Pharmacological conversion of atrial fibrillation in the patients of Graves' disease.

BACKGROUND: Hyperthyroidism is one of the common causes of atrial fibrillation (AF), and AF is associated with increased morbidity and mortality due to thromboembolism. The sinus rhythm maintenance rate of hyperthyroidism-induced AF patients after conversion to sinus rhythm is excellent. The present study was undertaken to assess the efficacy and safety of bepridil, a multichannel blocker, in patients with hyperthyroidism-induced persistent AF. METHODS AND RESULTS: Sixty-two patients with hyperthyroidism-induced persistent AF were treated with bepridil. Oral bepridil therapy resulted in conversion to sinus rhythm in 32 (51.6%) of the 62 patients. There were no significant differences in clinical characteristics between the responders and non-responders. At the observation period of an average of 23.9 months, the sinus rhythm maintenance rate was found to be 81.3%. Adverse effects consisted of abnormal QTc prolongation in 3 patients and sinus bradycardia in 10 patients. There was one death in which a causal association with bepridil could not be ruled out. CONCLUSIONS: Bepridil is as beneficial treatment to convert AF for the patients with hyperthyroidism-induced persistent AF as it is for the patients with AF due to other causes. However, bepridil should be used with caution to avoid serious side effects.

Systematic evaluation of nitric oxide, tetrahydrobiopterin, and anandamide levels in a porcine model of endotoxemia.

PURPOSE: Using a lipopolysaccharide (LPS)-treated porcine model, we examined: (1) whether nitric oxide (NO), anandamide, and tetrahydrobiopterin (BH4) increased or not in early endotoxic shock; and (2) the location of the major site of production of these molecules, by comparing their concentrations in arteries and the portal and hepatic veins. METHODS: Ten pigs received an infusion of LPS at 1.7 microg x kg(-1)x h(-1) via the portal vein for 240 min. Consecutive changes in systemic hemodynamics, hepatosplanchnic circulation, and oxygen delivery were measured. Furthermore, the variable changes in the concentrations of nitrite and nitrate (NOx), anandamide, and BH4 were measured. To access the effects of surgery, anesthesia, and fluid management on BH4, an experiment without LPS infusion was performed in two other animals. RESULTS: Mean arterial pressure and cardiac index started to decrease at 60 min after LPS infusion. However, systemic vascular resistance remained unchanged. Total hepatic blood flow and hepatic oxygen delivery also decreased significantly. NOx and anandamide did not change during LPS infusion. BH4 values did not change without LPS infusion. However, BH4 values increased significantly in the arterial, portal, and hepatic circulation during LPS infusion, especially in the hepatic vein (from 136.8 +/- 27.5 to 281.3 +/- 123.2 mol/ml; P < 0.01). CONCLUSION: Our data suggest that the BH4 values were significantly increased in several organs, especially in the liver during endotoxic shock. Impaired cardiac output and decreased blood pressure appeared in the early phase of porcine endotoxemia. Longer-term observation of these parameters after LPS treatment should be performed as the next step in future studies.

Validation of the Friedewald Equation for Evaluation of Plasma LDL-Cholesterol.

