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asic and Translational Science

echanical Function andene Expression of �1-Adrenoceptorubtypes in Dog Intravesical Ureter

hinya Kobayashi, Yoshitaka Tomiyama, Yuji Hoyano, Yoshinobu Yamazaki,iroshi Kusama, Yasue Kubota, Shoichi Sasaki, and Kenjiro Kohri

BJECTIVES To characterize the contractile functions and gene expression of the �1-adrenoceptor (AR)subtypes present in the dog intravesical ureter.

ETHODS In a functional study, �1-AR antagonists were evaluated against phenylephrine (�1-AR agonist)-induced contractions in dog isolated intravesical ureteral preparations. The quantitative expres-sion of �1-AR subtype mRNA in this tissue was determined using real-time quantitative reversetranscriptase-polymerase chain reaction.

ESULTS In the isolated intravesical ureter, prazosin (nonselective �1-AR antagonist), silodosin (selective�1A-AR antagonist), naftopidil (selective �1D-AR antagonist), and BMY-7378 (selective�1D-AR antagonist) all shifted the concentration-contractile response curve for phenylephrineto the right. The rank order of potencies (pKB value) was silodosin (9.45 � 0.14), prazosin (8.16 �0.08), naftopidil (7.39 � 0.19), and BMY-7378 (6.78 � 0.20). The �1A-AR antagonist silodosinwas much more potent than the 2 �1D-AR antagonists. The rank order of mRNA expressionlevels among the �1-AR subtypes was �1d (72.68%), �1a (24.14%), and �1b (3.18%).

ONCLUSIONS In the dog intravesical ureter, �1A-AR plays a major role in contraction, despite the prevalence

of �1D-AR. UROLOGY 74: 458–462, 2009. © 2009 Elsevier Inc.

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rolithiasis represents a common and importantproblem in daily urologic practice, affecting 8%-15% of the population in Europe and North

merica,1 with the stones frequently located in the distalortion of the ureter.2 Morita et al.3 noted that the densityf �1-adrenoceptors (ARs) was significantly greater thanhat of �2- and �-ARs in the dog ureter, and a similarnding has been reported for the human ureter, particularlyhe lower segment.4 Moreover, �1-AR antagonists haveeen found to inhibit ureteral contractions,5 an effect thateemed to accelerate expulsion of a ureteral stone. Severallinical trials have shown that �1-AR antagonists do pro-ote stone passage in patients with distal ureteral stones.6,7

hese reports have suggested that inhibition of distal ure-eral contractions was important for ureteral stone passagend that �1-ARs played a role in contraction in that portionf the ureter. Therefore, we focused on �1-AR subtypes inhe intravesical ureter (a part of the distal ureter) in theresent study. Three subtypes of �1-AR mRNA (�1a, �1b,nd �1d) have been reported to be expressed in the human

rom Pharmacology Research and Development, Kissei Pharmaceutical Company,imited, Nagano, Japan; and Department of Nephro-urology, Nagoya City Universityraduate School of Medical Sciences, Nagoya, Aichi, JapanReprint requests: Shinya Kobayashi, Pharmacology Research and Development,

issei Pharmaceutical Company, Limited, 4365-1, Kashiwabara, Hotaka, Azumino,

magano 399-8304 Japan. E-mail: shinya_kobayashi@pharm.kissei.co.jpSubmitted: July 10, 2008, accepted (with revisions): January 2, 2009

58 © 2009 Elsevier Inc.All Rights Reserved

istal ureter.8,9 Concerning the ureteral contractile func-ions of these various �1-AR subtypes, it has recently beeneported that stimulation of the �1A-AR subtype is mainlyesponsible for mediating ureteral contractions in hamstersnd humans.10,11 Although it is known that �1-AR agonistsnduce contractions in the porcine isolated intravesical ure-er,12 the identities of both the functional and the predom-nant �1-AR subtypes in this tissue remain unknown. It islso unclear which �1-AR subtypes participate in intraves-cal ureteral contractions in dogs. In the present study, weought to characterize the functional �1-AR subtypes me-iating contractions in the dog intravesical ureter and touantify the mRNA level for each subtype.

