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We investigated the bladder and urethral function in a rat model lacking the protein lysyl oxidase-like 1 (Loxl1). Female nulliparous rats of Loxl1-/- or age-matched wild type (WT) rats had leak-point pressure testing, cystometry, histopathological analyses of lower urinary tract, and contractile response of isolated detrusor strips to carbachol and electric field stimulation. The Loxl1-/- rats showed increased looseness and redundancy of the skin, the decreased intercontraction interval and voided volume in cystometry, the lower leak-point pressure, thinner elastic fibers of the mesentery, bladder, urethra and vagina, and smaller contractile response of detrusor strips to carbachol when compared to the WT rats. Thus, the insufficient hydrostatic mechanism of urethra via submucosal impaired elastin synthesis might reduce the resting urethral closure pressure and the diminished cholinergic contractile response of detrusor smooth muscle might be involved in bladder activity in the Loxl1-/- rats.

(Purpose) Ingestion of hydrogen is said to prevent oxidation in the body, but hydrogen is produced by intestinal bacterial flora and excreted in the exhaled breath. We investigated how breath hydrogen concentrations change with the diurnal cycle and under various conditions, including after consuming food or drink, and in people with urological disease. (Subjects and methods) Participants were healthy volunteers (40 men, 45 women; 30-83 years old) and urological outpatients (40 men with benign prostatic hyperplasia, 30 women with overactive bladder; 60 years or older). Breath hydrogen levels were measured before and after eating and drinking in three volunteers, and its diurnal variation was examined in one. The relationship between breath hydrogen and age or urological disease status was also analyzed by gender. Additional measurements were taken in the person with the highest breath hydrogen concentration and the person with the lowest; in these two people, breath hydrogen was measured at the same time for 10 or more days to determine the fluctuation range. (Results) Breath hydrogen concentration increased temporarily after ingestion of tap water, hydrogen water or food. It also increased with food intake and in cases of flatulence with intestinal gas accumulation, but decreased after defecation. In the person with the highest breath hydrogen, concentrations were 11.2-188.6 ppm, whereas in the person with the lowest, they were 0.4-2.3 ppm. Breath hydrogen increased significantly with age in healthy female volunteers. There was no association between breath hydrogen and benign prostatic hyperplasia, overactive bladder or constipation. (Conclusion) Breath hydrogen concentration increases with eating, drinking and aging, and is not associated with benign prostatic hyperplasia, overactive bladder or constipation. Breath hydrogen concentration varies widely between individuals, which may be due to differences in intestinal flora.

INTRODUCTION: Alpha/beta-hydrolase domain 6 (ABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol, a high-efficiency endogenous cannabinoid. Although the endocannabinoid system has been suggested to be involved in regulation of bladder function, the roles of ABHD6 in the control of micturition remain unknown. To elucidate the physiological and pathological roles of ABHD6 in vivo, we examined phenotypes of ABHD6 knockout rats (Abhd6(−/−)) generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins system. MATERIALS AND METHODS: Age-matched knockout and wild-type (WT) rats of both sexes were used. RESULTS: Expression of ABHD6, assessed by quantitative real-time polymerase chain reaction and Western blot analysis, was clearly diminished in Abhd6(−/−) rats compared with WT rats. Mutant rats had a normal appearance, and the body weight and food consumption were similar to those of WT rats. The interval between bladder contractions assessed by continuous cystometry was significantly shorter in ABHD6 knockout rats than in WT rats when the bladder was stimulated with acetic acid. Mechanical paw withdrawal thresholds measured by von Frey testing were significantly lowered in the knockout rats than in WT rats. The plasma levels of prostaglandin E(2) (PGE(2)) and the stable metabolite of PGE(2) in Abhd6(–/–) rats were twice as high as that in WT rats. CONCLUSIONS: Deletion of the ABHD6 gene in rats causes more frequent urination in the stimulated bladder and hyperalgesia to non-noxious mechanical stimuli along with increased plasma PGE(2).

Objectives The scent of vanilla has a relaxing effect and is used to treat sleep disorders. Sleep disorders can both cause and be caused by nocturia. Therefore, we examined whether vanilla inhalation would reduce the frequency of urination in rats under light urethane anesthesia. Methods Twenty-four rats were anesthetized with 0.6 g/kg urethane subcutaneously (half the usual dose) to induce a sleep-like state. In 12 rats, continuous cystometry was performed via a transurethral catheter before, during and after inhalation of vanilla (n = 7) or the citrus fruit shiikuwasa (n = 5) for 60 minutes. The remaining 12 rats did not undergo cystometry but underwent vanilla inhalation treatment for 60 minutes (n = 6), or no inhalation treatment (n = 6); blood was then collected from these two groups and serum monoamine levels were compared. Results Intervals between bladder contractions were significantly longer after vanilla inhalation than before. However, baseline bladder pressure, maximum bladder contraction pressure, and residual volume remained unchanged. During shiikuwasa inhalation, the body movement of each rat increased but cystometric parameters did not change. Serum concentrations of adrenaline, noradrenaline and dopamine, but not serotonin, were significantly lower in rats that had inhaled vanilla than in those that had not. Conclusions Vanilla scent decreased serum catecholamine levels and urination frequency in rats under light urethane anesthesia. These results suggest that the scent of vanilla may reduce nocturia.

