Kazunori Kimura

Photomanipulation of Vasodilation with a Blue-Light-Controllable Nitric Oxide Releaser

Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluorogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.

Upregulation of KCa3.1 K+ channel in mesenteric lymph node CD4+ T lymphocytes from a mouse model of dextran sodium sulfate-induced inflammatory bowel disease

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1/KCNN4 plays an important role in the modulation of Ca(2+) signaling through the control of the membrane potential in T lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in a mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5% dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4(+) T lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp, and flow cytometric analyses. Concomitant with an upregulation of KCa3.1a and nucleoside diphosphate kinase B (NDPK-B), a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4(+) T lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results: 1) a significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4(+) T lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the upregulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4(+) T lymphocyte and, therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.

Chronic vardenafil treatment improves erectile function via structural maintenance of penile corpora cavernosa in rats with acute arteriogenic erectile dysfunction.

INTRODUCTION Chronic phosphodiesterase type 5 inhibitor treatment may be useful in reversing erectile dysfunction (ED). However, the mechanisms of this improvement remain unknown. AIM The aim of this article was to determine the mechanisms of the improvement by chronic vardenafil treatment for acute arteriogenic ED in rats. METHODS Eight-week-old male Wistar-ST rats were divided into four groups: sham-operated rats (Control group) and rats with acute arteriogenic ED induced by ligating bilateral internal iliac arteries (Ligation group), subsequently treated with low-dose (0.4 mg/kg/day; VL group) or high-dose (4.0 mg/kg/day; VH group) vardenafil for 20 days from 1 week after ligature. MAIN OUTCOME MEASURES Erectile function was assessed based on changes of intracavernous pressure (ICP) followed by electrostimulation of the cavernous nerves and was evaluated by the area under the curve of ICP/area under the curve of mean arterial pressure (area of ICP/MAP). Transforming growth factor (TGF)-β(1), vascular endothelial growth factor-A, endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS mRNA expression levels in penile corpus cavernosum were determined by real-time PCR. Western blotting for TGF-β(1) protein levels and Masson trichrome staining of penile tissues were performed in each at group 4 weeks after surgery. RESULTS In the VH group, area of ICP/MAP was significantly improved when compared with the Ligation group (P < 0.01). The smooth muscle (SM)/collagen ratio in the VH group was significantly higher than in the Ligation group (P < 0.05), and was comparable with that in the Control group. TGF-β(1) mRNA and protein levels in the VH group were significantly lower when compared with the Ligation group (P < 0.05). CONCLUSIONS Chronic vardenafil administration ameliorates impairment of penile hemodynamics and maintains normal SM to collagen ratio in cavernous tissues after acute arterial injury in rats.

Oral l‐Citrulline Supplementation Improves Erectile Function in Rats with Acute Arteriogenic Erectile Dysfunction

INTRODUCTION Oral L-citrulline supplementation increases serum L-arginine levels more efficiently than L-arginine itself and increases nitric oxide (NO) production. AIM To investigate whether oral L-citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction (ED). METHODS We divided 8-week-old male Wistar-ST rats into 3 groups: sham-operated rats (control group), arteriogenic ED rats who underwent ligation of both internal iliac arteries (ligation group), and arteriogenic ED rats receiving oral 2% L-citrulline water supplementation (citrulline group). Citrulline water was given to arteriogenic ED rats for 3 weeks from 1 week after surgery. Erectile function was evaluated by maximum intracavernous pressure/mean arterial pressure (ICP/MAP) ratios via cavernous nerve stimulation at 4 weeks after surgery. Then, the penises were resected, stained with Massons trichrome, and observed microscopically. Serum nitrogen oxides (NOx) levels were measured by high-performance liquid chromatography. Bonferronis multiple t-test was used for statistical analysis. MAIN OUTCOME MEASURES The main outcome measures were changes in ICP/MAP, smooth muscle (SM)/collagen ratios, and NOx levels following L-citrulline supplementation. RESULTS The ICP/MAP ratio in the ligation group was significantly lower than that in the control group (P<0.05), denoting ED. The ICP/MAP ratio of the citrulline group was significantly higher than that of the ligation group (P<0.05), indicating ED amelioration. Levels of NOx in the ligation group were significantly lower than in the control group (P<0.05), while those in the citrulline group were significantly higher than in the ligation group (P<0.05). SM/collagen ratios in the ligation group were significantly lower than in the control group (P<0.05), while ratios in the citrulline group were significantly higher than those in the ligation group (P<0.05). CONCLUSIONS Oral L-citrulline supplementation improved ICP/MAP and SM/collagen ratios and increased NOx. Therefore, oral L-citrulline supplementation might be a useful novel therapy for acute arteriogenic ED.

