estradiol caused acute endothelium-dependent relaxations in both groups. Relaxations to 17β-estradiol were inhibited byl-NMMA and 1- H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but not ICI-182780. Tetraethylammonium inhibited relaxations only in veins with endothelium from intact females. Results indicate that 17β-estradiol causes acute endothelium-dependent relaxations in femoral veins. The relative contribution of nitric oxide and K+channels as mechanisms involved in relaxations to 17β-estradiol in femoral veins is modulated by ovarian status.

We previously reported that α2-adrenergic receptor (α2-AR) stimulation in Purkinje fibers in vitro prolongs action potential duration and suppresses β-adrenergic-induced delayed afterdepolarizations and sustained triggered activities. We examined the effects of α2-AR stimulation on reperfusion-induced ventricular arrhythmias [ventricular tachycardia/ventricular fibrillation (VT/VF)] in vivo. Arterial blood pressure, heart rate, surface electrocardiogram, and renal sympathetic nerve activities were recorded simultaneously in Sprague-Dawley rats. The incidence of VT/VF was 87.5% for controls, 50% for the β-blocker group, 72% for the α1-blocker group, and 12.5% for the α1 + β-blockers group (unopposed α2-adrenergic activation). Direct α2-AR stimulation with UK-14304 also prevented VT/VF. These effects were reversed by the α2-adrenergic antagonist yohimbine. Increases in renal sympathetic nerve activity were associated with left anterior descending coronary artery ligation and reperfusion (33 ± 1.5 and 62 ± 1.7% over baseline, respectively) in controls. Similar patterns were observed among all experimental groups irrespective of the incidence of VT/VF on reperfusion. We conclude that α2-AR stimulation has a potent antiarrhythmic effect on ischemia-reperfusion-induced VT/VF in vivo and that this effect is not centrally mediated.

Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGFABand PDGFBB), transforming growth factors (TGF-β1and TGF-β2), and vascular endothelial growth factor (VEGF165) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGFBB, but not PDGFAB, TGF-β1, and TGF-β2, increased with ovariectomy. VEGF165was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGFBBin platelets. Higher levels of PDGFBBin platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy.

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-Piand creatine phosphate-to-Piratios. Postischemic AK1 knockout hearts maintained ∼40% of β-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ERα and ERβ), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ERβ and ERα receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.

K+ channel openers have been recently recognized for their ability to protect mitochondria from anoxic injury. Yet the mechanism responsible for mitochondrial preservation under oxidative stress is not fully understood. Here, mitochondria were isolated from rat hearts and subjected to 20-min anoxia, followed by reoxygenation. At reoxygenation, increased generation of reactive oxygen species (ROS) was associated with reduced ADP-stimulated oxygen consumption, blunted ATP production, and disrupted mitochondrial structural integrity coupled with cytochrome c release. The prototype K+ channel opener diazoxide markedly reduced mitochondrial ROS production at reoxygenation with a half-maximal effect of 29 μM. Diazoxide also preserved oxidative phosphorylation and mitochondrial membrane integrity, as indicated by electron microscopy and reduced cytochrome c release. The protective effect of diazoxide was reproduced by the structurally distinct K+ channel opener nicorandil and antagonized by 5-hydroxydecanoic acid, a short-chain fatty acid derivative and presumed blocker of mitochondrial ATP-sensitive K+ channels. Opener-mediated mitochondrial protection was simulated by the free radical scavenger system composed of superoxide dismutase and catalase. However, the effect of openers on ROS production was maintained in nominally K+-free medium in the presence or absence of the K+ ionophore valinomycin and was mimicked by malonate, a modulator of the mitochondrial redox state. This suggests the existence of a K+ conductance-independent pathway for mitochondrial protection targeted by K+ channel openers. Thus the cardioprotecive mechanism of K+ channel openers includes direct attenuation of mitochondrial oxidant stress at reoxygenation.