Pelvic venous congestion (PC) is thought to be related to several diseases of the lower urinary tract (LUT). We examined the characteristics of the LUT in rats with PC. To create PC, female rats were anesthetized with isoflurane, and the bilateral common iliac veins and bilateral uterine veins were ligated. At 1-8 weeks after either ligation or sham surgery, we performed cystometry with or without administration of carbazochrome sodium sulfonate hydrate or propiverine hydrochloride, histologic examination of the bladder, blood flow imaging, assessment of locomotor activity, measurement of urinary 8-hydroxydeoxyguanosine (8-OHdG) and nitric oxide metabolites (NOx), and the Evans blue dye extravasation test. PC elevated frequency of urination after 2-6 weeks, and caused a decrease of spontaneous locomotor activity. In addition, there was a decrease of bladder blood flow, an increase of bladder vascular permeability, an increase of urinary 8-OHdG, a decrease of urinary NOx, and mild inflammatory changes of the bladder. In rats with PC, frequency of urination was normalized by administration of propiverine or carbazochrome. Rats with PC may be used as a model of PC associated with high frequency of urination, and this model may be useful when developing treatment for LUT symptoms associated with PC.

Nitric oxide (NO) is synthesised not only from l-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, but whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency would induce the metabolic syndrome in mice. To this end, we prepared a low-nitrite/nitrate diet (LND) consisting of an amino acid-based low-nitrite/nitrate chow, in which the contents of l-arginine, fat, carbohydrates, protein and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. Three months of the LND did not affect food or water intake in wild-type C57BL/6J mice compared with a regular diet (RD). However, in comparison with the RD, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia and glucose intolerance. Eighteen months of the LND significantly provoked increased body weight, hypertension, insulin resistance and impaired endothelium-dependent relaxations to acetylcholine, while 22 months of the LND significantly led to death mainly due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS downregulation, adiponectin insufficiency and dysbiosis of the gut microbiota. These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in the metabolic syndrome and its vascular complications.

Objectives To examine whether electrical stimulation of the perineum inhibited urinary frequency in rats with pelvic venous congestion, and whether electrical stimulation influences spinal glycinergic/gamma-aminobutyric acid-ergic neurons. Methods Bilateral common iliac veins and bilateral uterine veins were ligated to create pelvic venous congestion rats. At 4 weeks after ligation, cystometry was carried out before and after electrical stimulation with/without intrathecal injection of strychnine (a glycine receptor antagonist) and/or bicuculline (a gamma-aminobutyric acid type A receptor antagonist). In addition, measurement of amino acid levels in the lumbosacral cord was carried out with/without electrical stimulation, and cystometry was carried out after oral administration of glycine. Results Continuous cystometry showed that the interval between bladder contractions was shorter in pelvic venous congestion rats than in sham rats. Electrical stimulation did not change cystometric parameters in sham rats, but the interval between bladder contractions was increased by electrical stimulation in pelvic venous congestion rats. Electrical stimulation increased the levels of glutamic acid, glycine, gamma-aminobutyric acid, and taurine in the lumbosacral cord of pelvic venous congestion rats. Intrathecal strychnine abolished the effects of electrical stimulation in pelvic venous congestion rats, and intrathecal administration of both strychnine and bicuculline shortened the interval between bladder contractions more than before electrical stimulation. Oral administration of glycine (3%) to pelvic venous congestion rats increased bladder capacity. Conclusions Electrical stimulation of the perineum inhibits urinary frequency mainly through activation of spinal glycinergic neurons, and partly through activation of gamma-aminobutyric acid-ergic neurons in a rat model of pelvic venous congestion.

The effects of solifenacin and mirabegron on vesical and urethral function were compared in rats with or without spinal cord injury (SCI). Isovolumetric cystometry and urethral pressure recording were initially performed in intact rats. Then, the bladder neck was ligated under urethane anesthesia, after which a catheter was inserted through the bladder dome for isovolumetric cystometry and another catheter was inserted into the urethra to measure urethral pressure. Solifenacin (0.03-3 mg/kg) or mirabegron (0.03-3 mg/kg) was injected intravenously, and bladder and urethral activity were recorded. To create rats with SCI, the spinal cord was transected at the lower thoracic level under isoflurane anesthesia. After 2 weeks, a catheter was inserted through the bladder dome for single cystometry and bladder activity was recorded without anesthesia following intravenous injection of solifenacin or mirabegron. Isovolumetric cystometry revealed a larger decrease in maximum bladder contraction pressure after injection of solifenacin, whereas prolongation of the interval between bladder contractions was greater with mirabegron. In SCI rats, single cystometry showed that solifenacin and mirabegron both increased bladder volume at the first non-voiding bladder contraction and decreased the maximum bladder contraction pressure. Mirabegron also increased the voided volume and decreased the percentage residual volume without altering bladder capacity. Solifenacin and mirabegron both inhibited bladder contractility, and mirabegron possibly also induced urethral relaxation. Mirabegron may be suitable for patients with overactive bladder and residual urine.