In most clinical laboratories, low density lipoprotein (LDL) cholesterol is usually estimated indirectly with the Friedewald equation or directly with the N-geneous assay. We assessed LDL-cholesterol values obtained by both methods to find an appropriate fasting period and to assess the influence of the energy content of the last meal. Blood samples were taken from 28 healthy volunteers who had consumed a standard meal (107 g of carbohydrate, 658 kcal) followed by a fasting period of 12 and 18 h, or a high-energy meal (190 g of carbohydrate, 1011 kcal) with a fasting period of 12 h. Prolongation of the fasting period from 12 h to 18 h decreased glucose level, but did not decrease triacylglycerol, total cholesterol, or high density lipoprotein (HDL) cholesterol. LDL-cholesterol levels measured with the N-geneous assay did not change (94.0 +/- 21.5 to 96.3 +/- 19.1 mg/dl). LDL-cholesterol levels calculated with the Friedewald equation were also similar after fasting periods of 12 h (98.5 +/- 21.4 mg/dl) and 18 h (99.7 +/- 20.2 mg/dl). The high-energy meal did not change the level of LDL-cholesterol measured with the N-geneous assay (96.1 +/- 21.2 mg/dl), or the glucose, triacylglycerol, total cholesterol, or HDL-cholesterol level, but LDL-cholesterol levels evaluated from the Friedewald equation (92.6 +/- 20.3 mg/dl) became significantly lower. A fasting time longer than 12 h is not necessary to obtain reasonable blood lipid levels. The Friedewald equation gave higher LDL-cholesterol levels than N-geneous assay in young Japanese females who had eaten a low-energy meal, and lower values when they had eaten a high-energy meal. Thus, it may be necessary to pay attention to energy of nigh meal prior to blood withdrawal.

Adenosine triphosphate-sensitive potassium channels prevent extension of myocardial ischemia to subepicardium during hemorrhagic shock.

Cardiac dysfunction during hemorrhagic shock (HS) is associated with myocardial ischemia, during which adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels can be activated. We investigated the role of K(ATP) channels in HS-induced myocardial ischemia. Canine HS was induced using an aortic reservoir to maintain the aortic pressure at a constant 40 mmHg. To visualize the myocardial ischemia as a nicotinamide adenine dinucleotide (NADH) - fluorescent area, the beating hearts were rapidly cross-sectioned (120 ms) and freeze-clamped (-190 degrees C) using a sampling device after 10 min of HS. The effect of a K(ATP) channel blocker, glibenclamide (1 mg/kg, i.v.), on myocardial ischemia was also quantified. Regional myocardial blood flow was measured using heavy element-loaded nonradioactive microspheres. Myocardial ischemia developed in the subendocardium in the HS alone group, whereas it extended through all the cardiac layers in the glibenclamide-treatment group. The coadministration of a K(ATP) channel opener, cromakalim (50 microg/kg, i.v.), with glibenclamide prevented the extension of myocardial ischemia to the subepicardium. Glibenclamide decreased the myocardial ATP concentration selectively in the subepicardium during HS. The HS decreased myocardial blood flow transmurally, and following the administration of glibenclamide, further decreased the blood flow selectively in the subepicardium. These results suggest that K(ATP) channels are activated during HS, enabling selective subepicardial coronary dilatation and protecting the myocardium from the extension of myocardial ischemia to the subepicardium.

Activation of inducible nitric oxide synthase increases MMP-2 and MMP-9 levels in ApoE-knockout mice.

OBJECTIVES: Atheroma with reduced collagen content becomes fragile, but the underlying mechanisms have not been established. We investigated the influence of inducible nitric oxide synthase (iNOS) induction upon matrix metalloproteinases (MMP)s and collagen content in atheroma. METHODS AND RESULTS: ApoE-/- x iNOS-/- double knockout and ApoE-/- x iNOS+/+ mice were fed a high-cholesterol diet for 15 weeks. Large atheromatous lesions of comparable size appeared in the roof of the aorta in both strains. Induction of iNOS mRNA was observed only in the atheroma of the ApoE-/-/iNOS+/+ mice. Collagen content was sparse and fat droplets were increased. Gelatin zymography and in situ zymography showed that pro- and active forms of MMP-2 and MMP-9 were more strongly expressed in ApoE-/-/iNOS+/+ than in ApoE-/-/iNOS-/- mice, nitrotyrosine and MMP-9 were co-expressed in the atheroma. CONCLUSION: We conclude that induction of iNOS in atheroma of high-cholesterol-fed ApoE-/-/iNOS+/+ mice leads to increased production and activation of MMPs, with a subsequent decrease in collagen content, affording fragile plaque.

