Mayuko Sakanashi

Cardioprotective Effects of Extracts from Psidium guajava L. and Limonium wrightii, Okinawan Medicinal Plants, against Ischemia-Reperfusion Injury in Perfused Rat Hearts

The aim of this study was to determine whether the medicinal herbs growing in Okinawa and possessing a radical-scavenging activity would exert cardioprotective effects against ischemia-reperfusion injury using isolated perfused rat hearts. Effects of the aqueous extracts from Psidium guajava L. and Limonium wrightii at concentrations having an equipotent radical-scavenging activity on myocardial injury produced by global ischemia followed by reperfusion were tested and were further compared with those of quercetin and gallic acid, major antioxidative components of P. guajava L. and L. wrightii, respectively. Both extracts significantly attenuated ischemic contracture during ischemia and improved myocardial dysfunction after reperfusion. Decreases in high-energy phosphates and increases in malondialdehyde in the reperfused hearts were significantly lessened with both plant extracts. Quercetin and gallic acid also exerted similar beneficial effects. These results indicate that P. guajava L. and L. wrightii both have cardioprotective effects against myocardial ischemia-reperfusion injury in isolated rat hearts, primarily through their radical-scavenging actions.

Effect of 1-week treatment with erythropoietin on the vascular endothelial function in anaesthetized rabbits

Chronic administration of erythropoietin (EPO) is often associated with hypertension in animals and humans. The aim of this study was to estimate whether 1‐week treatment with EPO can affect the vascular endothelial function. Rabbits were given with EPO (400 iu kg−1 s.c.) or saline each other day for 1 week. Hypotensive responses to intravenously given acetylcholine (ACh), endothelium‐independent nitric oxide donors (NOC7, nitroprusside and nitroglycerin) and prostaglandin I2 were tested before and after administration of NG‐nitro‐L‐arginine methyl ester (L‐NAME), a specific nitric oxide synthase inhibitor, under pentobarbitone anaesthesia. Blood haemoglobin concentration in EPO group was significantly higher than that in control group, whereas baseline values of aortic pressure, heart rate and femoral vascular resistance were similar. The dose of ACh (172 ng kg−1) requiring for a 15 mmHg hypotension from the baseline in EPO group was apparently higher than that (55 ng kg−1) in control group. On the contrary, hypotensive responses to NOC7, nitroprusside, nitroglycerin and prostaglandin I2 were comparable between two groups. The extent of ACh‐induced hypotension did not correlate with haemoglobin concentration. L‐NAME significantly inhibited the ACh‐induced vasodilating response in control group but did not in EPO group. In another set of rabbits, the same treatment with EPO also decreased vasodilating responses to carbachol, bradykinin and substance P besides ACh as compared with control group. These results indicate that 1‐week treatment with EPO selectively attenuates depressor responses to endothelium‐dependent vasodilators in anaesthetized rabbits, most likely due to inhibition of endothelial nitric oxide synthase.

Increasing dihydrobiopterin causes dysfunction of endothelial nitric oxide synthase in rats in vivo

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

Haemodynamic effects of the crude venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rabbits

Haemodynamic effects of saline-extracted venom from nematocysts isolated from tentacles of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) were investigated. In anaesthetized rabbits, i.v. injections of the venom produced hypotension following a transient hypertension. Mean femoral arterial blood flow markedly decreased immediately after the injection and femoral vascular resistance increased. Left ventricular dP/dt remarkably decreased after a transient and small increase, and heart rate decreased. Left ventricular end-diastolic pressure markedly elevated. All of the above changes by 0.2-5 microg/kg of the venom expressed as the amount of protein were seen dose-dependently and occurred without tachyphylaxis. In five of seven animals received an injection of the venom at 10 microg/kg, irreversible cardiac arrest occurred. Changes produced by 1 or 2 microg/kg of the venom were significantly attenuated either by heating the venom at 40 degrees C for 10min or by pretreatment with diltiazem. These results indicate that the venom from Habu-kurage has both vasoconstrictor and cardiodepressive effects, and suggest that these thermolabile actions may be due partly to activation of voltage-dependent calcium channels and probably subsequent calcium-overload.

Uneven Changes In Circulating Blood Cell Counts With Adrenergic Stimulation To The Canine Spleen

1. Responses of splenic diameter measured by sonomicrometry to α‐ and β‐adrenoceptor stimulants were estimated together with simultaneously measured systemic arterial and splenic venous concentrations of red blood cells (RBC), white blood cells (WBC) and platelets (PLT) in anaesthetized dogs.

Effect of caffeine contained in a cup of coffee on microvascular function in healthy subjects.

Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

Improvement of impaired endothelial function by tetrahydrobiopterin in stroke-prone spontaneously hypertensive rats

To investigate the role of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, in endothelial function in a model of genetic hypertension, acetylcholine- and sodium nitroprusside (SNP)-induced vasodilator responses were examined in the absence and presence of BH4 in age-matched adult stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. Acetylcholine-induced depressor responses attenuated significantly in SHRSP compared with those in WKY rats. Acetylcholine-induced relaxations in phenylephrine-precontracted aortic rings of SHRSP were also significantly impaired as compared to those of WKY rats, while SNP-induced relaxations were similar between both strains. In SHRSP, intravenous infusion of BH4 (0.12 mg/kg per min for 20 min following a bolus injection of 0.48 mg/kg) significantly improved vasodilator responses to acetylcholine without affecting those to SNP, but in WKY rats BH4 did not influence those to acetylcholine. BH4 infusion itself had no hemodynamic effect in both strains. However, BH4 levels in plasma and thoracic aorta as well as plasma concentrations of nitrite plus nitrate, metabolites of NO, in SHRSP were all significantly greater than those in WKY rats, suggesting the occurrence of compensatory upregulation of NO synthesis in SHRSP. These results demonstrate that the impaired endothelial function in SHRSP cannot be explained simply by the decrease in absolute amount of BH4.

Crucial vasculoprotective role of the whole nitric oxide synthase system in vascular lesion formation in mice: Involvement of bone marrow-derived cells.

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism.

Effects of glutathione S‐transferase inhibitors on nitroglycerin action in pig isolated coronary arteries

1. The present study was designed to clarify the role of glutathione S‐transferase (GST) in the vasorelaxation response and development of tolerance to nitroglycerin (GTN) using GST inhibitors.

Development of an experimentally useful model of acute myocardial infarction: 2/3 nephrectomized triple nitric oxide synthases-deficient mouse

We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy (NX) was performed on male triple NOSs(-/-) mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs(-/-) mice as early as 4months after the surgery. The 2/3NX triple NOSs(-/-) mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs(-/-) mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin-angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs(-/-) mouse is a new experimentally useful model of AMI. Renin-angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.