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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 Ca2+ overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca2+ 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 Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential (ΔΨm) 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 μM) prevented ouabain-induced hypercontracture (from 40 ± 2 to 24 ± 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca2+]m overload (from 149 ± 6 to 121 ± 5% of the baseline value, P < 0.05) but did not affect ΔΨm. These results indicate that cariporide attenuates the [Ca2+]m overload without the accompanying depolarization of ΔΨm. 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 [Ca2+]m overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

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 Ca2+ transients were measured after exposing the cells to ouabain. STC1 alone did not affect cell shortening or the Ca2+ transient. Exposure to ouabain significantly increased the fraction of hypercontractured cells (40.5±1.4% vs 3.5±1.7% in the control, p

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.

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.

血中LDLコレステロール値測定にはFriedewaldの式による計算法に対し, 近年直接測定する方法 (直接法) が開発されたがその普及率はいまだ高くない。また, 健診前夜の夕食の種類による血清脂質濃度変化の詳細な検討はない。そこで直接法の有用性および前夜摂取した夕食による変動を検討した。健康な男女35人に全員同時に3回の試験を行った。第1回目の試験は午後7時に被検者全員が同一の普通食 (658 kcal) を摂取し以後は禁食とした。翌朝午前8時に採血を行い糖質と脂質濃度を測定した。LDL-コレステロール値は直接法と計算法とで求めた。1週後に第2回目として夕食に高エネルギー食 (普通食に比し糖質のみを増量したもの, 1, 011 kcal) を用い, 次の1週後に第3回目として夕食を絶食とし, 1回目と同様に8時に採血し糖質と脂質濃度を測定した。中性脂肪値, 総コレステロール値およびHDL-コレステロール値は3種の食餌問に有意差はみられなかったが, グルコース値は, 絶食 (84.4±8.6mg/dl) で普通食 (87.1±7.4mg/dl) , 高エネルギー食 (87.2±8.4mg/dl) に比べて有意な低下が見られた。LDL-コレステロール値 (直接法) は, 普通食に比べ高エネルギー食 (101.5±22.1, 104.3±24.1mg/dl) で高い傾向を示し, 絶食で有意な上昇がみられた (105.5±23.3mg/dl) 。しかし計算法値では, 絶食での有意な上昇は検出されず, 高エネルギー食 (99.7±24.7mg/dl) では逆に普通食 (105.1±23.9mg/dl) および絶食 (108.7±26.0mg/dl) よりも有意に低下する結果となった。また, 普通食および絶食では直接法値よりも計算法値が高くなる人が多かったのに対し, 高エネルギー食では逆に計算法値が低くなる人が多かった。以上より, 健診においてはLDL-コレステロール測定を直接法へ迅速に移行することが必要であり, 前日の夕食については標準的カロリーの食事を勧める記載が必要と考えられた。

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.

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.

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 ( p0.001). Nicorandil (100 microM) significantly attenuated the ouabain-induced mitochondrial Ca2+ overload (129+/-4% of baseline; p0.001 vs. ouabain). Nicorandil decreased the DeltaPsim during application of ouabain, thereby reducing the intensity of JC-1 fluorescence to 89+/-2% of baseline ( p0.05). 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%, p0.05 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.

The hypotheses that nitrotyrosine formation is a downstream mechanism following TNF-α production in lipopolysaccharide (LPS)-induced liver injury and its inhibition reduces the injury were examined. Under anesthesia, experiments were performed 6h after the intravenous administration of LPS (10mg/kg) or saline using male Sprague-Dawley rats (300-400g). Liver injury was evaluated in 4 groups: control (n=4), LPS alone (n=5), LPS+quercetin (n=5), LPS+clodronate (n=5) by plasma alanine aminotransferase (ALT) level, nitrotyrosine concentration in liver and histological changes. Quercetin, an inhibitor of nitrotyrosine formation was injected at a dose of 50mg/kg i.p. 24h before the LPS injection and clodronate, a Kupffer cell remover was injected i.v. at a dose of 0.2ml (0.23M) 24h before the LPS injection. Nitrotyrosine was measured by enzyme-linked immunosorbent assay and high performance liquid chromatography-electrochemical detection methods. In the LPS group, the liver contained 1, 403±240ng nitrotyrosine/g protein (control: 21.2±1.7ng/g) and plasma ALT was elevated (1, 781±455IU/liter vs. control 55±5IU/liter). Nitrotyrosine formation was decreased in the LPS+quercetin and LPS+clodronate groups (240.6±47.6 and 301.4±26.2ng/g protein, respectively) and the increase of plasma ALT was attenuated (486±20.7 and 79.8±9.4IU/liter, respectively). The plasma TNF-α level was markedly elevated in the LPS group (1, 124.1±67.6pg/ml). It remained high in quercetin groups (1, 292.4±159.9pg/ml) but was very low in the clodronate group (34.8±5.3pg/ml). LPS-induced liver injury involves nitrotyrosine formation and treatments to decrease its formation attenuated the injury. The protection was observed without affecting TNF-α level; suggesting that the nitrotyrosine formation is a downstream mechanism for the injury following TNF-α production.

We investigated the effectiveness of supportive therapy with a fish-oil extract called repair tuberculosis (RTB) in anti-tuberculosis treatment, and the underlying mechanism of action. The active component of RTB is the unsaturated fatty acid docosatetraenoic acid (C 22 H 36 O 2 ), which was reported to induce the resorption and healing of pulmonary lesions in patients with severe pulmonary tuberculosis. We administered RTB to a rat model of CFA-induced pulmonary tuberculous granuloma (RTB group), and compared the results with those in a control group, which did not receive RTB. Histological examination of the lungs showed a significantly smaller area of granuloma in the RTB group than in the control group. IFN-γ levels in bronchoalveolar lavage fluid (BALF) were higher in the RTB group than in the control group, suggesting that Th1-type immune reaction is activated in the RTB group. Moreover, significantly enhanced expression of inducible nitric oxide synthase mRNA in lung tissue was observed in the RTB group. Superoxide production by cells recovered from BALF was attenuated in the RTB group. There were no difference in IL-4 levels in BALF, or in expression of TNF-α mRNA in lung tissue between the RTB and control groups. The above results suggest that RTB activates Th1-type cellular immune reaction, promotes absorption of lesions, and inhibits the generation of cytotoxic substances.