Pelvic venous congestion (PC) is thought to be related to several diseases of the lower urinary tract (LUT). We examined the characteristics of the LUT in rats with PC. To create PC, female rats were anesthetized with isoflurane, and the bilateral common iliac veins and bilateral uterine veins were ligated. At 1-8 weeks after either ligation or sham surgery, we performed cystometry with or without administration of carbazochrome sodium sulfonate hydrate or propiverine hydrochloride, histologic examination of the bladder, blood flow imaging, assessment of locomotor activity, measurement of urinary 8-hydroxydeoxyguanosine (8-OHdG) and nitric oxide metabolites (NOx), and the Evans blue dye extravasation test. PC elevated frequency of urination after 2-6 weeks, and caused a decrease of spontaneous locomotor activity. In addition, there was a decrease of bladder blood flow, an increase of bladder vascular permeability, an increase of urinary 8-OHdG, a decrease of urinary NOx, and mild inflammatory changes of the bladder. In rats with PC, frequency of urination was normalized by administration of propiverine or carbazochrome. Rats with PC may be used as a model of PC associated with high frequency of urination, and this model may be useful when developing treatment for LUT symptoms associated with PC.

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.

Aims Ligation of the urethra to create partial bladder outlet obstruction has widely been used as an animal model of bladder obstruction, although obstructive bladder dysfunction may be due to both mechanical and functional obstruction. Previous studies in rodents have demonstrated that long-term nitric oxide (NO) deficiency can lead to detrusor overactivity, and lack of NO may thus cause impairment of bladder outlet relaxation. The aim of this study was to define the characteristics of bladder and urethral dysfunction induced by chronic NO deficiency through both in vivo and in vitro investigations. Main methods Rats were divided into two groups, and one group received an NO synthase inhibitor (Nω-nitro-L-arginine methyl ester hydrochloride: L-NAME) in the drinking water for 4 weeks. Bladder and urethral function were evaluated by continuous cystometry and isovolumetric cystometry. In vitro functional studies of detrusor strips and measurement of the mRNA and protein expression of an ischemic marker and a gap junction protein were also performed in separate rats. Key findings L-NAME administration raised blood pressure and decreased plasma nitrite/nitrate level compared to the control group. L-NAME treatment increased the frequency of bladder contractions and the residual volume, and elevated urethral pressure and bladder contraction pressure. In addition, carbachol-induced contraction was reduced in isolated detrusor strips from the L-NAME group, and bladder expression of HIF-1 and connexin 43 showed upregulation. Significance These findings suggest that chronic administration of L-NAME to rats induces bladder hyperactivity with residual urine, and may provide a useful model of functional bladder obstruction.

Background Hormone replacement therapy has failed to reduce ischemic cardiovascular events in climacteric women. To explore alternative therapy, we examined whether san'o-shashin-to (TJ-113), a kampo medicine, ameliorates cardiac ischemia-reperfusion (IR) injury in a climacteric rat model. Methods and results Cardiac function and infarct size after IR were significantly exacerbated in ovariectomized rats as compared with sham-operated rats, whereas long-term treatment with a clinical dosage of TJ-113 for 4 weeks markedly improved these functional and morphological changes. Myocardial inducible nitric oxide synthase (iNOS) expression and peroxynitrite levels were significantly higher in ovariectomized rats compared with sham-operated rats, and long-term TJ-113 treatment significantly reduced these oxidative changes. Furthermore, myocardial manganese superoxide dismutase (Mn-SOD) activity was significantly lower in ovariectomized than in sham-operated rats, and long-term TJ-113 treatment significantly restored antioxidant activity. Importantly, those beneficial actions of TJ-113 were significantly inhibited by the estrogen receptor antagonist, fulvestrant, and the phytoestrogen, emodin, a TJ-113 ingredient, mimicked the actions of TJ-113, suggesting involvement of emodin in the effects of TJ-113. Conclusions These results provide the first evidence that long-term treatment with a clinical dosage of TJ-113 markedly ameliorates cardiac IR injury in ovariectomized rats via inhibition of iNOS expression, suppression of peroxynitrite formation, and restoration of Mn-SOD activity. TJ-113 may be a novel therapeutic option in the treatment of ischemic heart disease in climacteric women.

