Yoshinobu Yamazaki

Functional and molecular biological evidence for a possible β3-adrenoceptor in the human detrusor muscle

The possible existence of a β3‐adrenergic receptor (β3‐AR) in the human detrusor muscle was investigated by in vitro functional studies and analysis of mRNA expression. Isoprenaline, noradrenaline and adrenaline each produced a concentration‐dependent relaxation of the human detrusor. The rank order for their relaxing potencies was isoprenaline (pD2 6.37±0.07) noradrenaline (pD2 6.07±0.12) adrenaline (pD2 5.88±0.11). Neither dobutamine (β1‐ and β2‐AR agonist) nor procaterol (β2‐AR agonist) produced any significant relaxation at concentrations up to 10−5 M. BRL37344A, CL316243 and CGP‐12177A (β3‐AR agonists), relaxed the preparations significantly at concentrations higher than 10−6 M. The pD2 values for BRL37344A, CL316243 and CGP‐12177A were 6.42±0.25, 5.53±0.09 and 5.74±0.14, respectively. CGP‐20712A (10−7–10−5 M), a β1‐AR antagonist, did not affect the isoprenaline‐induced relaxation. On the other hand, ICI‐118,551, a β2‐AR antagonist, produced a rightward parallel shift of the concentration‐relaxation curve for isoprenaline only at the highest concentration used (10−5 M) and its pKB value was 5.71±0.19. Moreover, SR58894A (10−7–10−5 M), a β3‐AR antagonist, caused a rightward shift of the concentration‐relaxation curve for isoprenaline in a concentration‐dependent manner. The pA2 value and slope obtained from Schild plots were 6.24±0.20 and 0.68±0.31. The β1‐, β2‐ and β3‐AR mRNAs were all positively expressed in detrusor smooth muscle preparations in a reverse transcription polymerase chain reaction assay. In conclusion, the present results provide the first evidence for the existence of the β3‐AR subtype in the human detrusor. They also suggest that the relaxation induced by adrenergic stimulation of the human detrusor is mediated mainly through β3‐AR activation.

Relaxant effects of isoproterenol and selective β3 : Adrenoceptor agonists on normal, low compliant and hyperreflexic human bladders

PURPOSE We compared the relaxant effects of the stimulation of beta-adrenoceptors with isoproterenol and of drugs selective for beta-adrenoceptor subtypes in detrusor preparations from patients with normal and neurogenic bladder, respectively. MATERIALS AND METHODS We studied in vitro preparations of a cystometrically normal, low compliant and hyperreflexic bladder from 45, 26 and 7 patients, respectively. RESULTS Isoproterenol relaxed concentration dependently and with the same potency as detrusor preparations obtained from normal and neurogenic bladders. In 37 normal detrusor, 25 low compliant and 7 hyperreflexic cases pD2 values were 6.36, 6. 25 and 6.38, respectively. Maximal relaxation did not differ significantly among the 3 groups (about 80% of 10-5 M. forskolin induced relaxation). Neither the beta1-/beta2-adrenoceptor agonist dobutamine nor the beta2-adrenoceptor agonist procaterol produced any significant relaxation of preparations from the 3 groups at a concentration of up to 10-5 M. At a concentration of 10-4 M. the preparations were relaxed but neither of these effects reached a maximum. BRL37344A and CL316243, selective beta3-adrenoceptor agonists and CGP-12177A (a selective beta3-adrenoceptor partial agonist and beta1-/beta2-adrenoceptor antagonist) relaxed detrusor preparations from the normal, low compliant and hyperreflexic groups when applied at concentrations greater than 10-6 M. For each agonist the pD2 value did not differ significantly among the 3 groups. CONCLUSIONS beta-adrenoceptor stimulation is an effective way of relaxing the human detrusor and the effect is similar in normal and neurogenic bladders. A major portion of the relaxant effect of isoproterenol is mediated via beta3-adrenoceptor stimulation. Clinical trials may reveal whether this method is useful for treating bladder overactivity.