Background— Heat-shock protein 70 (HSP70) plays a major role in the pathophysiology of inflammation, and the induction of HSP70 before the onset of inflammation can reduce organ damage through a self-protective system. Glutamine is known to be an inducer of HSP70, and its preoperative administration seems useful in attenuating cardiopulmonary bypass (CPB)–induced inflammatory response. Methods and Results— Adult male Sprague-Dawley rats (group G, received 100 mg/kg of glutamine via the right jugular vein 3 times per day for 1 week and just before the initiation of CPB; group C served as control) underwent CPB (60 minutes, 100 mL/kg per minute, 34°C) and were killed 3 hours after the termination of CPB. Group G showed significantly lower plasma concentrations of interleukin-6 and interleukin-8 after CPB termination. Myocardial and respiratory damages were significantly attenuated in group G, as evidenced by Langendorff perfusion, respiratory index, and neutrophil adherence. HSP70 expressions in the heart, lung, and liver were detected only in group G before CPB and were markedly stronger in group G 3 hours after CPB termination. Although plasma nitrate+nitrite concentrations were not significantly different between the groups, endothelial-constitutive nitric oxide synthase (NOS) activity was markedly preserved and inducible NOS activity was markedly attenuated in the tissues of group G. Conclusions— These results suggest that preoperative glutamine administration induces HSP70 expression before CPB and attenuates CPB-induced inflammation by regulating NOS activity, which may be a prospective management for conferring tolerance to CPB-induced inflammatory response through a self-protective mechanism.

Atrial fibrillation is often induced in patients with hyperthyroidism and may trigger heart failure. Its prevalence and outcome were examined to obtain up-to-date information. Persistent atrial fibrillation was observed in approximately 1.7% of new hyperthyroid patients. It occurs more frequently in males (2.86%) than in females (1.36%), even though the number of male hyperthyroid patients is only one fifth of female patients. The rate increased with age, being 8% in the patients older than 70 years old. The initial treatment is to control the heart rate with routine pharmacologic therapy and to start antithyroid therapy as quickly as possible. Attempted cardioversion should be deferred until approximately the fourth month of maintaining a euthyroid state, because more than 56% of atrial fibrillation spontaneously reverts to sinus rhythm when the thyroid hormone levels start to decline. Elective cardioversion for those whose atrial fibrillation persists is highly effective and sinus rhythm maintenance rates were 56.7% and 47.6% at the 10th and the 14th year, respectively, even though the duration of atrial fibrillation prior to cardioversion was extremely long (35.0 +/- 29.0 months).

Peroxynitrite is responsible for nitration in vivo, whereas myeloperoxidase can also catalyze protein nitration in the presence of high NO2− levels. Recent reports of myeloperoxidase-mediated enzyme inactivation or lipid peroxidation have suggested a role of myeloperoxidase in various pathological conditions. To clarify the role of myeloperoxidase in ischemic brain injury, the authors measured nitrotyrosine formation and infarct volume in myeloperoxidase-deficient or wild-type mice subjected to 2-hour focal cerebral ischemia-reperfusion. Twenty-four hours after reperfusion, infarct volume was significantly larger in myeloperoxidase-deficient mice than in wild-type mice (81 ± 20 mm3 vs. 52 ± 13 mm3, P < 0.01), and nitrotyrosine levels in the infarct region were higher in myeloperoxidase-deficient mice than in wild-type mice (13.4 ± 6.1 μg/mg vs. 9.8 ± 4.4 μg/mg, P = 0.13). Fourteen hours after reperfusion, the nitrotyrosine level was significantly higher in myeloperoxidase-deficient mice than in wild-type mice (3.3 ± 2.9 μg/mg vs. 1.4 ± 0.4 μg/mg, P < 0.05). The authors conclude that the absence of myeloperoxidase increases ischemic neuronal damage in vivo, and that the myeloperoxidase-mediated pathway is not responsible for the nitration reaction in cerebral ischemia-reperfusion.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Hemoperfusion with a column of polymyxin B immobilized on fibers (PMX) has been used to adsorb endotoxin in-patients with septic shock. PMX hemoperfusion (PMX-H) increases blood pressure (BP) too rapidly for the effect to be attributable to endotoxin removal. Since inducible NO synthase (iNOS) is known to be involved in the profound hypotension, we hypothesized that a decrease of tetrahydrobiopterin (BH(4)), an essential cofactor of iNOS, might account for the rapid effect of PMX-H on BP, if plasma BH(4) is increased concomitantly with NO in septic shock patients and if PMX can decrease BH(4). BH(4) was evaluated by measuring total biopterin, which can include derivatives of BH(4) by using high-performance liquid chromatography (HPLC). We confirmed that PMX fabric time dependently decreased total biopterin concentration in vitro. The plasma level of total biopterin in shock patients was indeed markedly elevated compared with that in volunteers (131.1+/-33.4 vs. 10.4+/-1.1 nM, n=5, P

To examine the effect of thyroid hormone-induced cardiac hypertrophy on PKC expression, changes in the expression of PKC isoforms were studied in hypertrophied cardiac ventricles induced by triiodothyronine (T3) injection in the rat. Injection with T3 for 8 days induced 49% increase in cardiac weight compared to controls. Immunoblot analysis of cardiac ventricular extracts showed the expression of PKC-delta, -epsilon, and -zeta in both control and T3-treated groups. The expression of PKC-epsilon decreased by 40% in hyperthyroid rat cardiac ventricles, while PKC-delta and -zeta expressions were barely affected. PKC-epsilon immunoreactivity decreased in both cytosol and membrane fractions. On the contrary, PKC-epsilon expression did not decrease in the extract of hypertrophied cardiac ventricles produced by aortic banding or aortocaval shunt. These results indicate that thyroid hormone down regulates PKC-epsilon expression in the hyperthyroid-mediated cardiac hypertrophy.