Vacuolar protein sorting 35 (VPS35) was recently reported to be a pathogenic gene for late- onset autosomal dominant Parkinson's disease (PD), using exome sequencing. To date, VPS35 mutations have been detected only in whites with PD. The aim of the present study was to determine the incidence and clinical features of Asian PD patients with VPS35 muta- tions. We screened 7 reported nonsynonymous mis- sense variants of VPS35, including p.D620N, known as potentially disease-associated variants of PD, in 300 Japanese index patients with autosomal dominant PD and 433 patients with sporadic PD (SPD) by direct sequencing or high-resolution melting (HRM) analysis. In addition, we screened 579 controls for the p.D620N mutation by HRM analysis. The p.D620N mutation was detected in 3 patients with autosomal dominant PD (1.0%), in 1 patient with SPD (0.23%), and in no con- trols. None of the other reported variants of VPS35 were detected. Haplotype analysis suggested at least 3 independent founders for Japanese patients with p.D620N mutation. Patients with the VPS35 mutation showed typical tremor-predominant PD. We report Asian PD patients with the VPS35 mutation. Although VPS35 mutations are uncommon in PD, the frequency of such mutation is relatively higher in Japanese than reported in other populations. In VPS35, p.D620N sub- stitution may be a mutational hot spot across different ethnic populations. Based on the clinical features, VPS35 should be analyzed in patients with PD, espe- cially autosomal dominant PD or tremor-predominant PD. V C 2012 Movement Disorder Society

The influence of extracellular Ca 2+ concentration on contractile responses of aortae isolated from diabetic rats to KCl and α-adrenoceptor agonists was compared with that of non-diabetic rat aortae. Diabetic rats 6 weeks after administration of streptozotocin showed significantly lower body weight and higher plasma glucose concentration, but the protein content per each aortic ring preparation was not significantly different from that of non-diabetic preparation. Both diabetic and non-diabetic aortae showed concentration-dependent contractile responses to norepinephrine, which were concentration-dependently inhibited by prazosin. On the other hand, Clonidine induced small contractions in both aortae, which tended to be more inhibited by prazosin than yohimbine. In non-diabetic aortae, the contractile responses to KCl, norepinephrine, methoxamine and Clonidine were significantly greater with 2.5 mmol/l of extracellular Ca 2+ than 1.25 mmol/l. In diabetic aortae, however, the contractile responses were not significantly influenced by changes in extracellular Ca 2+ concentration. Additionally, the contractile responses to each agonist were markedly greater in non-diabetic aortae than diabetic ones. The present results indicate that the contractions of diabetic vasculature do not respond to changes in extracellular Ca 2+ concentration, suggesting that there may be some impairment of Ca 2+ related mechanisms.

An elevation of oxidized forms of tetrahydrobiopterin (BH4), especially dihydrobiopterin (BH2), 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 BH2in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH2concentration causes endothelial dysfunction in rats. To increase vascular BH2levels, the BH2precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH2to BH4. MTX/SEP treatment did not significantly affect aortic BH4levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH2levels (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 BH4levels but decreased the BH4-to-BH2ratio. 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 BH2causes eNOS dysfunction in vivo even in the absence of BH4deficiency, demonstrating a novel insight into the regulation of endothelial function.

Vascular endothelial dysfunction has been demonstrated in obesity, but the molecular basis for this link has not been clarified. We examined the role of free fatty acids (FFA) on vascular reactivity in the obese fa/fa Zucker diabetic fatty (ZDF) rat. Addition of acetylcholine produced a dose-dependent relaxation in aortic rings of ZDF and lean +/+ rats, but the ED50 value was higher in ZDF (−6.80 ± 0.05 vs. −7.11 ± 0.05 log10 mol/liter, P = 0.033). A 2-wk treatment with a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pitavastatin (3 mg/kg/d) or a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin (5 mmol/liter in drinking water), improved the response in ZDF (ED50, −7.16 ± 0.03 and −7.14 ± 0.05 log10 mol/liter, P = 0.008 and P = 0.015 vs. vehicle, respectively). Vasodilator response to sodium nitroprusside was identical between ZDF and +/+ rats. Vascular reactive oxygen species (ROS) levels and NADPH oxidase activity in aorta were increased in ZDF rats but were decreased by pitavastatin. In in vitro cell culture, intracellular ROS signal and NADPH oxidase subunit mRNA were increased by palmitate, but this palmitate-induced ROS production was inhibited by NADPH oxidase inhibitor or pitavastatin. In conclusion, FFA-induced NADPH oxidase subunit overexpression and ROS production could be involved in the endothelial dysfunction seen in obese ZDF rats, and this could be protected by pitavastatin or NADPH oxidase inhibitors.