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening.

The Na(+)/H(+) exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca(2+) overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca(2+) overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and the mitochondrial membrane potential (DeltaPsi(m)) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 microM) prevented ouabain-induced hypercontracture (from 40 +/- 2 to 24 +/- 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca(2+)](m) overload (from 149 +/- 6 to 121 +/- 5% of the baseline value, P < 0.05) but did not affect DeltaPsi(m). These results indicate that cariporide attenuates the [Ca(2+)](m) overload without the accompanying depolarization of DeltaPsi(m). Moreover, cariporide increased the time taken to induce the MPT (from 79 +/- 11 to 137 +/- 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 +/- 3 to 50 +/- 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca(2+)](m) overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

Stanniocalcin 1 prevents cytosolic Ca2+ overload and cell hypercontracture in cardiomyocytes.

BACKGROUND: The aim of the present study was to examine whether stanniocalcin 1 (STC1) affects cardiomyocytes under physiological or pathophysiological conditions. METHODS AND RESULTS: Using fresh isolated rat cardiomyocytes, the effects of STC1 on cell hypercontracture, cell shortening and Ca(2+) transients were measured after exposing the cells to ouabain. STC1 alone did not affect cell shortening or the Ca(2+) transient. Exposure to ouabain significantly increased the fraction of hypercontractured cells (40.5+/-1.4% vs 3.5+/-1.7% in the control, p<0.01). However, treatment with STC1 decreased the percentage of cell hypercontracture that was induced by ouabain, in a concentration-dependent manner (17.4+/-2.6% at 2.5 nmol/L STC1, p<0.01). Moreover, STC1 prevented the increase in diastolic intracellular Ca(2+) level that was induced by ouabain (-5.3+/-2.7% vs 7.9+/-3.7% induced by ouabain, p<0.05; -15.3+/-5.1% in the control) in the cardiomyocytes. CONCLUSIONS: STC1 prevented the increase in diastolic Ca(2+) overload and ouabain-induced cell hypercontracture, which suggests that STC1 could effectively prevent cytosolic Ca(2+) overload and protect cardiomyocytes from pathophysiological conditions such as in the failing heart.

Kurozu moromimatsu inhibits tumor growth of Lovo cells in a mouse model in vivo.

OBJECTIVE: In Japan, rice vinegar that has been matured and fermented for years in earthenware jars is considered a health food with anticolon cancer action. It is divided into the liquid component (Kurozu) and the sediment (Kurozu moromimatsu), which contains large amounts of organic materials and minerals. The effect of Kurozu moromimatsu (Kurozu-M) on cancer has not yet been examined. In this study, we examined the activity of Kurozu-M on colon cancer and investigated the mechanisms involved, focusing on active oxygen generation, apoptosis, and metalloproteinases (MMPs). METHODS: We used Lovo cells transplanted into nude mice as an experimental model. We measured the tumor volume and MMP levels and conducted hematoxylin-eosin staining (for polymorphonuclear leukocytes), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining (for apoptosis), and immunostaining for nitrotyrosine (a marker of active oxygen generation) in control, Kurozu-treated, and Kurozu-M--treated groups. RESULTS: The tumor volume was the same in the control group (231 +/- 36 mm(3)) and Kurozu group (238 +/- 52 mm(3)), but was significantly reduced in the Kurozu-M group (152 +/- 28 mm(3), P < 0.001 versus control). Apoptosis of tumor cells and accumulation of polymorphonuclear leukocytes were not observed. Nitrotyrosine production, total MMP levels, and MMP activation were significantly reduced in the Kurozu-M group. CONCLUSION: The administration of Kurozu-M prolonged the lifespan of cancer cell-transplanted mice, inhibited tumor progression, and reduced nitrotyrosine production and MMP activation, but did not induce apoptosis.