We examined whether transmyocardial revascularization (TMR) relieves myocardial ischemia by increasing regional perfusion via the transmural channels in acute canine experiments. Regional blood flow during transient coronary ligation (2 min) was compared before and 30 min after TMR, and at the third transient ischemia the mid-left ventricle (LV) was cut and immediately frozen along the short axis for the analysis of NADH fluorescence in the regions around the TMR channels. In low-resolution analysis (2–4 g tissue or 2–3 cm2 area), regional perfusion was not significantly altered after TMR, and NADH fluorescence was observed throughout the ischemic region without significant spatial variation. High-resolution analysis (2.8 mg, 1 mm × 1 mm) revealed that the flow after TMR was lower, and NADH fluorescence was higher in the regions close to the channels (1–2 mm) than in the regions 3–4 mm away from them. Creating TMR channels did not improve the regional perfusion and rather aggravated the local ischemia in the vicinity of the channels in the immediate phase.

We previously reported that MET-88, 3-(2,2,2-trimethylhydrazinium) propionate, improved left ventricular diastolic dysfunction induced by congestive heart failure (CHF) in rats. The present study was designed to investigate the mechanism by which MET-88 improved the cardiac relaxation impaired in CHF rats. The left coronary artery of the animals was ligated, and the rats were then orally administered vehicle (control), MET-88 at 50 or 100 mg/kg or captopril at 20 mg/kg for 20 days. Myocytes were isolated from the non-infarcted region in the left ventricle, and cell shortening and [Ca2+]i transients were measured with a video-edge detector and by fluorescence analysis, respectively. In CHF control rats, the diastolic phase of cell shortening was prolonged compared with that of the sham-operated (sham) rats. This prolongation was prevented by treatment with MET-88 at 100 mg/kg or captopril at 20 mg/kg. CHF control rats also showed an increase in the decay time of [Ca2+]i transients compared with sham rats. MET-88 at 100 mg/kg and captopril at 20 mg/kg attenuated the increase in decay time of [Ca2+]i transients. Ca2+ uptake activity of the sarcoplasmic reticulum (SR) isolated from the non-infarcted region in the left ventricle was measured, and Lineweaver-Burk plot analysis of the activity was performed. CHF control rats revealed a decrease in the Vmax for SR Ca2+ uptake activity without alteration in Kd. MET-88 at 100 mg/kg significantly prevented the decrease in Vmax, but had no effect on Kd. Also, treatment with MET-88 at 100 mg/kg improved myocardial high-energy phosphate levels impaired in CHF rats. These results suggest that one of the mechanisms by which MET-88 improved cardiac relaxation in CHF rats is based on the amelioration of [Ca2+]i transients through increase of SR Ca2+ uptake activity.

Background. Recent studies have shown that nitrio oxide interacts with superoxide to form peroxynitrite a potent oxidant that modifies cellular proteins pro ducing 3-L-nitrotyrosine (N-Tyr). This study was de signed to evaluate N-Tyr quantitatively with high-per formance liquid chromatography (HPLC) during cardiac allograft rejection. Methods. Rat transplanted hearts (allogeneic or syngeneic grafts) were examined with HPLC analysis, immunohistochemistry for N-Tyr, and histological studies on 0, 1, 3, and 7 days after transplantation. Results. No histological rejection was found in syngeneic grafts, or day 0 or 1 allografts. HPLC demonstrated that N-Tyr in allografts increased on day 1 and continued to increase through day 7, while N-Tyr was not detected in any syngeneic grafts. Immunostaining of the allografts did not show N-Tyr on day 1. Conclusion. These results demonstrate that N-Tyr shows a time-dependent accumulation in cardiac allografts during acute rejection. N-Tyr detection using HPLC may be an useful maker for early diagnosis of acute rejection before pathological rejection occurs.

Nitric oxide (NO) may either protect against or contribute to inflammatory lung injury. In this study we investigated whether inhalation of 20 ppm NO alters tyrosine nitration, and we assessed the degree of lung inflammation and edema in rats after lipopolysaccharide (LPS) instillation. The amount of nitrotyrosine relative to the total amount of tyrosine was measured in lung homogenates, and lung tissue sections were stained for nitrotyrosine and aminotyrosine (a reduced form of nitrotyrosine). Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, and myeloperoxidase activity was measured in lung homogenate. Lung edema and inflammatory cell accumulation in lung tissue were estimated by extravascular lung water weight (EVLW) and extravascular dry lung weight (EVDW), respectively. LPS instillation caused increases in nitrotyrosine concentration and immunohistochemical staining of nitrotyrosine and aminotyrosine in the lungs. LPS instillation increased the BALF leukocyte count, myeloperoxidase activity in lung tissue, and both EVLW and EVDW. Inhalational exposure to 20 ppm NO induced nitrotyrosine and aminotyrosine formation only in bronchial epithelial cell surface of the lungs not instilled with LPS. NO inhalation reduced the increases in nitrotyrosine and aminotyrosine in LPS-instilled lung tissue as well as the leukocyte count in BALF and myeloperoxidase activity in lung tissue, but it did not significantly change EVLW or EVDW. Leukocyte depletion in LPS-instilled rats reduced interstitial inflammatory cells, which were stained with nitrotyrosine and aminotyrosine, and attenuated the nitrotyrosine staining of alveolar capillaries. These results suggest that inhalation of 20 ppm NO reduces leukocyte accumulation in the lungs and inhibits tyrosine nitration caused by LPS instillation.

To clarify the pathophysiological cascade leading to lipopolysaccharide- (LPS) induced myocardial dysfunction, we measured sarcoplasmic reticulum (SR) function, expression of inducible nitric oxide synthase (iNOS), and left ventricular (LV) function in a rat whole heart model. The LV function was evaluated by peak LV pressure and SR function was evaluated by the mechanical restitution (MR) curve, a physiological parameter of SR function. The mechanical restitution curve was constructed by plotting extrasystolic potentiation of LV dP/dt during extrasystoles (100-700 ms) under fixed pacing. Functions were evaluated using the perfusion apparatus at 6 or 24 h after LPS administration. In the 6 h group, LV pressure was depressed to 62% of the control, the SR function was impaired, and iNOS protein was expressed. In the 24 h group LV pressure and SR function remained at the control levels, iNOS was not detected. In the 6 h group dexamethasone co-administration normalized the LPS effect and iNOS was not expressed. LPS-induced myocardial dysfunction appeared to be caused by impaired SR function and NO expression suggesting that NO may act as a trigger.