Background: The role of each nitric oxide synthase (NOS) isoform in cerebral infarction has been studied in individual NOS isoform-deficient mice. It has been reported that, in a model of middle cerebral artery occlusion (MCAO), neuronal and inducible NOSs exacerbate cerebral infarction, whereas endothelial NOS conversely alleviates it. Although the role of the whole NOSs system in cerebral infarction has been examined in pharmacological studies with non-selective NOS inhibitors, the results are quit inconsistent, possibly because of non-specificity of the agents. In this study, we addressed this point in mice in which all three NOS genes are completely disrupted. Method and Results: We newly generated triple NOSs -/- mice and wild-type littermates by crossbreeding single NOS -/- mice. Transient (1 hour) or permanent MCAO was performed in male triple NOSs -/- and wild-type mice at 8-12 weeks of age (n=8-11). There was no anatomical difference in the structure of cerebral arteries between the triple NOSs -/- and the wild-type mice. Reductions in cerebral blood flow during MCAO were also comparable between the two genotypes. However, cerebral infarct size at 24 hours after transient MCAO was markedly reduced in the triple NOSs -/- genotype as compared with the wild-type genotype ( P -/- than in the wild-type genotype ( P -/- genotype compared with the wild-type genotype (each P -/- genotype as compared with the wild-type genotype ( P Conclusions: These results provide the first evidence that complete disruption of all NOS genes markedly reduces cerebral infarct size after MCAO in mice, demonstrating a novel injurious role of the entire NOSs system in the pathogenesis of cerebral infarction. Inhibition of the NOSs system may be a new therapeutic option in the treatment of cerebral infarction.

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.

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.

In order to protect the heart from unfavorable conditions such as myocardial ischemia or heart failure, it seems necessarily to investigate what components are related to such conditions or what drugs affect them. For this purpose, at symposium No. 40 "Recent Advances in Cardiac Protection" at the 75th Annual Meeting of The Japanese Pharmacological Society held in Kumamoto City, the following 6 studies were presented: (1) The Role of Sarcoglycan Complex in Chronic Heart Failure (H. Yoshida et al.); (2) Heart Failure and Proinflammatory Cytokines (T. Kubota et al.); (3) Fundamental Strategies and Current Topics for Cardiac Protection During Open-Heart Surgery (R. Tominaga et al.); (4) Role of Tetrahydrobiopterin in Myocardial Ischemia/ Reperfusion Injury (K. Noguchi et al.); (5) Cardiac Protection by Nitric Oxide (T. Obata); (6) Reconsideration of Anti-oxidants as Protective Drugs on Ischemic Myocardium (K. Ichihara et al.). This review gathers the reports of studies presented at this symposium.

SUMMARY 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. 2. Intravenous and intrasplenic arterial injections of adrenaline, noradrenaline and phenylephrine all produced profound decreases in splenic diameter. Increases in systemic arterial concentrations of RBC produced by these stimulants were observed immediately following the splenic contraction and marked increases in splenic venous concentration of RBC. 3. Adrenaline and noradrenaline both caused rapid and transient decreases in WBC release from the spleen and these agonists then, in addition to phenylephrine, caused gradual and small increases in systemic arterial WBC concentrations. The increases in arterial RBC and WBC concentrations were abolished by transient isolation of the spleen from the systemic circulation. These three agents did not significantly modify differences between splenic venous and systemic arterial concentrations of PLT. 4. Intra-arterial injections of isoprenaline caused an increase in splenic diameter and a significant decrease in the splenic venous concentration of WBC, but barely influenced the veno-arterial differences in RBC and PLT concentrations across the spleen. 5. The present results indicate that contraction of the spleen through stimulation of splenic α1-adrenoceptors contributes to supplying RBC but not PLT into the systemic circulation, whereas splenic β-adrenoceptor activation leads to splenic dilatation with increased sequestration of WBC to the spleen despite no changes in the release of RBC and PLT from the spleen.

Background: The role of each nitric oxide synthase (NOS) isoform in the pathogenesis of cerebral infarction has been studied in individual NOS isoform-deficient mice. It has been reported that, in a model of middle cerebral artery occlusion (MCAO), neuronal and inducible NOSs exacerbate cerebral infarction, whereas endothelial NOS conversely alleviates cerebral infarction. Although the role of the whole NOSs system in cerebral infarction has been examined in pharmacological studies with non-selective NOS inhibitors, the results are quit inconsistent, possibly because of non-specificity of the agents. In this study, we addressed this point in mice in which all three NOS genes are completely disrupted. Method and Results: We newly generated triple NOSs-deficient mice and wild-type littermates by crossbreeding single NOS -/- mice. Transient (1 hour) and permanent MCAO was performed in male triple NOSs -/- and wild-type mice at 8-12 weeks of age (n=9-11). Cerebral infarct size was evaluated by triphenyltetrazolium chloride (TTC) staining. There was no anatomical difference in the structure of cerebral arteries between the triple NOSs -/- and wild-type mice (n=3 each). Reductions in cerebral blood flow during MCAO were identical between the two mice. However, cerebral infarct size at 24 hours after transient MCAO was markedly smaller in the triple NOSs -/- than in the wild-type mice ( P -/- mice as compared with the wild-type mice ( P -/- mice compared with the wild-type mice (both P Conclusions: These results provide the first evidence that complete disruption of all NOS genes markedly reduces cerebral infarct size after MCAO in mice, demonstrating a novel injurious role of the entire NOSs system in cerebral infarction. Inhibition of the NOSs system may be a novel therapeutic option in the treatment of cerebral infarction.

