Masuo Akahane

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.

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.

β-Adrenoceptor subtypes in the ureteral smooth muscle of rats, rabbits and dogs

Abstract We investigated the β-adrenoceptor subtypes mediating ureteral relaxation in rats, rabbits and dogs. The relaxing effects of β-adrenoceptor agonists were evaluated on KCl-induced ureteral contractions. The rank order of potency of the catecholamines tested was isoprenaline>noradrenaline>adrenaline in rat ureter; isoprenaline>adrenaline>noradrenaline in rabbit ureter; only isoprenaline was effective in canine tissues. The β1-adrenoceptor agonist, dobutamine, produced relaxation of rat ureter. The β2-adrenoceptor agonist, procaterol, produced more significant relaxation of rabbit ureter than did dobutamine. CL-316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethylamino]propyl]-1,3-benzodioxole-2,2-dicarboxylate] and CGP-12177A [(±)[4-[3[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride], β3-adrenoceptor agonists, were more effective in relaxing canine ureter than were dobutamine and procaterol. Isoprenaline-induced relaxation was antagonized by a β1-adrenoceptor antagonist, CGP-20712A [2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazole-2-yl)phenoxy)propyl)amino)ethoxy)-benzamide monomethane sulphonate], in rats and by a β2-adrenoceptor antagonist, ICI-118,551 [(±)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride], in rabbits. The non-selective β-adrenoceptor antagonist, bupranolol, antagonized isoprenaline-induced relaxation in all species tested. In conclusion, β-adrenoceptor agonists may relax ureter by stimulating mainly β1-adrenoceptors in rats, β2-adrenoceptors in rabbits and mainly β3-adrenoceptors in dogs.

Participation of thromboxane A2 in the cough response in guinea-pigs: antitussive effect of ozagrel

The purpose of this study was to investigate the involvement of thromboxane A2 (TXA2) in the cough response in a guinea‐pig cough model. Here, we describe results obtained using a selective TXA2 synthetase inhibitor, ozagrel, and a selective TXA2 agonist, U‐46619. Guinea‐pigs were anaesthetized and exposed to an aerosol of capsaicin (100 μM) to elicit coughing. The number of coughs was 20.0±5.8 during capsaicin provocation (5 min), but only 2.8±0.4 during a 5‐min inhalation of phosphate‐buffered saline (PBS) (P<0.05). TXB2 levels in BAL were 101.4±8.0 and 58.4±8.7 pg ml−1 following capsaicin and PBS inhalation, respectively (P<0.01), but there was no intergroup difference in the cell populations in BAL. Inhalation of U‐46619 did not induce a cough response by itself at concentrations of 100 ng ml−1 to 10 μg ml−1. However, it caused a 2 fold increase in the number of capsaicin‐induced coughs. To explore the source of the TXA2, BAL cells were stimulated with capsaicin and the supernatants collected for analysis. The TXB2 concentration in BAL was increased dose‐dependently, indicating that TXA2 is released from BAL cells in response to capsaicin. Ozagrel was administered orally 1 h before a 5 min capsaicin provocation and the number of coughs was counted during the capsaicin inhalation. Ozagrel decreased the number of coughs dose‐dependently (ED50 value, 26.3 mg kg−1). These results show that TXA2 modulates the capsaicin‐induced cough response by increasing capsaicin‐sensitivity.

β3-Adrenoceptor agonists for the treatment of frequent urination and urinary incontinence: 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acid

In a search for novel analogues of beta(3)-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl)phenoxy]-2-methylpropionic acids (1a-e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective beta(3)-AR agonist in functional assays using the ferret detrusor (beta(3)-AR), rat uterus (beta(2)-AR), and rat atrium (beta(1)-AR); beta(3): EC(50)=7.8 nM, beta(2): IC(50)=7,300 nM, beta(1): EC(20)=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved beta(3)-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED(50)=31 microg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b-e), possess beta(3)-AR agonistic activity in the absence of undesirable beta(1)- or beta(2)-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.

KRH-594, A New Angiotensin At1 Receptor Antagonist, Prevents End-Organ Damage In Stroke-Prone Spontaneously Hypertensive/Izm Rats

1. In the present study, we examined whether KRH‐594, a new angiotensin AT1 receptor antagonist, would stop the progression of renal failure and end‐organ damage and improve the survival rate in salt‐loaded stroke‐prone spontaneously hypertensive rats (SHRSP/Izm).