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 ± 1.4m −1 s−1 at 25 °C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and iron-sulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no “very rapid” spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes.

Haloperidol (HPL), a widely used antipsychotic drug, is known to induce serious ventricular arrhythmias. However, the mechanism underlying their induction is not clear. We therefore examined the effects of HPL on the intracellular Ca(2+) ([Ca(2+)](i)) transient and on cell motion in cultured cardiac myocytes, as well as the pathways involving the HPL-induced abnormality of Ca(2+) homeostasis. HPL prolonged the diastolic phase of the Ca(2+) transient, with a mid-diastolic re-elevation of [Ca(2+)](i). The re-elevation of [Ca(2+)](i) was shown to be provoked by Ca(2+) release from sarcoplasmic reticulum (SR), which can trigger delayed afterdepolarization, the major arrhythmogenic factor. The re-elevation of [Ca(2+)](i) coincided with cell re-contraction during diastole. The induction of this abnormality by HPL appears to be independent of the mechanisms of the antipsychotic action.

Myocardial stunning is characterized by the impairment of excitation-contraction coupling via a decrease in myofilament Ca2+ responsiveness, thought to be triggered by hydroxyl radicals (·OH) generated upon reperfusion. Since peroxynitrite is also expected to be produced during reperfusion, we examined whether it can induce a stunned myocardium-like impairment of cardiac myocytes. Its effect on cultured cardiac myocytes was compared with that of hydrogen peroxide (H2O2), ·OH source. Infusion of peroxynitrite (0.2 mM) induced a decrease in cell motion and a complete arrest in diastole at 2.9 ± 0.3 min, which coincided with an elevation in [Ca2+]i. Arrest induced by infusion of H2O2 (10 mM) was not associated with an increase in [Ca2+]i. The ATP content was unaffected by peroxynitrite (control, 34.3 ± 3.4: + peroxynitrite, 32.9 ± 3.5 nmol/mg protein) and the cells remained viable. Sulfhydryl (SH) content was decreased by peroxynitrite, but not by H2O2. The membrane fluidity (a measure of peroxidation of the membrane lipids) was not affected by peroxynitrite, but was decreased by H2O2. Onset time of arrest was unaffected by deferoxamine (0.2 mM), but was delayed by DTT (10 mM) (from 2.9 ± 0.3 to 19.2 ± 1.6 min). Nitrotyrosine content was unchanged by peroxynitrite, and its augmentation with Fe3+/EDTA (1 mM) was not associated with a shortened onset time of arrest. The function of the Na+/Ca2+ exchanger was impaired by peroxynitrite, but not by H2O2. Peroxynitrite and H2O2 each induce arrest, but only the former increases [Ca2+]i. One of the mechanisms of the increase in [Ca2+]i is Na+/Ca2+ exchanger dysfunction. The impairments were induced through SH oxidation by peroxynitrite, but through lipid peroxidation by H2O2. Myocardial stunning may be induced by both species in concert.

Adhesion molecules are key molecules for inflammatory cardiovascular diseases and are known to be up-regulated by inflammatory cytokines. However, the role of adhesion molecules in the cytokine-induced myocardial dysfunction in vivo remains unclear. This role was examined in our novel canine model, in which chronic treatment of the heart with IL-1 beta-bound microspheres (MS), but not control MS, causes sustained myocardial dysfunction in vivo. The expression of P-selectin (mRNA and immunoreactivity) was more prominent in the IL-1 beta group than in the control group (treated with control MS alone) after MS injection. The extent of neutrophil infiltration and myocardial myeloperoxidase (MPO) activity were significantly increased in the IL-1 beta group (P0.01). Pre-treatment with SLeX-OS (a novel oligosaccharide analog of sialyl LewisX) or PB1.3 (a monoclonal antibody to P-selectin) prevented the myocardial dysfunction and significantly suppressed the neutrophil infiltration and the increase in myocardial MPO activity induced by IL-1 beta (P0.01 each). These results indicate that adhesion molecules play an important role in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in dogs in vivo.

The induction and distribution of 3-l-nitrotyrosine (NO2-Tyr) were examined with HPLC and immunohistochemistry in rabbit spinal cords after 15 minutes of transient ischemia until 7 days of the reperfusion. After the 15-minute ischemia, there was a significant decrease of neurologic scores in the ischemic group compared with the sham-operated control group at 7 days of reperfusion ( P = 0.0017), and the majority of motor neurons was selectively lost at 7 days of reperfusion ( P = 0.0039). NO2-Tyr was transiently induced at 8 hours of reperfusion in the ventral part of the spinal cord (0.47% ± 0.86%, NO2-Tyr/total tyrosine; P = 0.0021), but was not induced at any time point of reperfusion in the dorsal part of the spinal cord. Strong immunoreactivity for NO2-Tyr was selectively induced in large pyramidal motor neurons at 8 hours of reperfusion and was still weakly present until 7 days of reperfusion. (There may be a difference in sensitivity between the two techniques.) These results suggested that protein tyrosine nitration by nitric oxide plays a role in the selective motor neuron cell damage after transient spinal cord ischemia.

Studies in vitro suggested that inflammatory cytokines could cause myocardial dysfunction. However, the detailed mechanism for the cytokine-induced myocardial dysfunction in vivo remains to be examined. We thus examined this point in our new canine model in vivo, in which microspheres with and without IL-1beta were injected into the left main coronary artery. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 wk. Immediately after the microsphere injection, LVEF decreased to approximately 30% in both groups. While LVEF rapidly normalized in 2 d in the control group, it was markedly impaired in the IL-1beta group even at day 7. Pretreatment with dexamethasone or with aminoguanidine, an inhibitor of inducible nitric oxide synthase, prevented the IL-1beta-induced myocardial dysfunction. Nitrotyrosine concentration, an in vivo marker of the peroxynitrite production by nitric oxide and superoxide anion, was significantly higher in the myocardium of the IL-1beta group than in that of the control group or the group cotreated with dexamethasone or aminoguanidine. There was an inverse linear relationship between myocardial nitrotyrosine concentrations and LVEF. These results indicate that IL-1beta induces sustained myocardial dysfunction in vivo and that nitric oxide produced by inducible nitric oxide synthase and the resultant formation of peroxynitrite are substantially involved in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in vivo.

