Mao-Hsiung Yen

Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age‐matched Wistar‐Kyoto rats (WKY). We subsequently hypothesized that the over‐expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg−1 day−1, p.o., antioxidant and nuclear factor‐κ B inhibitor) and aminoguanidine (15 mg kg−1 day−1, p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti‐oxidant and/or nuclear factor‐κ B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Reduction in Lipopolysaccharide-Induced Thrombocytopenia by Triflavin in a Rat Model of Septicemia

BACKGROUND Thrombocytopenia frequently occurs early in the course of Gram-negative bacterial infections. Triflavin, an Arg-Gly-Asp-containing disintegrin, has been suggested to interfere with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. The present study was undertaken to determine whether triflavin could prevent thrombocytopenia in lipopolysaccharide (LPS)-treated rats. METHODS AND RESULTS In this study, 51Cr-labeled platelets were used to assess blood and tissue platelet accumulation after LPS challenge. The administration of LPS (4 mg/kg IV bolus) for 4 hours induced a reduction in radiolabeled platelets in blood and an obvious accumulation of platelets in liver. Triflavin (500 microg/kg) but not GRGDS (20 mg/kg) significantly prevented the alteration of radiolabeled platelet distribution in blood and liver when induced by LPS. Furthermore, triflavin but not GRGDS markedly suppressed the elevation in plasma thromboxane B2 concentration within the 4-hour period of LPS administration. In LPS-treated rats, the 5-hydroxytryptamine level was lower in the blood and higher in the liver compared with levels in normal saline-treated rats. Pretreatment with triflavin (500 microg/kg) significantly reversed the 5-hydroxytryptamine concentration in blood and liver of LPS-treated rats. In histological examinations and platelet adhesion assay, triflavin markedly inhibited the adhesion of platelets to subendothelial matrixes in vivo and in vitro. CONCLUSIONS The results indicate that triflavin effectively prevents thrombocytopenia, possibly through the following 2 mechanisms: (1) Triflavin markedly inhibits platelet aggregation, resulting in decreased thromboxane A2 formation. (2) It inhibits the adhesion of platelets to subendothelial matrixes, thereby leading to a reversal in the distribution of platelets in blood and liver in LPS-treated rats.

Abnormal activation of K+ channels in aortic smooth muscle of rats with endotoxic shock: electrophysiological and functional evidence

This study examined the role of K+ channels in vascular hyporeactivity of rats with endotoxic shock ex vivo. At the end of the in vivo experiments, thoracic aortas were removed from endotoxaemic and control rats. After removal of the endothelium, aortic segments were mounted in myographs for recording of isometric tension and smooth muscle membrane potential. Membrane potentials recorded from endotoxaemic rats were hyperpolarized compared to those of the controls. This hyperpolarization was partially reversed by tetraethylammonium, charybdotoxin or glibenclamide, but not significantly affected by apamin. The hyperpolarization was also partially attenuated by Nω‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐l‐one (ODQ). In phenylephrine‐contracted aortic rings, both agonists of K+ channels, NS1619 and pinacidil, induced greater relaxations and re‐polarizations in the preparations obtained from endotoxaemic rats. The NS1619‐induced relaxation and re‐polarization in arteries from endotoxaemic rats were partially inhibited by tetraethylammonium and completely inhibited by charybdotoxin, L‐NAME or ODQ, but not significantly affected by apamin. Similarly, the greater relaxation and re‐polarization induced by pinacidil in arteries from endotoxaemic rats were also inhibited by glibenclamide, L‐NAME or ODQ. However, these inhibitors had no significant effect on relaxations and re‐polarizations induced by NS1619 and pinacidil in arteries from controls. This study provides the electrophysiological and functional evidence showing an abnormal activation of K+ channels in vascular smooth muscle in animals with endotoxic shock. Our observations suggest that overproduction of nitric oxide causes an activation of large conductance Ca2+‐activated K+ channels and ATP‐sensitive K+ channels which contributes to endotoxin‐mediated vascular hyporeactivity.

Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments

In this study, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Rutaecarpine (200 µg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules when it was intravenously injected. Rutaecarpine (200 µg/g) prolonged occlusion time by approximately 1·5‐fold (control 127 ± 29 vs. taecarpine 188 ± 23 s). Furthermore, aspirin (250 µg/g) also showed a similar prolongation of the occlusion time in this experiment. On a molar basis, rutaecarpine was approximately twofold more potent than aspirin at prolonging the occlusion time. Furthermore, rutaecarpine was also effective in reducing the mortality of ADP‐induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 25 and 50 µg/g. Intravenous injection of rutaecarpine (50 µg/g) significantly prolonged the bleeding time by approximately 1·5‐fold compared with normal saline in the severed mesenteric arteries of rats. Continuous infusion of rutaecarpine (5 µg/g/min) also significantly increased the bleeding time 1·5‐fold, and the bleeding time returned to baseline within 60 min after cessation of rutaecarpine infusion. These results suggest that rutaecarpine has an effective anti‐platelet effect in vivo and that it may be a potential therapeutic agent for arterial thrombosis, but it must be assessed further for toxicity.

