Michiko Oka

Increased bladder activity is associated with elevated oxidative stress markers and proinflammatory cytokines in a rat model of atherosclerosis-induced chronic bladder ischemia.

To further characterize, in a rat model, the effects of atherosclerosis‐induced chronic bladder ischemia on bladder function and associated changes in oxidative stress markers and proinflammatory cytokines.

Suppression of Bladder Oxidative Stress and Inflammation by a Phytotherapeutic Agent in a Rat Model of Partial Bladder Outlet Obstruction

PURPOSE Ischemia/reperfusion injury is a major etiological factor in the progression of bladder dysfunction after partial bladder outlet obstruction and it is partly mediated by the generation of free radicals. The phytotherapeutic agent Eviprostat, a popular treatment for benign prostatic hyperplasia in Japan and Germany, has antioxidant and anti-inflammatory activity. We investigated the effect of Eviprostat on oxidative stress and inflammation in bladder dysfunction in a bladder outlet obstruction rat model. MATERIALS AND METHODS Bladder outlet obstruction was surgically induced in male rats by placing a rubber ring around the urethra. Rats with bladder outlet obstruction were administered daily oral Eviprostat or vehicle, while sham operated animals were treated with vehicle. On day 6 after surgery bladder weight, oxidative stress markers and proinflammatory cytokine levels as a measure of bladder inflammation, were determined and histological alterations noted. Functional contractility studies were performed with longitudinal bladder strips. RESULTS Bladder outlet obstruction led to a significant increase in bladder weight, oxidative stress markers and proinflammatory cytokine levels. Eviprostat significantly suppressed these increases without affecting bladder weight. Histological analysis showed increased detrusor muscle hypertrophy and increased numbers of collagen fibers with accompanying inflammatory infiltration in the bladder of vehicle treated bladder outlet obstruction animals. Eviprostat treatment was associated with suppression of these changes. Decreased responses of obstructed bladder strips to electrical stimulation and KCl were ameliorated by Eviprostat treatment. CONCLUSIONS Eviprostat mediated decrease of the increased oxidative stress and bladder inflammation caused by bladder outlet obstruction may contribute to the protection of bladder function.

Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture.

Abstract : The effect of 4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy)pyrimidine hydrochloride (NS‐7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)‐stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and ω‐agatoxin IVA (ω‐Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS‐7 blocked KCl‐stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS‐7 on L‐type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (±)‐Bay K 8644, whereas NS‐7 inhibited the maximal response without affecting the responses induced by low concentrations of (±)‐Bay K 8644. The effects of NS‐7 on L‐type and P/Q‐type Ca2+ channels involving KCl‐stimulated cyclic GMP formation were subsequently examined. NS‐7 suppressed the KCl‐stimulated cyclic GMP formation measured in the presence of ω‐Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS‐7 had no influence on ionomycin‐induced enhancement of cyclic GMP formation. Finally, NS‐7 reversed KCl‐induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS‐7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L‐type and P/Q‐type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.

Involvement of peroxynitrite and hydroxyradical generated from nitric oxide in hypoxia/reoxygenation injury in rat cerebrocortical slices

The changes in nitric oxide (NO) formation during hypoxia and reoxygenation were measured in slices of rat cerebral cortex, and the possible involvement of NO and its decomposition products, including peroxynitrite and hydroxyradical, in the hypoxia/reoxygenation injury was subsequently investigated. NO formation estimated from cGMP accumulation in the extracellular fluids was enhanced during hypoxia and to a lesser extent in the reoxygenation period. The mRNA for inducible NO synthase (NOS) was detected 3-5 h after reoxygenation, although neuronal NOS mRNA decreased after reoxygenation. Several NOS inhibitors such as N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine blocked not only the NO formation but also the hypoxia/reoxygenation injury as determined by lactate dehydrogenase (LDH) leakage. The hypoxia/reoxygenation injury was prevented by peroxynitrite scavengers including deferoxamine and uric acid, or several hydroxyradical scavengers such as dimethylthiourea, 2-mercaptopropionylglycine and D(-) mannitol. In addition, the hypoxia/reoxygenation injury was attenuated by poly(ADP-ribose)synthetase inhibitors such as banzamide, 3-aminobenzamide and 1,5-isoquinolinediol. On the other hand, both N-morpholinosidnonimine, a peroxynitrite generator, and hydroxyradical-liberating solution containing FeCl(3)-ADP and dihydroxyfumarate caused a marked LDH leakage in normoxic slices. These findings suggest that the enhanced formation of NO causes hypoxia/reoxygenation injury after degradation to peroxynitrite and hydroxyradical and the resultant activation of poly(ADP-ribose)synthetase.