In hemorrhagic shock (HS), nitric oxide synthase (NOS) inhibitor is known to increase blood pressure and prolong survival time. On the other hand, NOS inhibitor decreases coronary flow and worsens myocardial ischemia. Therefore, we hypothesized that the beneficial effect of NOS inhibitor is attributable to the increased coronary perfusion pressure and that it outcompetes the coronary vasodilating effects of nitric oxide. To investigate the direct effect of NOS inhibitor on the regulation of coronary circulation and the induction of myocardial ischemia in HS, we used a canine model at a constant aortic pressure of 40 mmHg with an aortic reservoir. In seven dogs, intravenous administration of Nomega-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) at 10 min after induction of HS increased both systemic and coronary vascular resistances and further increased the serum catecholamine concentration at 10 min after L-NAME. In another 14 dogs, the beating hearts were rapidly cross-sectioned (120 ms) and freeze clamped (-190 degrees C) by a specially developed sampling device after 10 min of HS. Transmurally distributed myocardial ischemia was visualized by the enhanced reduced nicotinamide adenine dinucleotide fluorescence, which was significantly increased with L-NAME (n=7). Chemical analysis revealed a decrease in the myocardial ATP concentration with L-NAME in the subendocardial ischemic region in HS. In conclusion, with the use of an aortic reservoir to keep the aortic pressure constant in HS, NOS blockade significantly worsened myocardial ischemia by decreasing coronary flow and augmenting the serum catecholamine concentration.

The purpose of this study is to determine if the polymorphonuclear leukocyte (PMN) is a major causative agent for lipopolysaccharide (LPS)-induced lung injury and responsible for the excess production of superoxide anion in the lung. We measured superoxide anion production from the lung and pulmonary capillary permeability in rats with and without PMN depletion. The superoxide anion production from the lung was measured using a purpose-built ex vivo chemiluminescence apparatus. Pulmonary capillary permeability was evaluated by the Evans blue dye extravasation method. PMN sequestration was determined by counting PMNs in histologic tissue specimens using microscopy. All rats received 3 mg/kg LPS intravenously. Examinations were undertaken at 2, 6, and 12 h after the LPS injection. The PMN-depleted group received cyclophosphamide 4 days before the LPS injection, which resulted in a PMN count of less than 200 cells/microliter. In rats without PMN depletion, Evans blue dye extravasation increased significantly at 12 h after the LPS injection; PMN sequestration increased at 2, 6, and 12 h after the LPS injection; and superoxide anion production increased at 6 h and remained elevated at 12 h after the LPS injection. The increased permeability, PMN sequestration, and superoxide anion production were not seen in the PMN-depleted group. The contribution of the xanthine/xanthine oxidase system and alveolar macrophages to the observed superoxide anion production was negligible. We conclude that, in rats, the PMN is a major causative agent in LPS-induced lung injury and is responsible for the excess production of superoxide anion in the lung.

ObjectiveIn the present study, we investigated the influence of dialysate glucose on superoxide (02) generation by peripheral and peritoneal phagocytes in continuous ambulatory peritoneal dialysis (CAPD) patients.DesignPeripheral polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL), and peritoneal cells were isolated from peripheral blood and peritoneal effluents, respectively, and their oxidative metabolism was assessed by measuring 02 generation after stimulation with a soluble stimulant [phorbol myristate acetate (PMA), 1 mg/mL, Sigma Chemical, St. Louis, MO, U.S.A.] using the chemiluminescence method. Dialysate glucose effect on 02 generation was also studied in vitro by exposing peripheral PMNL and MNL from healthy controls to peritoneal dialysis fluid (PDF) containing glucose or amino acids at a neutral pH for different time periods.ResultsThe amount of 02 generation by both peripheral and peritoneal phagocytes in CAPD patients was significantly higher than that in the control, and the response was greater in patients who were dialyzed with high glucose dialysate than those using low glucose dialysate. In an in vitro study, all incubated cells, except the control, showed suppression of 02 generation in the early dwell time (2 hr), and subsequently showed increased responses (peaking at 6 hr), although lower in degree than those observed in vivo. In contrast, amino acid-based PDF exhibited no such effect on 02 generation at identical pH with similar or lower osmolality. Furthermore, the respective increased or decreased oxidative responses with the increased or decreased PDF glucose concentrations in the same patient confirmed the positive effect of PDF glucose on phagocyte 02 generation.ConclusionIt is suggested that increased 02 generation by peritoneal and circulating phagocytes in CAPD patients is at least partly due to the enhancement of hexose monophosphate shunt activity by increasing glucose metabolism in phagocytes, and the increased 02 generation might be involved in long-term complications of CAPD. Therefore, a suitable alternative osmotic agent is needed to provide a more physiological environment to minimize the adverse effects of glucose on cell functions.

To characterize the mechanisms for myocardial ischemia induced by hemorrhagic shock, 29 dogs were subjected to hemorrhage at a mean aortic pressure (MAoP) of 30-60 mmHg. After 10 min of hemorrhage, the beating hearts were rapidly cross sectioned and freeze clamped to visualize the two-dimensional distribution of myocardial ischemia with NADH fluorescence (NADH-F) in 22 dogs. NADH-F was developed at an MAoP of 40 mmHg or less and involved both the subendocardial half and the subepicardial half of the left ventricle [34 +/- 14 vs. 20 +/- 14% (P < 0.05) and 65 +/- 16 vs. 52 +/- 15% (not significant) of the cross-sectional area of the left ventricular slice at MAoP levels of 40 and 30 mmHg, respectively]. Magnified NADH-F photography demonstrated heterogeneously distributed microischemic lesions with a columnar shape (mode of short-axis length, 60-80 microns). NADH-F-guided microsamplings revealed higher NADH and lactate concentrations in a positive NADH-F area than those in a negative NADH-F area. The ratio of endocardial to epicardial blood flow was maintained at a relatively high level (1.07 +/- 0.07 and 0.88 +/- 0.07 at MAoP levels of 40 and 30 mmHg, respectively; n = 7 dogs), and the reactive hyperemia was preserved as well. In conclusion, myocardial ischemia in early hemorrhagic shock was characterized by minimal transmural heterogeneity and marked heterogeneity between contiguous small regions.