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 4 months 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.

Convergent synthesis of HIJK ring model compound 1 of ciguatoxin is described. The present synthesis relied on a palladium-catalyzed Suzuki cross-coupling reaction between eight-membered ketene acetal phosphate 2 and seven-membered alkylborane 6.

SUMMARY 1. The haemodynamic effects of cilnidipine, a new calcium channel blocker, were examined in a canine model of acute congestive heart failure and were compared with those of nicardipine at equihypotensive doses. 2. The model was prepared by injections of saponin into coronary arteries of anaesthetized open-chest dogs followed by volume loading and continuous i.v. infusion of methoxamine. After the treatment, aortic blood flow (AoF) and left ventricular dP/dt markedly decreased, while left ventricular end-diastolic pressure (LVEDP), right atrial pressure and systemic vascular resistance (SVR) increased. Cilnidipine (0.3,1.0 and 3.0 μ/kg per min), nicardipine (0.3, 1.0 and 3.0 (μ/kg per min) or the respective vehicle was given i.v. after accomplishment of heart failure. 3. These drugs both produced a comparable reduction in aortic pressure and an increase in AoF associated with profound decreases in LVEDP, SVR and coronary vascular resistance. In contrast, administration of nicardipine was associated with significant increases in heart rate and cardiac contractility but that of cilnidipine was not. 4. These results indicate that cilnidipine as well as nicardipine can exert beneficial haemodynamic effects in a model of acute heart failure probably through lessening afterload and cilnidipine may moderate reflex-induced sympathetic stimulation.

The current situation and practice of undergraduate and postgraduate education regarding clinical pharmacology at university and college hospital were investigated using questionnaires which were sent to 80 departments of pharmacy in university and college hospitals. The questionnaires were returned by 40 departments of pharmacy (50%). The major findings were as follows:1. Undergraduate clinical pharmacology education for medical students has been established at 27 out of 40 departments (67.5%), and the main persons responsible were the department of pharmacy staff members in 25 out of 27 university and college hospitals (93%).2. Postgraduate education for medical doctors and pharmacists has been established at 9 out of 40 departments (22.5%), and the main persons responsible were also the department of pharmacy staff members in 7 out of 9 university and college hospitals (78%).3. The course curriculum for clinical pharmacology tended to include: the prescription of drugs, pharmacokinetics, therapeutic drug monitoring, law and rules etc. in undergraduate programs, while it was seminars in postgraduate programs.4. Difficult issues in both undergraduate and postgraduate programs for clinical pharmacology were lacks of staff and time for sufficient education, etc.5. The most important factors for the future of clinical pharmacology education were sufficient equipment for the academic requirements and the establishment of a department of clinical pharmacology in the school of medicine etc.These results show that undergraduate education for clinical pharmacology has not yet been universally established at the departments of pharmacy in university and college hospitals, especially postgraduate education was poor, and therefore further efforts are called for to improve clinical pharmacology education.

1. Platelet-activating factor (PAF) is a phospholipid mediator with potent cardiovascular and haematological actions. But its mechanisms of action in vivo have not been fully elucidated, probably due to difficulties arising from previous findings that the effects of PAF are largely mediated by the release of a variety of other autacoids. In the present study, the roles of nitric oxide and eicosanoids in the effects of PAF (0.01-0.25 microgram kg-1 i.v.) on systemic and pulmonary vasculatures and circulating blood cell count were pharmacologically evaluated in anaesthetized dogs. 2. Higher doses of PAF (> 0.1 microgram kg-1) produced a biphasic systemic hypotension. The first hypotension seen 30 s after the injection was accompanied by a decrease in systemic vascular resistance, thrombocytopenia and leukopenia, while the second hypotension seen 1-2 min after PAF was accompanied by a marked rise in pulmonary vascular resistance and decreases in aortic blood flow and cardiac contractility. Lower doses of PAF (0.01 - 0.05 microgram kg-1) caused only the first responses in a dose dependent manner. 3. Pretreatment with indomethacin inhibited the second responses to PAF without affecting the first responses. The thromboxane A2/prostaglandin H2 (TP)-receptor antagonist vapiprost blocked the PAF-induced rise in pulmonary vascular resistance. AA-861, a 5-lipoxygenase inhibitor, attenuated the PAF-induced cardiac depression. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester inhibited the PAF-induced early decrease in systemic vascular resistance. 4. All observed changes in haemodynamics and blood cell count after PAF were almost abolished by TCV-309, a PAF-receptor antagonist. 5. Reproducible hypotension and thrombocytopenia produced by a lower dose of PAF (0.05 microgram kg-1) were respectively attenuated and potentiated by pretreatment with NG-nitro-L-arginine, another nitric oxide synthase inhibitor. Administration of L-arginine reversed the effects of the nitric oxide synthase inhibitor. 6. These results indicate that PAF-receptor-mediated production of not only eicosanoids but also nitric oxide may contribute to the cardiovascular and haematological responses to PAF in the dog.

