Phenotypic Characterization of the Endocannabinoid-Degrading Enzyme Alpha/Beta-Hydrolase Domain 6 Knockout Rat.

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 E2 (PGE2) and the stable metabolite of PGE2 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 PGE2.

Deletion of the lysyl oxidase-like 1 gene induces impaired elastin fiber synthesis and inefficient urethral closure in rats.

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.

[ASSOCIATION OF BREATH HYDROGEN CONCENTRATION WITH ORAL INTAKE AND URINARY DISEASES].

(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.

Vanilla scent reduces frequency of urination in urethane-anesthetized rats.

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.

Mirabegron causes vesical and urethral relaxation in rats with spinal cord injury.

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.

Spinal glycinergic and gamma-aminobutyric acid-ergic neurons inhibit the micturition reflex after electrical stimulation of the perineum in rats with pelvic venous congestion.

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.

Evaluation of a rat model of functional urinary bladder outlet obstruction produced by chronic inhibition of nitric oxide synthase.

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.

Pelvic venous congestion induces lower urinary tract dysfunction in rats.

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.

Long-term dietary nitrite and nitrate deficiency causes the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

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.

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.

Long-term treatment with san'o-shashin-to, a kampo medicine, markedly ameliorates cardiac ischemia-reperfusion injury in ovariectomized rats via the redox-dependent mechanism.

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.

VPS35 mutation in Japanese patients with typical Parkinson's disease.

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 mutations. We screened 7 reported nonsynonymous missense 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 controls. 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 substitution may be a mutational hot spot across different ethnic populations. Based on the clinical features, VPS35 should be analyzed in patients with PD, especially autosomal dominant PD or tremor-predominant PD.

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.

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.

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.

Effect of decreased levels of intrinsic tetrahydrobiopterin on endothelial function in anesthetized rats.

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.

Vascular lipotoxicity: endothelial dysfunction via fatty-acid-induced reactive oxygen species overproduction in obese Zucker diabetic fatty rats.

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 ED(50) value was higher in ZDF (-6.80 +/- 0.05 vs. -7.11 +/- 0.05 log(10) 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 (ED(50), -7.16 +/- 0.03 and -7.14 +/- 0.05 log(10) 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.

Prostacyclin causes splenic dilation and haematological change in dogs.

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 E(2), 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.

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats.

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2-5 microg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 microg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET(A) receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 microg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 microg/kg venom and 6 of 6 rats at 10 microg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 microg/kg) exerted only modest effects and the rats survived. Heating venom (50 degrees C for 10 min) before injection practically abolished the haemodynamic effects of 10 microg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.