Higher level of plasma nitric oxide in spontaneously hypertensive rats.

We had detected a slightly, but significantly, higher level of plasma nitrite/nitrate in the spontaneously hypertensive rat (SHR) by using the nitric oxide (NO) analyzer (Sievers 280 NOA), which converts nitrate (including nitrate converted from nitrite) to NO. Here, we examined whether the release of NO from protein-bound dinitrosyl nonheme iron complexes (DNIC) contributes to the elevated plasma nitrate level in the SHR. The SHR and their genetic normotensive controls, Wistar-Kyoto rats (WKY), were anesthestized and cannulized for monitoring blood pressure, collecting a blood sample, and the administration of endotoxin (lipopolysaccharide [LPS]). The nitrate levels (an indicator of NO formation) in the plasma and the aorta were measured by an NO analyzer. In addition, the relaxation of acetylcholine (ACh) in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME) was also examined in thoracic aortae obtained from both strains. The slight, but significant, increase of basal nitrate levels in the plasma and aorta were observed, and the former was further enhanced in SHR treated with LPS for 3 h. In vitro, the ACh-induced relaxation was attenuated in the aortae obtained from SHR. However, this difference between SHR and WKY (without LPS treatment) was abolished by treatment of rings with L-NAME (30 micromol/L), suggesting that an impairment of NO formation was observed in the SHR. After rats were treated with LPS for 3 h, the ACh-induced relaxation was reduced in the WKY, but not in the SHR. In addition, a 10-fold increase of L-NAME was needed to abolish the difference in ACh-induced relaxation between SHR and WKY, indicating an expression of inducible NO synthase in both strains treated with LPS. We suggest that the elevated plasma NO level in SHR may be due to the release of NO from DNIC in the vascular bed to combat the hypertensive state.

Exercise training activates large-conductance calcium-activated K(+) channels and enhances nitric oxide production in rat mesenteric artery and thoracic aorta.

Exercise training has reversible beneficial effects on cardiovascular diseases, e.g. hypertension, which may result from a decrease in systemic vascular resistance. The purpose of this study was to investigate possible mechanisms associated with the changes in vascular reactivity in large and small arteries with vasoconstrictors and vasodilators in rats after exercise. Wistar-Kyoto rats were trained for 8 weeks (Ex group) on a treadmill and compared with sedentary counterparts (Sed group). After the measurement of blood pressure and heart rate at 8 weeks, rat mesenteric arteries and thoracic aortas were excised and prepared as rings for this study. In addition, special care was taken not to damage the endothelium of the preparations. Our results showed that exercise training for 8 weeks (1) not only prevented an increase in blood pressure but also caused a fall in heart rate, (2) attenuated the contractions induced by both prostaglandin F(2alpha) (PGF(2alpha)) and high K(+) in the mesenteric artery, but reduced the PGF(2alpha)-induced contraction in the aorta only, (3) enhanced the relaxation elicited by acetylcholine (ACh) in both mesenteric arteries and aortas, and (4) increased nitrate [an indicator of nitric oxide (NO) formation] in plasma. The enhancement of ACh-induced relaxation in the mesenteric arteries in the Ex group was suppressed by pretreatment with N(omega) -nitro-L-arginine methyl ester (L-NAME), tetraethylammonium (TEA; a nonselective inhibitor of K(+) channels) or charybdotoxin [CTX; a selective inhibitor of large-conductance calcium-activated K(+) (BK(Ca)) channels], whereas in the aorta that response was attenuated by TEA or CTX and almost completely abolished by L-NAME. However, with a combination of L-NAME plus CTX in the mesenteric artery, ACh-induced relaxation was completely abolished in the Sed group, but not in the Ex group. These results suggest that in addition to NO, activation of BK(Ca) channels in the vascular beds, at least in part, also contributes to vasodilatation in animals with exercise training.

Pentoxifylline improves circulatory failure and survival in murine models of endotoxaemia.