Assessment of Androgen Replacement Therapy for Erectile Function in Rats with Type 2 Diabetes Mellitus by Examining Nitric Oxide-Related and Inflammatory Factors

INTRODUCTION Type 2 diabetes mellitus (T2DM) has become a major public health issue and is considered a risk factor for erectile dysfunction (ED). T2DM is also associated with androgen deficiency. However, there have been few basic studies on androgen replacement therapy (ART) for ED treatment in T2DM animal models, and the mechanism underlying the effect of ART on T2DM-induced ED is unclear. AIM To investigate the effect of ART on ED in T2DM rats by examining inflammatory and nitric oxide (NO)-related factors. METHODS Otsuka Long-Evans Tokushima Fatty (OLETF) rats and their controls, Long-Evans Tokushima Otsuka (LETO) rats, were distributed into three groups: LETO, OLETF, and ART. In the ART group, OLETF rats were treated daily with testosterone (3 mg/kg/day, subcutaneously) from 20 to 25 weeks of age; LETO and OLETF rats received vehicle only. MAIN OUTCOME MEASURES We measured erectile function by using measurements of the ratio between intracavernosal pressure (ICP) and mean arterial pressure (MAP) following electrical stimulation of the cavernous nerve and by evaluating the endothelial function of the corpus cavernosum in an isometric tension study. Expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS), sirtuin-1 (Sirt1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA was detected using polymerase chain reaction. RESULTS The ICP/MAP ratio in the OLETF group was significantly decreased and that in the ART group was significantly improved (P < 0.01). The response to acetylcholine was significantly decreased in the OLETF group and improved in the ART group (P < 0.01). Although expression of eNOS and Sirt1 mRNA was decreased and that of iNOS, IL-6, and TNF-α mRNA was increased in the OLETF group, ART improved mRNA expression. CONCLUSIONS ART suppressed inflammation in rats with T2DM and metabolic disorders and improved their endothelial and erectile functions. ART could be effective for T2DM-induced ED and may be considered a potential ED treatment method.

Oral L-citrulline supplementation improves erectile function and penile structure in castrated rats.

To investigate the efficacy of oral l‐citrulline for erectile dysfunction and penile structure disruption in a rat model.

Effects of Chronic Vardenafil Treatment Persist after End of Treatment in Rats with Acute Arteriogenic Erectile Dysfunction

INTRODUCTION In our previous study, chronic vardenafil treatment improved erectile function soon after the end of the treatment in rats with acute arteriogenic erectile dysfunction (ED). AIM The aim of this study is to evaluate whether the effects of chronic vardenafil treatment persist after the end of treatment using rats with acute arteriogenic ED. METHODS Rats were randomly divided into three groups: (i) control; (ii) ligation; and (iii) vardenafil + no treatment. Rats in the ligation and vardenafil + no treatment groups underwent ligature of the bilateral internal iliac arteries to induce acute arteriogenic ED and were subsequently treated with vehicle or vardenafil (4.0 mg/kg/day), respectively, for 3 weeks. Subsequently, all rats were kept for a further 2 weeks with no treatment. Rats in the control group underwent sham surgery. MAIN OUTCOME MEASURES Erectile function was assessed by changes in intracavernous pressure (ICP). Smooth muscle (SM)/collagen ratios in corpus cavernosum were analyzed by Masson trichrome staining. Transforming growth factor-β1 (TGF-β1 ) mRNA and protein levels in corpus cavernosum (CC) were, respectively, evaluated by real-time polymerase chain reaction (PCR) analysis and Western blotting analysis. RESULTS ICP/mean arterial pressure (MAP) in the ligation group remained significantly lower than that in control group (P < 0.01). Despite no treatment for 2 weeks, ICP/MAP in the var + no treatment group remained significantly higher than that in ligation group (P < 0.05). SM/collagen ratio in the ligation group remained significantly lower when compared with the control group (P < 0.01). The ratio in the var + no treatment group remained significantly higher when compared with the ligation group at 2 weeks after the end of treatment (P < 0.05). TGF-β(1) mRNA and protein levels did not differ among the groups. CONCLUSIONS The effects of chronic vardenafil treatment on erectile function and penile structure persist, even after the end of treatment, in acute arteriogenic ED rats.

Kinetic and molecular orbital analyses of dicarboxylic acylcarnitine methylesterification show that derivatization may affect the screening of newborns by tandem mass spectrometry

Newborns are routinely screened for organic acidemias by acylcarnitine analysis. We previously reported the partial catalytic methylesterification of dicarboxylic acylcarnitines by benzenesulfonic acid moiety in the solid extraction cartridge during extraction from serum. Since the diagnosis of organic acidemias by tandem mass spectrometry is affected by the higher molecular weight of these derivatized acylcarnitines, we investigated the methylesterification conditions. The kinetic constants for the methylesterification of carboxyl groups on the acyl and carnitine sides of carnitine were 2.5 and 0.24h(-1), respectively. The physical basis underlying this difference in methylesterification rates was clarified theoretically, illustrating that methylesterification during extraction proceeds easily and must be prevented.

Mineralocorticoid receptor stimulation induces urinary storage dysfunction via upregulation of epithelial sodium channel expression in the rat urinary bladder epithelium

We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10-12-week-old, male Sprague-Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms.

035 The mechanisms of stress-induced erectile dysfunction; a focus on regulators of contraction and relaxation in the corpus cavernosum by using a rat model of water immersion-restraint stress

application of sildenafil-np may represent a significant improvement because of potentially lowered doses for efficacy, improved safety profile, speed of efficacy and the potential to treat patients that are refractory to existing treatments. This new data shows a statistically improved erectile function when sildenafil-np is topically applied on the corpora (local) but no improvement when applied to the abdomen(distal) suggesting the lack of systemic effect on local application. Disclosure: Work supported by industry: no.