Background Excess amounts of NO produced by an inducible NO synthase (iNOS) in response to cytokines may be cytotoxic and can be destructive to tissue. We investigated the role of NO in the development of myocardial damage and the effects of aminoguanidine (AG), an inhibitor of iNOS, on experimental autoimmune myocarditis in rats. Methods and Results Autoimmune myocarditis was induced in 20 Lewis rats by injection of porcine cardiac myosin. Ten of the 20 rats were administered AG. The severity of myocarditis was evaluated by measuring the size of myocarditic lesion and serum levels of CK-MB. Serum NO levels were determined using the Cd/Cu method. Tissue specimens were immunohistochemically examined for iNOS and nitrotyrosine. Histopathological study revealed extensive myocardial destruction and massive inflammatory cell infiltration in AG-untreated rats but only focal mononuclear cell infiltration in AG-treated rats. The mean percent areas of inflammatory lesions in the untreated and treated rats were 56±13% and 3±2%, respectively ( P P P Conclusions Excess amounts of NO produced by iNOS appear to contribute to the progression of myocardial damage in myocarditis. AG may prove to be useful in the treatment of myocarditis.

To evaluate the usefulness of a radiographic system with monochromatic synchrotron radiation to depict small vessels and peripheral secretory ducts.Radiography of various organs was tested in 14 anesthetized dogs and pancreatography was performed in an excised human pancreas by using the following system: monochromatic synchrotron radiation with an energy level just above the k absorption edge of iodine as an x-ray source and a high-definition TV system with a high-light-sensitivity image pick-up tube camera coupled with a fluorescent screen as a detector.This system allowed depiction of small vessels (diameter50-100 microns) of the heart (penetrating transmural artery), brain (perforating arteries that arise directly in the circle of Willis), and intestinal organs (vasa recta and their submucosal communications) and of small branches (down to the fifth order) of the pancreatic duct.The synchrotron radiation system may be useful for evaluating microcirculatory disorders and early-stage malignant tumors in various human organs.

Superoxide dismutase (SOD) in renal tissue biopsy specimens obtained from patients with immunoglobulin A nephropathy (13 cases) and non-immunoglobulin A mesangial proliferative glomerulonephritis (nine cases) was studied at the protein level by an enzyme-linked immunosorbent assay method and at the mRNA level by the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Total SOD activity in the tissue supernatant was measured by applying an electron paramagnetic resonance/spin trapping method. Normal renal tissues obtained from kidneys removed for malignancies (six cases) were included as healthy controls. The copper and zinc form of SOD (Cu,Zn-SOD) activity at both the protein and mRNA levels was lower in the moderately or severely damaged tissues compared with that in the normal or mildly damaged tissues. On the other hand, manganese SOD (Mn-SOD) values at either the protein level or the mRNA level did not differ significantly between control and patient samples. In the histochemical study using a polyclonal rabbit anti-Cu,Zn-SOD antibody, the staining intensity for Cu,Zn-SOD antigen was lower in the areas with advanced histologic damage than in the intact tissues. A follow-up study showed that renal function deterioration was proportionately slower in patients whose SOD activity was within the range of healthy tissue levels at the time of biopsy. Our data suggest that a lower level of SOD activity, whether as a cause or a consequence of the disease process, might induce a decrease in the scavenger reaction of superoxide (O2-) thus causing the tissue to become more vulnerable to oxidative stress.

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank's balanced salt solution (pH 7.4) with HPA (1 mM). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 microM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.

To investigate the genetic response to oxidative stress in eukaryotic cells, we have applied the method RT-PCR differential display to the small, free-living nematode Caenorhabditis elegans (C. elegans) which was cultured under atmospheric or high oxygen concentrations. As a result, a new gene was cloned whose expression increased at high oxygen concentration.

MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a newly developed antioxidant which has been shown to reduce brain edema in cerebral ischemia through inhibition of the lipoxygenase pathway of arachidonic acid. However, its effect on myocardial reperfusion injury after prolonged ischemia has not yet been demonstrated. We compared the mode of the effect of MIC-186 and recombinant human CuZn superoxide dismutase (rh-SOD) in isolated perfused rat hearts subjected to 60-min ischemia followed by 60-min reperfusion. Left ventricular developed pressure (LVDP), necrotic area and the release of creatine phosphokinase (CPK) and endogenous CuZn superoxide dismutase (endoge-SOD) were measured to evaluate myocardial damage. The decrease in left coronary flow (CBF) was measured as an index of the damage of left coronary circulation. MCI-186 (14.5 mg/L) was perfused for 10 min in the MCI group and rh-SOD (70 mg/L) was perfused during the reperfusion period in the SOD group starting 5 min prior to reperfusion. The release patterns of CPK and endoge-SOD were analyzed to elucidate the difference in the mode of protection of MCI-186 and rh-SOD. The LVDP remained higher in both MCI and SOD groups than that of control (76 +/- 1, 77 +/- 2 and 69 +/- 1% of preischemic value, respectively). The necrotic area was significantly attenuated in both MCI and SOD groups compared with that in the control group (16 +/- 1, 14 +/- 1 and 32 +/- 1%, respectively, p0.05). Total CPK release was lower in both MCI and SOD groups than in the control (78 +/- 7, 100 +/- 2 and 116 +/- 4 x 10(3) units/g myocardium respectively). The decrease in CPK release was more marked in the MCI group than that in the SOD group (p0.05). The reduction in CBF was significantly attenuated by the treatment with rh-SOD or MCI-186, but the effect was much higher in the SOD group than in the MCI group (69 +/- 5, 58 +/- 2, and 48 +/- 2% in SOD, MCI and control groups, respectively). The release pattern of endoge-SOD was identical to that of CPK and thus this did not distinguish the mode of effect of MCI-186 from that of rh-SOD. These results indicate that MCI-186 reduces reperfusion injury in isolated perfused hearts with prolonged ischemia and the effect is more closely related to the reduction of myocyte damage than the preservation of the coronary circulation.