Brain pertussis toxin-sensitive G proteins are involved in the flavoxate hydrochloride-induced suppression of the micturition reflex in rats

The effect of flavoxate hydrochloride (flavoxate), an anti-pollakiurea agent, on cyclic AMP (cAMP) formation was investigated in the rat brain and a possible involvement of brain G proteins in the action of flavoxate on the bladder function was subsequently examined. Flavoxate (10(-8)-10(-5) M) inhibited cAMP formation in a concentration-dependent manner, an action which was completely abolished by pretreating the membranes with pertussis toxin (PTX). The inhibitory effect of flavoxate was also completely antagonized by combined treatment with any two antagonists for adenosine A1 (8-cyclopentyl-1,3-dipropylxanthine), dopamine D2 (sulpiride) or adrenergic alpha 2 (yohimbine) receptors, although each antagonist alone did not significantly block the flavoxate-induced inhibition of cAMP formation. Radioligand binding studies indicated that flavoxate at micro- or submicromolar concentrations has affinity for Gi-coupled receptors such as A1, D2 and alpha 2 receptors. Therefore, flavoxate may inhibit cAMP formation by the stimulation of A1, D2 and alpha 2 receptors. To clarify the involvement of brain Gi proteins in the flavoxate-induced inhibition of the micturition reflex, the effect of pretreatment with PTX (i.c.v.) on the flavoxate-induced inhibition of isovolumetric rhythmic bladder contractions was examined in rats. Flavoxate (3 mg/kg, i.v.) completely abolished rhythmic bladder contractions in vehicle-pretreated rats, but not in PTX-pretreated rats. These findings suggest that signal transduction via Gi-coupled receptors is involved, at least in part, in the inhibition of the micturition reflex by flavoxate in rats. These results also provide the first evidence suggesting a negative role of brain PTX-sensitive G proteins in the micturition reflex.

Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na+ and Ca2+ channels in cultured human astrocytes.

We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 μM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin‐dependent NOS activity estimated from cGMP formation. The functional expression of voltage‐gated Na+ channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage‐gated Ca2+ channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl‐evoked NO synthesis was totally reversed by L‐type Ca2+ channel blockers such as nifedipine and verapamil, but not by ω‐conotoxin GVIA, an N‐type Ca2+ channel blocker, or ω‐agatoxin IVA, a P/Q‐type Ca2+ channel blocker. In addition, verapamil abolished the KCl‐induced increase in the intracellular free Ca2+ concentration. RT‐PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the α1 subunit of Ca2+ channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage‐gated L‐type Ca2+ channel as well as Na+ channel.

Eviprostat suppresses urinary oxidative stress in a rabbit model of partial bladder outlet obstruction and in patients with benign prostatic hyperplasia.