ATERIAL AND METHODS

nimalshis study was conducted according to guidelines approved by

he Laboratory Animal Committee of Kissei Pharmaceutical,nd it conformed to current Japanese Law. A total of 22 maleeagle dogs (10.5-13.2 kg, Nihon Nosan, Yokohama, Japan)ere maintained with a 12-hour light/12-hour dark cycle with

ree access to water and standard laboratory food until the dayf the experiment.

solation of Intravesical Ureterhe dogs were anesthetized with sodium pentobarbital (30

g/kg intravenously) and then killed with an overdose of the

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ame agent. The intravesical ureters were carefully dissectedree from the surrounding fatty tissues and fibrous tissues.

unctional Experimentntravesical ureters were cut into circular layer preparations (15 �

mm). The tissues were suspended longitudinally in a 10-mLrgan bath containing Krebs solution (NaCl, 118.1 nM; KCl,.7 nM; CaCl2, 2.5 nM; MgSO4 7H2O, 1.2 nM; NaHCO3, 25.0M; KH2PO4, 1.2 nM; glucose, 11.1 nM). This solution wasaintained at 37°C and continuously gassed with a mixture of

5% oxygen and 5% carbon dioxide. The tissue’s response toarious maneuvers was assessed using an isometric force-trans-ucer and measuring system (TB-611T, AP-601G, and RPM-004, Nihon Kohden, Tokyo, Japan) connected to a polygraphRecti-Horiz-8K, GE Marquette Medical System Japan, Tokyo,apan). An initial resting tension of 0.5 g was placed on eachegment, which was allowed to equilibrate with the solution forhour. Phenylephrine (�1-AR agonist) was cumulatively added

n 0.5-log increments. In the competition experiments, eachissue was equilibrated with 1 of the �1-AR antagonists for 2ours before the addition of any phenylephrine, with the phen-lephrine-induced contractions expressed as a percentage of theaximal response achieved in each preparation. Each prepara-

ion was exposed to only 1 antagonist and/or 1 agonist.

eal-Time Quantitative Reverseranscriptase-Polymerase Chain Reactionotal RNA was isolated from the tissues using Isogen (Nipponene, Tokyo, Japan), with DNase I treatment. cDNA was

ynthesized from 2 �g of total RNA using random 6 primer andrimeScript (Takara Bio, Otsu, Japan) in a final volume of 100L. The mRNA expression levels corresponding to a couple of

arget genes were determined by real-time quantitative reverseranscriptase-polymerase chain reaction using an Applied Bio-ystems 7500 real-time polymerase chain reaction system (Ap-lied Biosystems, Foster City, CA). Table 1 lists the sequencesf the oligonucleotides used as primers and probes. Primers andAM/TAMRA double-dye probes for dog �1-ARs were de-igned using Primer express, version 2.0 (Applied Biosystems).he polymerase chain reaction mix contained 5 �L cDNA

emplate, 10 �L of 2 � Premix Ex Taq (Takara Bio), a 3 �Moncentration of each of the primers for �1a-, �1b-, or �1d-AR,nd 2 �M �1a-, �1b-, or �1d-AR probe in a total volume of 20L. Gene expression was calculated by interpolation of the

hreshold cycle number values on standard curves generated

Table 1. Primers and double-dye probes used for real-time

Gene (Accession No.)

�1a (XM_534567)Forward primerReverse primerDouble-dye probe

�1b (XM_536449)Forward primerReverse primerDouble-dye probe

�1d (AY994052)Forward primerReverse primerDouble-dye probe

rom dilutions of plasmid DNAs containing dog �1a-, �1b-, or c

ROLOGY 74 (2), 2009

1d-AR. Reverse transcriptase-polymerase chain reaction waserformed in duplicate. All procedures were performed accord-ng to the manufacturer’s instructions, unless otherwise speci-ed.

rugsilodosin [KMD-3213, which is chemically (–)-1-(3-hydroxypropyl)--[(2R)-2-({2-[2-(2,2,2-trifluoro-ethoxy)-phenoxy]ethyl}amino)ropyl]-2,3-dihydro-1H-indole-7-carboxamide] and naftopi-il were synthesized in our laboratory (Kissei Pharmaceuti-al, Nagano, Japan). Phenylephrine hydrochloride, prazosinydrochloride, and BMY-7378 hydrochloride were purchased