Krh-594, A New Angiotensin At1 Receptor Antagonist, Ameliorates Nephropathy And Hyperlipidaemia In Diabetic Spontaneously Hypertensive Rats

1. We examined whether KRH‐594, a new angiotensin AT1 receptor antagonist, ameliorates the progression of diabetic nephropathy and hyperlipidaemia in streptozotocin (STZ)‐induced diabetic unilateral nephrectomized spontaneously hypertensive rats (DM‐1K‐SHR) or not.

Prophylactic Effects Of Pilocarpine Hydrochloride On Xerostomia Models Induced By X-Ray Irradiation In Rats

1. In the present study, we investigated the prophylactic effects of pilocarpine hydrochloride on xerostomia models induced by either single (15 Gy) or repeated (8.6 Gy ×3 days) X‐ray irradiation in rats. Pilocarpine hydrochloride was administered orally 90 min before each irradiation session. Then, 7 days later, salivary volume, amylase activity and protein concentration in the saliva secreted from the right parotid gland were measured before and after a subsequent administration of pilocarpine hydrochloride (intraduodenal).

Comparison between CL-316243- and CGP-12177A-Induced Relaxations in Isolated Canine Ureter

We compared the effects of CL-316243, a selective β3-adrenoceptor agonist, and CGP-12177A, a nonconventional partial β-adrenoceptor agonist, on the KCl-induced contraction in the isolated canine ureter. CL-316243 concentration dependently relaxed the ureteral contraction, the pD2 value being 7.75 ± 0.11. This relaxation was competitively antagonized by the selective β3-adrenoceptor antagonist SR58894A and by the nonselective β-adrenoceptor antagonist bupranolol, their pA2 values being 7.08 ± 0.08 and 6.43 ± 0.09, respectively. CGP-12177A concentration dependently reduced the KCl-induced contraction, the pD2 value being 6.30 ± 0.25. Even at 1 × 10–5 mol/l, CGP-20712A (a selective β1- adrenoceptor antagonist) did not shift the concentration-response curves for CL-316243 or CGP-12177A. SR58894A did not induce a parallel rightward shift in the concentration-response curve for CGP-12177A, but bupranolol did produce such a shift, pA2 and slope values in the Schild plot being 7.15 ± 0.77 and 0.60 ± 0.15, respectively. Hence, the competition characteristics for SR58894A and bupranolol differed between the CL- 316243-induced and CGP-12177A-induced relaxations. Our results suggest that CGP-12177A produces ureteral relaxation in the dog via an atypical β-adrenoceptor (possibly, an atypical site/state of the β3-adrenoceptor) as well as via the typical β3-adrenoceptor.

KTO-7924, a Beta3-adrenergic Receptor Agonist, Reduces Hyperglycemia, and Protects Beta-cells in the Islets of Langerhans of db/db Mice

Introduction. The effect of beta3-adrenergic receptor agonists on beta cells in the islets of Langerhans is not yet clear. This study examined the beta3-adrenergic receptor agonist on beta cells in the islets of Langerhans. Methods. Obese diabetic C57BL/KsJ-db/db mice were treated with KTO-7924, a newly-developed beta3-adrenergic receptor agonist for 28-day. We analyzed plasma parameters, insulin resistance, and insulin-positive areas among beta-cells in the islets of Langerhans. Results and Conclusion. After a 28-day oral administration period, plasma levels of hemoglobin (Hb) A1c, glucose, triglyceride (TG), and free fatty acid (FFA) were all significantly reduced in KTO-7924 treatment groups compared with controls. Plasma adiponectin levels decreased with age in the control group, but were significantly higher in a treatment group throughout the study period. Furthermore, sequential administration of KTO-7924 led to an improvement in insulin resistance in the OGTT (Oral glucose tolerance test (OGTT)), and an increase in the percentage of insulin-positive areas among beta-cells in the islets of Langerhans compared with controls. This is the first study to show islet histology after treatment of a beta3-adrenergic receptor agonist, and reveals that KTO-7924 reduces hyperglycemia, and protects beta-cells in the islets of Langerhans of db/db mice.