Intravenous injection of phagocytes transfected ex vivo with FGF4 DNA/biodegradable gelatin complex promotes angiogenesis in a rat myocardial ischemia/reperfusion injury model.

Conventional gene therapies still present difficulties due to poor tissue-targeting, invasiveness of delivery, method, or the use of viral vectors. To establish the feasibility of using non-virally ex vivo transfected phagocytes to promote angiogenesis in ischemic myocardium, gene-transfection into isolated phagocytes was performed by culture with positively charged gelatin impregnated with plasmid DNA. A high rate of gene transfection was achieved in rat macrophages and human monocytes, but not in mouse fibroblasts. The efficiency was 68 +/- 11% in rat macrophages and 78 +/- 8% in human monocytes. Intravenously injected phagocytes accumulated predominantly in ischemic tissue (13 +/- 8%) and spleen (84 +/- 6%), but negligibly in other organs in rodents. The efficiency of accumulation in the target ischemic tissue reached more than 86% on direct local tissue injection. In a rat model of myocardial ischemia-reperfusion, intravenous injection of fibroblast growth factor 4 (FGF4)-gene-transfected macrophages significantly increased regional blood flow in the ischemic myocardium (78 +/- 7.1 % in terms of flow ratio of ischemic/non-ischemic myocardium) compared with intravenous administration of saline (36 +/- 11%) or nontransfected macrophages (42 +/- 12 %), or intramuscular administration of naked DNA encoding FGF4 (75 +/- 18 %). Enhanced angiogenesis in the ischemic tissue we confirmed histologically. Similarly, intravenous injection of FGF4-gene-transfected monocytes enhanced regional blood flow in an ischemic hindlimb model in mice (93 +/- 22 %), being superior to the three other treatments described above (38 +/- 12, 39 +/- 15, and 55 +/- 12%, respectively). Phagocytes transfected ex vivo with FGF4 DNA/gelatin promoted angiogenesis. This approach might have potential for non-viral angiogenic gene therapy.

Treatment of natto, a fermented soybean preparation, to prevent excessive plasma vitamin K concentrations in patients taking warfarin.

The purpose of this study is to find a method of cooking natto that prevents the appearance of high-plasma vitamin K concentrations after the consumption of natto, so that patients taking warfarin can benefit from eating natto. Five cooking methods were examined to determine which could most effectively decrease the count of the living Bacillus subtilis in natto. Volunteers ate natto or treated natto, and their plasma vitamin K level was measured at 5, 8, 24 and 48 h thereafter. One gram of natto contained 9.7+/-0.1 Log cfu/mL of Bacillus subtilis. Boiling significantly reduced the Bacillus subtilis count to 5.1+/-0.3 Log cfu/mL, and concomitantly reduced the content of menaquinone-7 (MK-7), which is a form of vitamin K synthesized by Bacillus subtilis, from 660.40+/-65.32 ng/mL to 78.50+/- 11.12 ng/mL. Untreated natto increased the MK-7 concentration in blood from 1.86+/-1.51 ng/mL to 14.54+/-4.12 ng/mL at 5 h after intake, and the MK-7 concentration remained elevated at 8, 24 and 48 h (7.29+/-2.20, 6.97+/-2.60, and 5.37+/-1.94 ng/mL, respectively). In contrast, boiled natto increased plasma MK-7 only mildly (from 1.61+/-1.11 to 4.02+/-0.82 ng/ mL at 5 h) and the concentration remained relatively stable up to 48 h (3.46+/-0.83, 4.22+/-1.51 and 2.77+/-0.75 ng/mL at 8, 24 and 48 h, respectively). In conclusion, boiled natto did not cause a marked increase in the plasma concentration of vitamin K in subjects who consumed it. Thus, patients on warfarin may be able to eat boiled natto without ill effects.

Triiodothyronine acutely increases blood flow in the ventricles and kidneys of anesthesized rabbits.