The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.

We examined humoral and/or locally produced vasoactive factors involved in modulating sympathetic coronary vasoconstriction via the ATP-sensitive K (KATP) channel in 42 anesthetized dogs. Glibenclamide (30 micrograms.kg-1.min-1 ic or 0.6 mg.kg-1.min-1 left atrial injection) augmented coronary vascular resistance (CVR) at baseline and during cardiac sympathetic nerve stimulation (2-20 Hz), with a greater increase seen in the subepicardial region than in the subendocardial region both during beta-adrenergic receptor blockade and alpha- and beta-receptor blockade [P < 0.05 and P < 0.05 (n = 6 and 18 dogs), analysis of variance]. In contrast, pinacidil (10 micrograms.kg-1.min-1; n = 8 dogs) suppressed CVR. Glibenclamide enhanced CVR response to locally administered norepinephrine of 0.001–0.1 microgram.kg-1.min-1 (P < 0.05, analysis of covariance; n = 5 dogs) but did not enhance norepinephrine or neuropeptide Y overflow (n = 18 dogs). CVR was not modified by calcitonin gene-related peptide (CGRP) antagonist [CGRP-(8–37)], 8-phenyltheophylline, or N omega-nitro-L-arginine (n = 11 dogs). Thus sympathetic coronary vasoconstriction is modified by coronary vascular KATP channels with a transmural difference. However, CGRP, adenosine, and endothelial nitric oxide production are not involved in the modulation.

This study sought to show that unintentional preconditioning can be induced in the isolated perfused heart during the preparation procedure. The following four groups were compared: hearts were placed in ice cold saline and cooled for 15 s and then mounted to the Langendorff apparatus (n = 5; cool immediate group); hearts were cooled for 60 s and mounted (n = 5; cool delay group); hearts were mounted directly to the apparatus within 15 s after the isolation without cooling (n = 5; noncool immediate group); hearts were mounted without cooling, but the mounting was delayed for 60 s after the isolation (n = 5; noncool delay group). All hearts were paced at a fixed rate of 300 bpm, and an occlusion of left coronary (LCA) for 60 min was performed, which was followed by reperfusion for another 60 min. Coronary flow (CBF), left ventricular developed pressure (LVDP), and creatine phosphokinase (CPK) release did not change among the four groups during ischemia. At the end of reperfusion the LVDP values were 70 +/- 1%, 66 +/- 2%, 62 +/- 3%, and 73 +/- 2% of preischemic values in cool immediate, cool delay, noncool immediate, and noncool delay groups, respectively. CPK values were 116 +/- 4, 121 +/- 7, 138 +/- 6, and 29 +/- 1 x 10(3) U/g myocardium, and percentage necrosis/risk areas were 24 +/- 1.0%, 21 +/- 1.7%, 38 +/- 2.6%, and 13 +/- 0.5% in cool immediate, cool delay, noncool immediate, and noncool delay groups, respectively. The noncool delay group demonstrated high LVDP, least amount of CPK release, and smallest size of necrosis. These results indicate that an unintentional preconditioning effect can be induced when the cooling procedure is not applied and perfusion is delayed.

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.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.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.

An NO‐selective electrode was developed as an easily applicable tool for a real‐time nitric oxide (NO) measurement. The working electrode (0.2 mm diam) was made from Pt/Ir alloy coated with a three‐layered membrane. The counterelectrode was made from a carbon fiber. When a stable NO donor, S‐nitroso‐N‐acetyl‐dl‐penicillamine, was applied, the electrode current increased in a dose‐dependent fashion. The current and calculated NO concentration showed a linear relationship in the range from 0.2 nM (S/N=1) to 1 μM of NO. The response of the electrode was 1.14±0.09 s. The effects of temperature, pH, and chemicals other than NO on the electrode current were also evaluated. Electrodes which were placed in the luminal side of rat aortic rings exhibited 30 pA of current due to NO generation induced by the addition of 10−6 M of acetylcholine. The current was eliminated in the presence of 50 μM NG‐monomethyl‐L‐arginine, an inhibitor of NO synthase. Thus, this NO‐selective electrode is applicable to real‐time NO assay in biological systems.

Mutations in mev-1 of the nematode Caenorhabditis elegans render animals hypersensitive to high oxygen concentrations. They also reduce life span. To further understand the effects of mev-1 on aging, accumulation of fluorescent material resembling lipofuscin was measured by biochemical and histological analyses. Fluorescent material accumulated in both wild type and mev-1 animals with increasing age. The mev-1 mutant accumulated more fluorescent material at a greater rate than dose wild type. Furthermore, the accumulation rates depended on concentration of oxygen. Since this phenotype has been widely used as an aging marker, these results validate mev-1's use as a model to study aging.

Objective: The aim was to investigate whether nicorandil suppresses the rise in coronary vascular resistance that occurs during stimulation of the sympathetic nerve supply to the heart and, if so, what are the mechanisms of action. Methods: The effects of nicorandil on coronary vascular resistance during ventrolateral cardiac nerve stimulation and on the reactivity of the coronary vasculature to intracoronary infusion of noradrenaline or neuropeptide Y (NPY) were examined under β receptor blockade. The effects of nicorandil on the overflow of noradrenaline and NPY during ansae subclaviae stimulation were compared with those in a control group and in a group treated with glyceryl trinitrate and Nω-nitro-L-arginine (L-NNA) under both α and β receptor blockade with vagotomy. Results: Intracoronary infusion of nicorandil decreased coronary vascular resistance prior to cardiac nerve stimulation, and during stimulation it suppressed the percentage increase in resistance from the prestimulation value. Nicorandil suppressed the reactivity of the coronary vasculature to exogenous noradrenaline and NPY. Intra-atrial infusion of nicorandil significantly reduced the overflow of NPY but not of noradrenaline during stimulation of the ansae subclaviae at 20 Hz. This suppressive effect was not observed in the glyceryl trinitrate + L-NNA group. Conclusions: Nicorandil reduces sympathetic coronary vasoconstriction by decreasing the reactivity of the vasculature to sympathetic neurotransmitters and by suppressing NPY overflow during cardiac sympathetic nerve stimulation. The suppressive action on NPY overflow is thought to be due to the opening of ATP sensitive potassium channels in the sympathetic nerve endings rather than to a glyceryl trinitrate-like action of nicorandil. Cardiovascular Research 1994; 28 :917-922