Eviprostat is a phytotherapeutic agent that has been used widely for more than 40 years in the treatment of benign prostatic hyperplasia (BPH) in Japan and Germany, and is known to have antioxidant activity. The present study investigated the effect of Eviprostat on the levels of the urinary oxidative stress marker 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in a rabbit model of surgical partial bladder outlet obstruction (PBOO) and in patients with lower urinary tract symptoms (LUTS) associated with BPH. In the rabbit model, 8‐OHdG levels in urine collected after 3 weeks of PBOO were 3.8‐fold higher than in the urine of sham‐operated rabbits. When twice‐daily Eviprostat was administered orally throughout the 3‐week PBOO period, the increase in urinary 8‐OHdG levels was suppressed by 70%. In the clinical study, nine patients who received Eviprostat for 4 weeks showed 2.5‐fold lower urinary 8‐OHdG levels than before treatment. During Eviprostat treatment, the total International Prostate Symptom Score (IPSS) decreased from 16.56 ± 2.74 to 13.67 ± 2.30 and the quality of life score from 4.22 ± 0.40 to 3.22 ± 0.46. The findings provide evidence that the antioxidant activity of Eviprostat is responsible for its beneficial effects in the treatment of BPH. Copyright

Inhibitions of urinary oxidative stress and renal calcium level by an extract of Quercus salicina Blume/Quercus stenophylla Makino in a rat calcium oxalate urolithiasis model.

Objectives:  To clarify the mechanism of Urocalun, an extract of Quercus salicina Blume/Quercus stenophylla Makino (QS), in the treatment of urolithiasis.

Calcium-dependent injury of human microvascular endothelial cells induced by a variety of iodinated radiographic contrast media

Rationale and objectivesThe aim of the present study was to determine the possible mechanisms underlying the endothelial cell damage induced by iodinated radiographic contrast materials (RCM). MethodsThe cultured human skin microvascular endothelial cells (HMVECs) were exposed to various contrast media, and the cell viability was measured by mitochondrial enzyme activity. Nuclear damage was assessed by Hoechst 33342 staining and a fluorescent single-cell gel electrophoresis. The effects of contrast materials on the cellular ATP content and intracellular free Ca2+ concentration were subsequently examined. ResultsAlthough the iodinated RCM tested all caused the cell injury in HMVECs, ionic RCM including amidotrizoate and ioxaglate were more potent in producing the cell damage than nonionic RCM. It is unlikely that the contrast material-induced cell damage is associated with hyperosmolality, since hyperosmolar solution of mannitol or NaCl had no marked influence on the endothelial cell viability. Nuclear damage was noted in cells exposed to amidotrizoate. Amidotrizoate lowered cellular ATP content while elevating the intracellular free Ca2+ concentration. It was notable that the RCM-induced endothelial cell damage was reversed by the chelation of intracellular Ca2+ with 1,2-bis(&ogr;-aminophenoxy)ethane-N, N, N ′, N ′-tetra-acetic acid but not by the removal of extracellular Ca2+. ConclusionsBoth ionic and nonionic contrast materials caused nuclear damage of endothelial cells. The decrease in tissue ATP content and elevation of intracellular Ca2+ are likely to contribute to the contrast materials-induced endothelial cell damage.

Release of endothelial nitric oxide in coronary arteries by celiprolol, a β1-adrenoceptor antagonist: possible clinical relevance

Mechanisms underlying celiprolol-induced vasodilatation were analyzed in isolated porcine coronary arteries. Celiprolol induced dose-related relaxation of the artery rings with endothelium, an effect which was suppressed by N(G)-nitro-L-arginine methylester (L-NAME), nitric oxide (NO) scavenger, guanylate cyclase inhibitor, endothelium denudation, and removal of Ca(2+). L-NAME contracted, and superoxide dismutase relaxed, the arteries only when the endothelium was preserved. Neither superoxide dismutase nor beta-adrenoceptor antagonists changed celiprolol-induced relaxations. Celiprolol increased the cyclic GMP content in the tissue. The release of NO from endothelium, estimated by the extracellular production of cyclic GMP in arteries incubated in medium containing guanylate cyclase and GTP, was augmented by celiprolol, and L-NAME abolished this action of celiprolol. It is concluded that celiprolol elicits relaxation by acting on sites other than beta-adrenoceptors in the endothelium and by releasing NO, which activates soluble guanylate cyclase in smooth muscle and produces cyclic GMP. Scavenging of superoxide anions from the endothelium does not seem to account for the induced relaxation.