Thyroid hormone (triiodothyronine [T(3)]) has various nongenomic effects, including alterations in glucose and fatty acid metabolism, augmentation of intracellular Ca(2+), enhancement of myocardial contractility, and vascular dilatation. However, its effect on regional blood flow remains to be established. We have measured the effect of T(3) on blood flow in major organs of anesthetized rabbits in vivo using the microsphere method. Under artificial respiration, nonradioactive microspheres (5 x 10(5)) labeled with barium were injected to measure blood flow at control level. Then, T(3) (50 microg/kg per milliliter) was administered and microspheres labeled with iodine (5 x 10(5)) were injected. The atria, ventricles, kidneys, and right upper limb were excised and their contents of microspheres were evaluated. Blood flow in the ventricles was significantly increased by T(3) (2.9 +/- 0.3 versus 3.4 +/- 0.3 mL/min per gram, vehicle versus T(3)). Similarly, blood flow in the kidneys was significantly higher after T(3) injection (4.3 +/- 0.5 versus 5.1 +/- 0.5 mL/min per, vehicle versus T(3)). The blood flow in the atria and skeletal muscles remained unchanged. These results indicate that the vasodilatory response to T(3) is not uniform and occurs preferentially in major organs such as cardiac ventricles and kidneys; this may be relevant to the T(3)-induced improvement of cardiac function.

Flow-independent myocardial ischemia induced by endothelin-1: an NADH fluorescence analysis.

The endothelin-1 (ET-1) is known to cause myocardial ischemia; however, whether this effect is entirely dependent on vasoconstriction is uncertain. The aim of this study was to characterize the myocardial ischemia after the intracoronary administration of endothelin-1, and compare it with that induced by coronary stenosis. In the left anterior descending coronary artery of 15 dogs, a mild inflow reduction (30%) was produced for 10 minutes using intracoronary endothelin-1 (46 +/- 33 pmol/min) or coronary stenosis. The hearts were rapidly cross-sectioned at short axial plane and freeze-clamped within 120 milliseconds using a specially developed device to visualize and quantify the area of ischemia (%IA) with NADH fluorescence photography. The %IA was larger in the endothelin-1 group than in the stenosis group (66 +/- 23 versus 18 +/- 18, P = 0.0005); furthermore, the ischemia was transmural in the ET-1 group, but limited to subendocardium in the stenosis group. ET-1 increased the coronary arterial resistance especially in subepicardial region and produced smaller ischemic foci in microcirculation. The mechanism of larger ischemia produced by ET-1 might depend on pro-ischemic effects on myocytes and vasoconstriction of the coronary microcirculation, predominantly in the subepicardium in vivo.

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria.

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.

Difference in propagation of Ca2+ release in atrial and ventricular myocytes.

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.

Deficiency of inducible nitric oxide synthase exacerbates hepatic fibrosis in mice fed high-fat diet.

The role of inducible nitric oxide synthase (iNOS) in the progression of fibrosis during nonalcoholic steatohepatitis remains to be elucidated. This study examined the role of iNOS in the progression of fibrosis during steatohepatitis by comparing iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice that were fed a high-fat diet. Severe fatty metamorphosis developed in the liver of iNOS(+/+) and iNOS(-/-) mice. Fibrotic changes were marked in iNOS(-/-) mice. Gelatin zymography showed that pro MMP-2 and pro MMP-9 protein expressions were more highly induced in iNOS(+/+) mice than in iNOS(-/-) mice. Active forms of MMP-2 and MMP-9 were clearly present only in the liver tissue of iNOS(+/+) mice. In situ zymography showed strong gelatinolytic activities in the liver tissue of iNOS(+/+) mice, but only spotty activity in iNOS(-/-)mice. iNOS may attenuate the progression of liver fibrosis in steatohepatitis, in part by inducing MMP-2 and MMP-9 expression and augmenting their activity.

Direct nitric oxide release from nipradilol in human coronary arterial smooth muscle cells observed with fluorescent NO probe and NO-electrode.