FcαR expression on polymorphonuclear leukocyte and superoxide generation in IgA nephropathy. Superoxide (O 2 − ) production and FcαR antigen expression of circulating polymorphonuclear leukocytes (PMNL) isolated from patients with IgA nephropathy (IgAN) and non-IgA mesangial proliferative glomerulonephritis (PGN) and healthy volunteers were investigated to establish their biological importance in the immunopathogenesis of mesangial proliferative glomerulonephritis. PMNL from both patient groups showed increased O 2 − production when stimulated with N-formyl methionyl leucyl phenylalanine (FMLP) and phorbol myristate acetate (PMA). The increased O 2 − generation demonstrated a positive correlation with the degree of proteinuria. Aggregated IgA caused enhanced O 2 − production only in patients with IgAN who also showed a significant correlation with proteinuria. Increased expression of FcαR on circulating PMNL was observed in IgAN patients as determined by flow cytometric analysis. The amount of FcαR on PMNL was positively correlated with O 2 − generation triggered with IgA aggregates. These results suggest that (1.) circulating PMNL may potentially be participating in the pathogenesis of glomerular injury in mesangial proliferative glomerulonephritis, and (2.) IgA aggregates/immune complexes may contribute to the immunopathogenesis of IgAN through augmenting the Fcα receptor-mediated generation of superoxide anion.

Objective: The study was designed to demonstrate the time course of neutrophil chemotactic factor (NCF) release from blood-free isolated rat hearts and to clarify the characteristics of NCF in order to facilitate its identification. Methods: Coronary effluents were collected every minute from Langendorff perfused rat hearts during ligation of the left coronary artery for 40 min and reperfusion for 60 min. The neutrophil chemotactic activity in the effluents was assayed using modified Boyden's chambers with rat neutrophils isolated from peripheral blood as the indicator cells. Results: The NCF release started at 10 min of coronary artery occlusion. During the reperfusion period, NCF release peaked at 5 min (230% of preischaemic value). To clarify the characteristics of NCF, the changes in chemotactic activity were examined using various inhibitors and inactivators of possible NCF candidates (LTB4, PAF, 5-lipoxygenase, and thromboxane synthase). The heat stability of NCF was also examined to exclude heat labile molecules such as adenosine or complements appearing as NCF. Among the various substances examined, only PAF antagonists (CV-6209 and TCV-309 at concentrations of 10−6 M and 10−6 M respectively) abolished the chemotactic activity. However direct measurement of PAF in the effluents was unsuccessful. Conclusion: NCF is released from the heart early after ischaemic insult, with the highest peak occurring at 5 min of reperfusion. PAF related substances might be the primary NCF in the effluent but this remains to be determined. Cardiovascular Research 1993; 27 :2194-2199

To determine the underlying mechanisms of the positive inotropic effect of sub-skinning concentrations of saponin, we studied changes in the intracellular Ca2+ ([Ca2+]i) and plasma membrane fluidity after exposure to digitonin (a representative saponin) in cultured cardiac cells. [Ca2+]i was measured by use of the fluorescent calcium indicator Calcium Green-1. The membrane fluidity was evaluated by measuring the diffusion coefficient using the method of fluorescence recovery after photobleaching. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate was used as the fluorescent probe. Digitonin at a sub-skinning concentration (0.1 to 1 microM) produced an increase in cell motion and an augmentation of [Ca2+]i. Membrane fluidity which is evaluated by the diffusion coefficient (from 0.34.10(-8) to 0.28.10(-8) cm2/s; P0.05), decreased in the presence of 0.2 microM digitonin while the cell maintained an augmented motion and an increased [Ca2+]i. The skinning concentration of digitonin (5 microM) produced a rapid contracture with a marked increase in [Ca2+]i. The membrane fluidity was further reduced (diffusion coefficient: 0.24.10(-8) cm2/s; P0.001). These results suggest that saponin at the sub-skinning concentration also causes holes in the plasma membrane by interaction with cholesterol, as was shown with the skinning concentration, and it increases [Ca2+]i, which thereby induces a positive inotropic effect.

The role of reperfusion injury in the progression to necrosis in pulmonary embolism was evaluated. To simulate this condition, we used a technique that enables occlusion and reopening of the pulmonary arterial branch supplying the right upper lobe in conscious rats. The rats were divided into five groups: the occlusion group (n = 12), in which the pulmonary artery (PA) branch was occluded without reperfusion; the reperfusion group (n = 12), in which the PA branch was reopened after a 2-h period of occlusion; and the reperfusion-SOD (n = 9), reperfusion-IM (n = 8), or reperfusion-IA-SOD (n = 6) groups, in which superoxide dismutase (SOD), indomethacin (IM), or inactivated SOD (IA-SOD), respectively, was administered during reperfusion. The lungs were removed 24 h after the PA occlusion, and histologic examination was performed. In the occlusion group, the alveolar structure of the right upper lobe was well preserved, and there was no erythrocyte or leukocyte accumulation. The only significant changes compared with the control lobe was the appearance of wavy internal elastic lamina of the PA and slight neutrophil adherence to the endothelial cells. In contrast, the right upper lobe of the reperfusion group disclosed numerous foci of hemorrhagic necrosis, with disrupted alveoli and leukocyte accumulation in all cases. With SOD treatment, the changes compatible with hemorrhagic necrosis were attenuated to the level of the control lobes. However, neither IM nor IA-SOD decreased these changes.(ABSTRACT TRUNCATED AT 250 WORDS)