Pentoxifylline, a methylxanthine derivative, has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with claudication and cerebrovascular disorders as well as in animals with sepsis. Here, we investigate the effects of pentoxifylline on the hypotension, vascular hyporeactivity to noradrenaline, release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), and inducible NO synthase protein expression in a rat model of circulatory shock induced by bacterial endotoxin (Escherichia coli lipopolysaccharide). In addition, we have evaluated the effect of pentoxifylline on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure and heart rate. Injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a significant fall in mean arterial pressure and an increase of heart rate. In contrast, animals pretreated with pentoxifylline (3 mg/kg, i.v., at 30 min prior to lipopolysaccharide) maintained a significantly higher mean arterial pressure but showed no effect on the tachycardia when compared to rats given only lipopolysaccharide (lipopolysaccharide-rats). The pressor effect of noradrenaline (1 microg/kg, i.v.) was also significantly reduced after the treatment of rats with lipopolysaccharide. Similarly, rings of thoracic aorta obtained from lipopolysaccharide-rats showed a significant reduction in the contractile responses elicited by noradrenaline (1 microM). Pretreatment of lipopolysaccharide-rats with pentoxifylline partially, but significantly, prevented this lipopolysaccharide-induced hyporeactivity to noradrenaline in vivo and ex vivo. The injection of lipopolysaccharide resulted in bell-shape changes in plasma TNF-alpha level which reached a peak at 60 min, whereas the effect of lipopolysaccharide on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increase of both TNF-alpha and nitrate levels induced by lipopolysaccharide was significantly reduced in lipopolysaccharide-rats pretreated with pentoxifylline. Endotoxaemia for 240 min caused a significantly increased protein expression of inducible NO synthase in the lung. In lipopolysaccharide-rats pretreated with pentoxifylline, inducible NO synthase protein expression in lung homogenates was attenuated by 48 +/- 5%. Treatment of conscious mice with a high dose of endotoxin (60 mg/kg, i.p.) resulted in a survival rate of only 10% at 36 h (n = 20). However, therapeutic application of pentoxifylline (3 mg/kg, i.p. at 0, 6, 15 and 24 h after lipopolysaccharide) increased the 36-h survival to 35% (n = 20). Thus, pentoxifylline protects against circulatory failure and improves survival in rodents with severe endotoxaemia. These effects may be due to inhibition of the release of TNF-alpha and of the induction of inducible NO synthase.

Terbutaline prevents circulatory failure and mitigates mortality in rodents with endotoxemia.

Septic shock is characterized by a decrease in systemic vascular resistance. Nevertheless, regional increases in vascular resistance can occur that may predispose mammals to organ dysfunction, including the acute respiratory distress syndrome. In the host infected by endotoxin (lipopolysaccharide, LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNFalpha) rapidly increases, and this cytokine production is regulated by agents elevating cyclic AMP. In this report, we present evidence that terbutaline, a beta2-agonist, inhibits TNFalpha production and enhances interleukin-10 (IL-10) release in the anesthetized rat treated with LPS. In addition, an overproduction of nitric oxide (NO, examined by its metabolites nitrite/nitrate) by inducible NO synthase (iNOS, examined by western blot analysis) is attenuated by pretreatment of LPS rats with terbutaline. Overall, pretreatment of rats with terbutaline attenuates the delayed hypotension and prevents vascular hyporeactivity to norepinephrine. In addition, pretreatment of mice with terbutaline also improves the survival in a model of severe endotoxemia. The infiltration of polymorphonuclear neutrophils into organs (e.g., lung and liver) from the surviving LPS mice treated with terbutaline was reduced almost to that seen in the normal controls. These findings suggest that the inhibition of TNFalpha and NO (via iNOS) production as well as the increment of IL-10 production contribute to the beneficial effect of terbutaline in animals with endotoxic shock.

Pharmacological Activity of DC−015, a Novel Potent and Selective α1-Adrenoceptor Antagonist

The pharmacological activity of 3−((4−(2−methoxyphenyl)piperazin−1−yl)methyl)−2,3−dihydroimidazo(1,2−c)quinazolin−5(6H)‐one (DC−015), a newly synthesized quinazoline derivative, was determined in rat isolated thoracic aorta and pressor responses were determined in spontaneously hypertensive rats (SHR).

Cardioprotective effects of long-term treatment with raloxifene, a selective estrogen receptor modulator, on myocardial ischemia/reperfusion injury in ovariectomized rats.

Objective: The aim of this study was to investigate the beneficial effect of long-term treatment with raloxifene (RAL), a selective estrogen receptor modulator, on myocardial ischemia/reperfusion (MI/R) injury in ovariectomized (Ovx) rats. Methods: Ovariectomy was performed in female Sprague-Dawley rats 8 weeks old. Ovx rats were treated with RAL 1 or 5 mg/kg (gavage, once daily) or 17&bgr;-estradiol (E2; 50 &mgr;g/kg SC, three times a week) for 8 weeks. The cardioprotective effect of RAL was evaluated in an open-chest anesthetized rat model of MI/R, which was induced by 40-minute left coronary artery occlusion and 100-minute reperfusion. Results: Long-term treatment with RAL 1 mg/kg significantly suppressed the duration of ventricular tachycardia elicited by MI. After MI/R, the levels of plasma creatine kinase-MB fraction and lactate dehydrogenase in Ovx rats were significantly higher than those in the sham group, which were significantly reduced by long-term treatment with RAL 1 mg/kg or E2. Neutrophil myeloperoxidase activity in ischemic myocardium markedly increased in the Ovx group, whereas long-term treatment with RAL 1 or 5 mg/kg or E2 significantly suppressed the elevation of myeloperoxidase activity. After MI/R, the protein expression of phosphorylated inhibitory &kgr;B&agr; and caspase-3 in ischemic myocardium pronouncedly increased in the Ovx group and was attenuated by long-term treatment with RAL 1 mg/kg or E2. Conclusions: Long-term treatment with RAL can reduce the severity of MI-induced arrhythmias and attenuate MI/R-induced damages and apoptosis in Ovx rats. This cardioprotective effect of RAL may be associated with inhibition of neutrophil infiltration and suppression of nuclear factor-&kgr;B activation.