BACKGROUND:: Nipradilol (3,4-dihydro-8-[2-hydroxy-3-isopropyl-amino]propoxy-3-nitroxy-2-H-1-benzopyran), a potent non-selective beta-adrenoceptor antagonist, has been shown to increase NO production. The mechanisms are up-regulation of nitric oxide synthase (NOS) and direct release of NO from nipradilol. The process of direct NO release from nipradilol requires a reductase, such as glutathione S-transferase (GST) in some cells but non-enzymatic NO release was reported in pig coronary arteries. Direct NO release from nipradilol in human coronary arteries has not been examined yet, though this information is of importance. PURPOSE:: To demonstrate direct NO release from nipradilol in human coronary arterial smooth muscle cells (HCASMC) by using a fluorescent NO probe (DAF-2) and an NO-electrode. METHODS AND RESULTS:: HCASMC were loaded with DAF-2 and images of fluorescence (515nm) were obtained under excitation at 488nm through an intensified CCD with an inverted phase-contrast microscope. Concomitantly, NO was measured using an NO-electrode (0.2mm o.d.; 501, Inter Medical Co. Ltd., Nagoya, Japan) after addition of various concentrations of nipradilol (1, 5 or 10microM) with or without ethacrynic acid (GST inhibitor). The cells showed no fluorescence at baseline, but intense fluorescence appeared at 30min after addition of 10microM nipradilol. The intensities of fluorescence at 30min in the control, nipradilol and nipradilol with ethacrynic acid groups were 98 +/- 6, 163 +/- 10 and 128 +/- 6% of the baseline level, respectively. Ethacrynic acid itself did not affect the fluorescence. Continuous measurements of NO by the electrode showed the NO generation peaked at about 30min, remained at the same level till about 45min and then gradually declined. Nipradilol did not produce NO at all in the absence of cells. The dose-dependency study of NO release from nipradilol showed 45 +/- 12, 72 +/- 24 and 157 +/- 23nM, respectively, at 1, 5 and 10microM nipradilol. All experiments were performed under conditions where endogenous formation of NO was inhibited by an NOS inhibitor (10(-4)M N(G)-monomethyl-l-arginine (l-NMMA)). CONCLUSION:: Nipradilol can release NO in the presence of human coronary arterial smooth muscle cells and the denitration reaction catalyzed by a reductase such as glutathione S-transferase contributes substantially to NO release from nipradilol.

Direct inhibition by a statin of TNFalpha-induced leukocyte recruitment in rat pial venules - in vivo confocal microscopic study.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to block leukocyte-endothelial interaction independently of their cholesterol-lowering properties. The effects of statins are generally attributed to a decrease in mevalonate caused by inhibition of HMG-CoA reductase, which results in an increase of nitric oxide (NO). However, a recent in vitro study demonstrated a novel effect which depended on the lipophilicity of statin and appeared to be unrelated to HMG-CoA reductase inhibition. The purpose of this study is to investigate whether the proposed mechanism actually operates in vivo. We examined the effects of simvastatin (lipophilic) and pravastatin (hydrophilic) on leukocyte behavior in a tumor necrosis factor alpha (TNFalpha)-induced leukocyte recruitment model. Leukocyte adhesion and rolling were examined in pial venules of rat brain by using confocal laser scanning microscopy after labeling leukocytes with rhodamine 6G. Experiments were conducted 4h after TNFalpha injection (0.5microg) in six groups: control, TNFalpha alone, TNFalpha + vehicle of simvastatin, TNFalpha + simvastatin (20mg/kg, 2ml/kg), TNFalpha + vehicle of pravastatin, and TNFalpha + pravastatin (40mg/kg, 2ml/kg). Statins and vehicles were injected subcutaneously for 3 days. TNFalpha caused a marked increase in rolling and adhered leukocytes. The number of adhered leukocytes in the simvastatin group was significantly less than in the vehicle group (276 +/- 38 cells/mm(2) versus 1155 +/- 89 cells/mm(2), P < 0.01), whereas pravastatin had little effect. Both simvastatin and pravastatin showed a tendency to decrease the number of rolling leukocytes, but there were no significant differences among TNFalpha-treated groups. Up-regulation of endothelial nitric oxide synthase (eNOS) mRNA or increased expression of P-selectin or intercellular adhesion molecule-1 (ICAM-1) was not observed, and therefore cannot account for the simvastatin-induced reduction of adhered leukocytes. Markedly different effect on leukocyte adhesion between simvastatin and pravastatin under comparable level of HMG-CoA reductase inhibitor was demonstrated in in vivo as was shown in in vitro study.