Species differences in the distribution of β-adrenoceptor subtypes in bladder smooth muscle

The β‐adrenoceptor (β‐AR) subtypes mediating relaxation of the rabbit, rat and canine detrusors were subjected to functional investigation using selective β‐AR agonists and antagonists. In all three species, isoprenaline, noradrenaline and adrenaline each produced a concentration‐dependent relaxation of the detrusor. The rank order for their relaxing potency was isoprenaline> adrenaline>noradrenaline in rabbits and rats, but isoprenaline>noradrenaline>adrenaline in dogs. Dobutamine did not produce relaxation of the detrusors at concentrations that are selective for β1‐AR. The selective β2‐AR agonist, procaterol, had a more potent relaxing effect on rabbit and rat detrusors than on the canine detrusor. CGP‐12177A, a selective β3‐AR agonist, was more effective in the rabbit than in the other two species. On the other hand, the relaxing effect of another β3‐AR agonist, CL316243, was more pronounced in dogs and rats than in rabbits. CGP‐20712A (10−9 to 10−7 M), a selective β1‐AR antagonist, caused a slight rightward shift of the concentration‐relaxation response curve for isoprenaline in the canine detrusor (pA2 9.41), but not in the rabbit and rat detrusors. ICI‐118,551, a selective β2‐AR antagonist, antagonized the isoprenaline‐induced relaxation in rabbits (pA2 9.45) and rats (pA2 9.05), but not in dogs. Bupranolol, a non‐selective β‐AR antagonist, caused a rightward shift of the concentration‐relaxation curve for isoprenaline in the rabbit (pA2 9.32) and rat (pA2 8.98). However, higher concentrations (3×10−8 to 10−5 M) were needed to induce a rightward shift of the curve for isoprenaline in the dog (pA2 8.19) than in the other two species. We have confirmed that the distribution of β‐AR subtypes in the detrusor muscle varies significantly from species to species and we provide here the first evidence of the presence of β3‐AR in the detrusor. It is suggested that the relaxation induced by adrenoceptor agonists in urinary bladder smooth muscle may be mediated mainly via β2‐AR in rabbits, via both β2‐ and β3‐AR in rats, but mainly via β3‐AR in dogs.

Effects of Selective β2 and β3-Adrenoceptor Agonists on Detrusor Hyperreflexia in Conscious Cerebral Infarcted Rats

ABSTRACTPurpose: We evaluated the effects of β-adrenoceptor agonists on detrusor hyperreflexia in cerebral infarcted rats.Materials and Methods: To produce cerebral infarction in Sprague-Dawley rats the left middle cerebral artery was occluded by introducing a monofilament nylon thread into the artery. In sham operated rats the same artery was exposed but not occluded. After these operations cystometric and cardiovascular experiments were performed with no anesthesia or restraint.Results: After the operation bladder capacity was significantly decreased and voiding pressure was significantly increased in cerebral infarcted but not in sham operated animals. The difference in cerebral infarcted and sham operated rats was significant for each parameter (p <0.01). Post-void residual urine volume was not affected in either group. In the cerebral infarction group intravenous administration of CL316243 ([R,R]-5–2-[[2-(3-chlorophenyl-2-hydroxyethyl]-amino]propyl] -1,3-benzodioxole-2,2-dicarboxylate) (Kissei Centra...

[Alpha1-adrenoceptor subtype selectivity and organ specificity of silodosin (KMD-3213)].