Changes in splenic diameter measured by sonomicrometry in response to various adrenergic stimulants were estimated together with simultaneously measured arterial haemoglobin content (HGB) and haematocrit (HCT) in anaesthetized dogs. Splenic diameter decreased following intravenous injections (i.v.) of adrenaline, noradrenaline and phenylephrine and splenic nerve stimulation associated with increases in arterial HGB and HCT, which were significantly attenuated by prazosin i.v. After prazosin i.v., adrenaline i.v. increased splenic diameter significantly, but noradrenaline i.v. did not. Isoprenaline i.v. increased splenic diameter transiently, followed by a decrease that was abolished by prazosin i.v. During occlusion of splenic arteries and veins, adrenaline i.v. and phenylephrine i.v. did not cause any change in arterial HGB and HCT. Injection to splenic artery (i.a.) of phenylephrine induced a significant decrease in splenic diameter that was attenuated by prazosin i.a. but not by yohimbine i.a. Clonidine i.a. did not change splenic diameter. The present results indicate that splenic contraction, which is mediated through alpha 1-adrenoceptor activation, causes a significant increase in arterial HGB and HCT.

The effects of "Kyushin" (KY), a Senso (toad venom)-containing drug, on the cardiovascular system were examined by intraduodenal administration of KY in anesthetized open-chest dogs. KY (3 or 10 mg/kg) dose-dependently increased the peak positive first derivative of left ventricular pressure ((+)LVdP/dt) and mean aortic pressure, and decreased the left ventricular end-diastolic pressure (LVEDP). Myocardial oxygen consumption (MVO2) and heart rate (HR) were not significantly influenced by KY. KY produced a cardiotonic effect without any increase in MVO2, because the increase in MVO2 due to the cardiotonic effect of KY may have been cancelled by a decrease in MVO2 due to reduction of preload and the lack of increase in HR. In order to clarify the relationship between the cardiovascular effects of KY and the drug concentration in plasma, the concentration of anti-bufalin IgG reactive substance (BRS) in plasma was measured by enzyme immunoassay. The maximum BRS concentrations 20 min after administration of 3 and 10 mg/kg KY were dose-dependent. From the relationship between changes in (+)LVdP/dt and changes in BRS concentration after administration of KY, it is inferred that the effective concentration of BRS in plasma at which KY produces a cardiotonic effect in dogs is approximately 2-3 ng/ml.

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.

Convergent synthesis of HIJK ring model compound 1 of ciguatoxin is described. The present synthesis relied on a palladium-catalyzed Suzuki cross-coupling reaction between eight-membered ketene acetal phosphate 2 and seven-membered alkylborane 6.

Cardiovascular effects of the crowns-of-thorns starfish ( Acanthaster planci ) venom were examined in rats. The crude venom extracted from the spines of A. planci caused systemic hypotension associated with an increase in heart rate and a decrease in renal cortical blood flow when given i.v. The hypotensive effect of the venom was not inhibited by pretreatment with atropine, indomethacin or aprotinin, but was significantly inhibited by SRI 63–441, a platelet activating factor (PAF) antagonist. The venom caused dose-dependent vasorelaxation of the isolated rat aortic ring preparation precontracted by noradrenaline, an effect which was significantly attenuated by pretreatment with SRI 63–441, methylene blue or parabromophenacyl bromide. Denudation of the endothelium also diminished the vasorelaxing effect of the venom. Both the vasorelaxing and the hypotensive effects showed tachyphylaxis. These results suggest the release of PAF or a PAF-like substance from the endothelium by the venom.

Effects of "Kyushin" (KY-2), a drug containing toad venom, on a low-output-type heart failure model produced in rabbits by protease treatment on the left ventricular anterior wall, were examined. Heart rate, aortic blood flow (AoF), left ventricular systolic pressure (LVP) and maximal rate of rise of LVP (max dP/dt) in this model were maintained at lower levels than those in normal rabbits, while left ventricular end-diastolic pressure (LVEDP) and systemic vascular resistance (SVR) were maintained at higher levels, and the mean blood pressure (MBP) was at a normal level. KY-2 was administered intraduodenally to the animal. KY-2 improved heart failure state by increasing the AoF, LVP and max dP/dt, and by decreasing the LVEDP and SVR without a significant change in MBP. These results suggest that the beneficial effects of KY-2 on this heart failure model originate from their cardiotonic activity.

Effects of a Senso (toad venom)-containing drug KY on systemic hemodynamics were examined, and participation of β-adrenoceptor in its action was evaluated by using propranolol in anesthetized dogs. KY produced a positive inotropic action, and decreased total peripheral (TPR) and coronary vascular resistances (CR), while renal vascular resistance (RR) was increased. After propranolol, KY significantly increased TPR, CR, vertebral vascular resistance and RR. KY-induced positive inotropic action was partly diminished but not abolished by β-blockade. These results indicate that the β-adrenergic action may be involved in the vasodilating effect of KY and partly in the positive inotropic action.