Peroxynitrite, a product between nitric oxide and superoxide anion, plays a cytotoxic role in the development of post-bypass systemic inflammatory response.

OBJECTIVE: Cardiopulmonary bypass (CPB) is known to induce post-bypass systemic inflammatory response. Peroxynitrite (ONOO-) is a potent oxidant formed by a rapid reaction between nitric oxide (NO) and superoxide anion. We hypothesized that ONOO- plays a role in the development of post-bypass systemic inflammatory response and examined the efficacy of ONOO- scavenger in a rat-CPB model. METHODS: Adult Sprague-Dawley rats underwent 60 min of CPB (100 ml/kg per min, 34 degrees C). Group-P (n = 10) received 50 mg/kg of ONOO- scavenger, quercetin, intraperitoneally 24 h before the initiation of CPB, and Group-C (n = 10) served as controls. RESULTS: There were significant time-dependent changes in plasma nitrate+nitrite (NOx), the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr: an indicator of ONOO- formation), interleukin (IL)-6, IL-8, and respiratory index (RI). There were significant differences in %NO2-Tyr between the groups both at CPB termination (Group-P vs C; 0.26+/-0.07 vs 0.55+/-0.11%, P < 0.01) and 3 h after CPB termination (0.65+/-0.14 vs 1.46+/-0.25%, P < 0.01); whereas there were no significant differences in NOx between the groups at any sampling point ((at CPB termination) Group-P vs C; 31.6+/-4.3 vs 32.7+/-4.1 micromol/l, (3 h after CPB termination) Group-P vs C; 47.8+/-4.9 vs 51.7+/-5.3 micromol/l). Group-P showed significantly lower plasma IL-6 (176.8+/-44.3 vs 302.4+/-78.1 pg/ml, P < 0.01), IL-8 (9.45+/-1.78 vs 16.42+/-2.53 ng/ml, P < 0.01) and RI (1.07+/-0.19 vs 1.54+/-0.25, P < 0.01) 3 h after CPB termination, though there were no significant differences between the groups at CPB termination. CONCLUSIONS: These results suggest that ONOO- plays a crucial role in the development of post-bypass systemic inflammatory response and the pretreatment with quercetin has a potential benefit to avoid deleterious effects of ONOO-.

Nicorandil attenuates the mitochondrial Ca2+ overload with accompanying depolarization of the mitochondrial membrane in the heart.