The selectivity of silodosin (KMD-3213), an antagonist of alpha(1)-adrenoceptor (AR), to the subtypes (alpha(1A)-, alpha(1B)- and alpha(1D)-ARs) was examined by a receptor-binding study and a functional pharmacological study, and we compared its subtype-selectivity with those of other alpha(1)-AR antagonists. In the receptor-binding study, a replacement experiment using [(3)H]-prazosin was conducted using the membrane fraction of mouse-derived LM (tk-) cells in which each of three human alpha(1)-AR subtypes was expressed. In the functional pharmacological study, the following isolated tissues were used as representative organs with high distribution densities of alpha(1)-AR subtypes (alpha(1A)-AR: rabbit prostate, urethra and bladder trigone; alpha(1B)-AR: rat spleen; alpha(1D)-AR: rat thoracic aorta). Using the Magnus method, we studied the inhibitory effect of silodosin on noradrenaline-induced contraction, and compared it with those of tamsulosin hydrochloride, naftopidil and prazosin hydrochloride. Silodosin showed higher selectivity for the alpha(1A)-AR subtype than tamsulosin hydrochloride, naftopidil or prazosin hydrochloride (affinity was highest for tamsulosin hydrochloride, followed by silodosin, prazosin hydrochloride and naftopidil in that order). Silodosin strongly antagonized noradrenaline-induced contractions in rabbit lower urinary tract tissues (including prostate, urethra and bladder trigone, with pA(2) or pKb values of 9.60, 8.71 and 9.35, respectively). On the other hand, the pA(2) values for antagonism of noradrenaline-induced contractions in rat isolated spleen and rat isolated thoracic aorta were 7.15 and 7.88, respectively. Selectivity for lower urinary tract was higher for silodosin than for the other alpha(1)-AR antagonists. Our data suggest that silodosin has a high selectivity for the alpha(1A)-AR subtype and for the lower urinary tract.

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim:  The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model.

Functional investigation of β-adrenoceptors in human isolated detrusor focusing on the novel selective β3-adrenoceptor agonist KUC-7322

This study aimed to characterize the β-adrenoceptor (β-AR) subtype mediating relaxation of isolated human bladder strips and to explore relaxation by the novel β3-AR-selective agonist KUC-7322 for its relaxant effect on the human isolated detrusor and for its effect on the carbachol (CCh)-induced contractile response. In two parallel studies, relaxation of isolated human bladder strips was tested for the β-AR agonists isoproterenol, clenbuterol, BRL 37344, and KUC-7322. For the isoproterenol and KUC-7322 responses, antagonism by CGP 20712A, ICI 118551, and SR59230A was determined. The potency and efficacy of the reference agonists for detrusor relaxation was in line with their known β3-AR activity. KUC-7322 relative to isoproterenol was a full agonist with a pEC50 of 5.95 ± 0.09 and 5.92 ± 0.11 in the two studies. SR59230A exhibited antagonism of the expected potency against isoproterenol (apparent pKB 7.2) but not against KUC-7322. Neither isoproterenol nor KUC-7322 nor forskolin significantly attenuated CCh-induced contraction. These results suggest that KUC-7322 displays full agonistic activity in relaxing the human detrusor without inhibiting the contraction induced by cholinergic stimulation. These characteristics, if proven in vivo, may be beneficial for the treatment of overactive bladder, as increased bladder capacity with a negligible effect on voiding contractions may be anticipated.

Effects of Ritobegron (KUC-7483), a Novel Selective β3-Adrenoceptor Agonist, on Bladder Function in Cynomolgus Monkey

We evaluated the pharmacological profile of ritobegron [KUC-7483; (−)-ethyl 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)-2,5-dimethylphenyloxy]acetate monohydrochloride] and its effects on the bladder in cynomolgus monkeys by in vitro and in vivo experiments. In vitro, ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC50 8.2 ± 2.3 × 10−7 M; maximal relaxation 88.7 ± 3.7%). The β3-adrenoceptor (AR) antagonist 3-(2-allylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol hydrochloride (SR58894A) produced a rightward shift of this concentration-response curve without altering the maximal response (pKB value 6.56 ± 0.35). In isolated atria, ritobegron increased the atrial rate only at high concentrations (EC50 6.5 ± 1.2 × 10−5 M). Ritobegron had no effect on tracheal contraction at concentrations from 10−9 to 10−4 M, and even at the highest concentration tested, 10−3 M, the maximal relaxation it induced was only 26.7 ± 8.1%. Tests of the selectivity of ritobegron for the bladder gave values of 79.3- and 1200-fold higher versus atria and trachea, respectively. In the in vivo study ritobegron significantly decreased intravesical pressure (ED50 1.44 mg/kg) without affecting either mean blood pressure or heart rate. In conclusion, ritobegron displayed potent and selective β3-AR agonistic activity and relaxed the monkey isolated bladder, and in vivo it decreased intravesical pressure without affecting cardiovascular parameters. These results suggest that ritobegron may be a promising potential agent for the treatment of overactive bladder.