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.

Effects of a Senso (toad venom)-containing drug, KY, on cardiovascular system were examined in anesthetized open-chest dogs. KY increased aortic pressure, peak positive first derivative of left ventricular pressure, stroke work index, percent segment shortening in left ventricular myocardium and myocardial oxygen consumption, and decreased heart rate and total peripheral vascular resistance (TPR). Propranolol augmented the increase in aortic pressure with KY, inhibited the increase in aortic flow with KY and reversed KY-induced decrease in TPR to an increase. These results indicate that KY has positive inotropic and vasodilating actions possibly originating from both digitalis- and adrenaline-like action of a Senso.

Tetrahydrobiopterin (BH4) deficiency has been suggested to be an important factor in vascular endothelial dysfunction. In this study, we investigated the influence of decreased BH4 level produced by administration of 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor of the rate-limiting enzyme of BH4 synthesis, on vascular endothelial function in anesthetized rats. Wistar rats were given DAHP (0.1 – 1.0 g/kg, i.p.) or the vehicle 5 h before the experiment. Depressor responses to the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator sodium nitroprusside were tested. After the experiment, blood and thoracic aorta were taken for estimating their BH4 levels and plasma concentrations of nitrite plus nitrate. DAHP produced marked decreases in BH4 levels in plasma and aorta in a dose-related manner. Baseline values for hemodynamics were not affected by DAHP. Depressor responses to acetylcholine were attenuated with the highest dose of DAHP (1.0 g/kg) but not with DAHP (0.3 g/kg), although similar decreases in BH4 levels were seen with these two doses of DAHP. Treatment with DAHP at each dose did not decrease plasma concentrations of nitrite plus nitrate. These findings suggest that a decrease in BH4 levels by acute inhibition of de novo BH4 synthesis does not necessarily cause endothelial dysfunction. Keywords:: tetrahydrobiopterin, 2,4-diamino-6-hydroxypyrimidine, endothelial function, acetylcholine, sodium nitroprusside

Effects of arotinolol, a combined alpha- and beta-adrenoceptor blocking agent, on regional myocardial dysfunction produced by severe coronary stenosis in anesthetized dogs were examined and compared with those of labetalol and propranolol. Doses of these three antagonists were selected to produce a comparable degree of the negative chrono- and inotropic effect but a different potency of alpha-adrenoceptor blockade (labetalol greater than arotinolol). Regional myocardial function measured as segment shortening (%SS) was decreased to around 2-3% by constriction of the left circumflex coronary artery (LCX), and then drug or saline was administered i.v. The stenosis of LCX was released 30 min after the administration. No significant alteration in hemodynamic and contractility parameters was seen as compared to the predrug value up to at least 30 min after saline i.v. Arotinolol and propranolol both reduced heart rate and peak positive left ventricular dP/dt (LVdP/dt) without a significant change in LCX flow. Concomitantly, %SS distal to a coronary stenosis was significantly improved by arotinolol and propranolol. On the other hand, labetalol significantly reduced LCX flow probably due to systemic hypotension and failed to improve %SS in the ischemic area, although the agent markedly decreased heart rate and LVdP/dt. These results indicate that arotinolol improves impaired regional myocardial function distal to a coronary stenosis in a similar manner with propranolol.

SUMMARY 1 The effect of vasodilators on spleen volume and the blood storage function is not yet well elucidated. To this end, in the present study the effects of prostacyclin, a potent vasodilator, on splenic diameter and blood cell concentrations in arterial and splenic venous blood were evaluated in anaesthetized dogs. 2 The main splenic artery and vein were dissected for measurement of splenic arterial blood flow and intra-arterial administration and for sampling of splenic venous blood, respectively. The diameter of the spleen was measured continuously by sonomicrometry. Counts of white blood cells (WBC), red blood cells (RBC) and platelets in blood sampling from the aorta and splenic vein were estimated by an automatic blood cell counter. 3 Bolus injections of prostacyclin (1–100 ng/kg) into the splenic artery produced dose-dependent increases in splenic arterial blood flow and splenic diameter associated with significant decreases in splenic venous concentrations of WBC, RBC and platelets. When splenic blood flow was kept constant, similar changes in splenic diameter and blood cell counts were observed with prostacyclin injection. 4 Splenic dilation and haematological changes induced by prostacyclin were relatively more potent than those induced by prostaglandin E2, acetylcholine, nitroglycerin or isoproterenol when doses producing a comparable increase in splenic blood flow were compared. 5 Infusion of prostacyclin (100 ng/kg per min) into the splenic artery caused a marked increase in splenic diameter, with immediate reductions in splenic venous concentrations of WBC, RBC and platelets, followed by significant reductions in these cell counts in the general circulation. 6 These results indicate that prostacyclin produces potent and flow-independent splenic dilation that may contribute to a decrease in circulating blood cell concentrations.