The anti-anginal drug nicorandil has been demonstrated to protect the myocardium against ischemic injury in both experimental and clinical studies. Although nicorandil seems to protect the myocardium via activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels, the mechanisms underlying its cardioprotection have remained elusive. We therefore examined whether nicorandil depolarizes the mitochondrial membrane and attenuates the mitochondrial Ca2+ overload. With the use of a Nipkow confocal system, the mitochondrial Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential (DeltaPsim) in rat ventricular myocytes were measured by loading cells with rhod-2 and JC-1 respectively. The number of cell hypercontractures resulting from mitochondrial Ca2+ overload was counted. Exposing cells to ouabain (1 mM) evoked mitochondrial Ca2+ overload and increased the intensity of rhod-2 fluorescence to 180+/-15% of baseline ( p<0.001). Nicorandil (100 microM) significantly attenuated the ouabain-induced mitochondrial Ca2+ overload (129+/-4% of baseline; p<0.001 vs. ouabain). Nicorandil decreased the DeltaPsim during application of ouabain, thereby reducing the intensity of JC-1 fluorescence to 89+/-2% of baseline ( p<0.05). Exposure of myocytes to ouabain eventually resulted in cell hypercontracture (51+/-2%). This ouabain-induced cell hypercontracture was blunted by application of nicorandil (37+/-2%, p<0.05 vs. ouabain). Moreover, these effects of nicorandil were abolished by 5-hydroxydecanoate (500 microM), a putative mitoKATP channel blocker, and by glibenclamide (10 microM), a nonselective KATP channel blocker. Our results suggest that nicorandil attenuates the matrix Ca2+ overload with accompanying depolarization of the mitochondrial membrane. Such effect might potentially be attributed to the mechanism of cardioprotection afforded by nicorandil.

Leukocyte-depleted terminal blood cardioplegia provides superior myocardial protective effects in association with myocardium-derived nitric oxide and peroxynitrite production for patients undergoing prolonged aortic crossclamping for more than 120 minutes.

OBJECTIVES: This study was designed to examine the myocardial protective effect of leukocyte-depleted terminal blood cardioplegia in association with nitric oxide and peroxynitrite production, especially for patients undergoing prolonged aortic crossclamping. METHODS: Fifty-four patients (34 men, 20 women, mean age 56.7 +/- 12.7 years) undergoing aortic valve replacement were randomly allocated to one of two groups; group LDTC (n = 27) received 10 minutes of leukocyte-depleted terminal blood cardioplegic solution, and group CONT (n = 27) served as controls. Each group was subdivided into 2 groups: aortic crossclamping for less than 120 minutes in groups LDTC-S (n = 13) and CONT-S (n = 14); aortic crossclamping for 120 minutes or more in groups LDTC-L (n = 14) and CONT-L (n = 13). RESULTS: After aortic unclamping, group LDTC-L showed higher incidence of spontaneous defibrillation (78.6% vs 30.8%, P =.0213), higher plasma nitrate + nitrite in the coronary sinus effluent (32.5 +/- 4.1 vs 28.7 +/- 3.0 micromol/L, P =.0013), lower differences between coronary sinus effluent and arterial blood in the percentage ratio of nitrotyrosine to tyrosine (myocardium-derived peroxynitrite; 2.987% +/- 0.576% vs 3.951% +/- 0.952%, P =.0036), and plasma polymorphonuclear-elastase (113.9 +/- 21.3 vs 155.5 +/- 41.6 microg/L, P =.0029) and malondialdehyde (2.75 +/- 0.67 vs 4.02 +/- 0.96 micromol/L, P =.0005) than group CONT did. Postoperatively, group LDTC-L showed lower human-heart fatty acid-binding protein (111.4 +/- 25.2 vs 156.4 +/- 38.6 IU/L, P =.0013), lower creatine kinase-muscle and brain (19.2 +/- 4.7 vs 24.8 +/- 6.5 IU/L, P =.0120), and smaller requirement of catecholamine (5.44 +/- 2.29 vs 8.45 +/- 3.42 microg x kg(-1) x min(-1), P =.0122). There were no significant differences in these parameters between groups LDTC-S and CONT-S. CONCLUSIONS: This study demonstrated that leukocyte-depleted terminal blood cardioplegia provided superior myocardial protective effects and regulated myocardial-derived nitric oxide and peroxynitrite production only for patients undergoing aortic crossclamping for more than 120 minutes. The results suggest that prolonged aortic crossclamping deteriorates the tolerance to leukocyte-mediated myocardial injury accompanied by endothelial dysfunction associated with nitric oxide and peroxynitrite production.