Characterization of β-adrenoceptor subtypes in the ferret urinary bladder in vitro and in vivo

Abstract In the present study, the β-adrenoceptor subtypes distributed in the detrusor of the ferret were investigated in functional experiments in vitro and in vivo using a variety of β-adrenoceptor agonists and antagonists. All the β-adrenoceptor agonists tested relaxed the isolated detrusor strip, the rank order of potency being (±)-( R *, R *)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]phenoxy]-acetic acid sodium (BRL 37344A)>(±)-4-(3- t -butylamino-2-hydroxypropoxy) benzimidazol-2-one (CGP-12177A), isoprenaline and ( R , R )-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethylamino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316,243)>dobutamine and procaterol. In antagonist experiment, 3-(2-allylphenoxy)-1-[(1 S )-1,2,3,4-tetrahydro-naphth-1-ylamino]-(2 S )-2-propanol hydrochloride (SR 58894A), but neither 2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1 H -imidazole-2-yl)-phenoxy)propyl)amino)ethoxy)-benzamide monomethane sulphonate (CGP-20712A) nor erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol hydrochloride (ICI-118,551), caused a rightward shift of the concentration-relaxation curve for isoprenaline. In in vivo experiments, isoprenaline and CL 316,243 each reduced bladder pressure in a dose-dependent manner. CL 316,243 was the only drug that did not produce any significant influences on blood pressure and heart rate at doses that reduced bladder pressure. The present functional study provides the first evidence that relaxation of the ferret detrusor by β-adrenoceptor activation is mediated mainly via the β 3 -adrenoceptor, as in the human detrusor.

Effects of silodosin and tamsulosin on the urethra and cardiovascular system in young and old dogs with benign prostatic hyperplasia

We examined whether the effects (efficacy on the urethra and hypotension) of silodosin (alpha(1A)-adrenoceptor antagonist) and tamsulosin (alpha(1A+1D)-adrenoceptor antagonist) in dogs with benign prostatic hyperplasia altered with age. We used young and old dogs, diagnosed as having benign prostatic hyperplasia by veterinarians palpation. Under anesthesia, the increase in intraurethral pressure evoked by hypogastric nerve stimulation was measured, together with the level of systemic mean blood pressure. Each drug was administered intravenously in progressively increasing doses. At the end of the experiment, the prostate was isolated from each dog, then weighed and investigated pathologically to confirm benign prostatic hyperplasia. The wet weight of the prostate was greater in old dogs with benign prostatic hyperplasia than in young dogs with benign prostatic hyperplasia. By light microscopy, hyperplasia in the prostatic epithelium was confirmed in both groups. Silodosin (0.3-300 microg/kg) dose-dependently inhibited the hypogastric nerve stimulation-induced increase in intraurethral pressure (without significant hypotensive effects) in both young and old dogs with benign prostatic hyperplasia. Tamsulosin (0.3-300 microg/kg) also dose-dependently inhibited the intraurethral pressure increase in both groups, but it had a hypotensive effect that was significantly greater in old than in young dogs with benign prostatic hyperplasia. In conclusion, as regards the effect of silodosin on intraurethral pressure, potency was similar between young and old dogs with benign prostatic hyperplasia, and it was without significant hypotensive effects. We therefore suggest that silodosin might be a good medication for lower urinary tract symptoms in patients with benign prostatic hyperplasia in all age groups.