rom Sigma-Aldrich (St. Louis, MO). Dimethyl sulphoxideas from Nacalai Tesque (Kyoto, Japan) and sodium pentobar-ital from Dainippon Pharmaceutical (Osaka, Japan). Silodo-in, naftopidil, and prazosin were dissolved in 100% dimethylulphoxide, followed by dilution with distilled water. The otherrugs were dissolved in distilled water. At the maximal con-entration reached in the bath (0.1%), dimethyl sulphoxidead no effect on the concentration-response curve for phen-lephrine. We used prazosin as a nonselective �1-AR antag-nist, silodosin as an �1A-AR antagonist, and naftopidil andMY-7378 as �1D-AR antagonists.13,14

tatistical Analysishe data are expressed as the mean � SEM. The pD2 value wasbtained by taking the negative logarithm of the molar halfaximal effective concentration value. The contractions in-

uced by phenylephrine were plotted as a percentage of theaximal increase to obtain each concentration-response curve.stimates of affinity are represented as the pKB value, calculatedith the following formula: pKB � log (CR � 1)-log (antago-ist), where CR is the ratio of phenylephrine concentrationshat induced a similar response (ie, half-maximal response)etween the presence and absence of the test drug. Tukey’s testas performed to assess the differences in pKB value amongetween all possible pairs of groups, with P � .05 consideredtatistically significant.

ESULTShenylephrine induced concentration-dependent tonicontractions in the dog intravesical ureter (Fig. 1), withhe pD2 value 5.09 � 0.11. The concentration-response

ntitative reverse transcriptase-polymerase chain reaction

Oligonucleotide Sequence

5=-TGGAAATGCCTCCGACAGTT-3=5=-AGAATGGCCTTGGATATGTTCAC-3=5=-CAACTGCACCCACCCGCCG-3=

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5=-TCGGAGGGCGTCTTCAAG-3=5=-GGGTAGATGAGCGGGTTCAC-3=5=-TCATCTTCTGGCTCGGCTACTTCAACAGC-3=

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urve for phenylephrine was shifted to the right by all 4

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f the �1-AR antagonists tested (Fig. 2). The rank orderf antagonist potencies (pKB value) was silodosin (�1A-ARelective, 9.45 � 0.14), prazosin (nonselective �1-ARntagonist, 8.16 � 0.08), naftopidil (�1D-AR selective,.39 � 0.19), and BMY-7378 (�1D-AR selective, 6.78 �.20; Table 2). The pKB value for the selective �1A-ARntagonist silodosin was significantly greater than thosebtained for the selective �1D-AR antagonists naftopidilnd BMY-7378.

Real-time quantitative reverse transcriptase-polymer-se chain reaction was performed using specific primersorresponding to the �1a-, �1b-, and �1d-AR sequencesTable 1). The expression of the various �1-AR mRNAubtypes was 5.0 � 1.9 copies/ng total RNA for �1a (n �), 0.7 � 0.2 copies/ng total RNA for �1b (n � 7), and5.0 � 5.5 copies/ng total RNA for �1d (n � 7). Theank order of mRNA expression levels among the �1-ARubtypes was �1d, �1a, and �1b (Fig. 3).

OMMENTecently, �1-AR antagonists have been reported to in-rease the stone expulsion rate in patients with a distalreteral stone.7 However, it is not known which �1-ARubtypes are involved in distal ureteral contractions inumans and other animal species. We therefore investi-ated the functional �1-AR subtypes mediating contrac-ions in the dog intravesical ureter (part of the distalreter) using pharmacologic techniques.In our mechanical functional experiment, the �1-AR

igure 1. (A) Typical tracing of concentration-dependentreparation from dog, with concentrations shown as log (ontractions induced by phenylephrine in such preparations

gonist phenylephrine produced tonic contractions of m

60

ntravesical ureters isolated from dogs, with the pD2 value5.09) similar to that reported for pigs (5.20).12 All 4 ofhe drugs tested inhibited the phenylephrine-inducedontractions in the dog intravesical ureter in a concen-ration-dependent manner, with each drug producing aightward shift in the concentration-response curve. TheKB value for the inhibitory effect of the �1A-AR antag-nist silodosin (9.45) was much greater than that ob-ained for the selective �1D-AR antagonists naftopidil7.39) and BMY-7378 (6.78) in this experiment. ThisKB value for silodosin was in good agreement with thealues reported for �1A-ARs in the human isolated pros-ate (9.44), dog prostate (9.64), and hamster ureter (pA2

alue 9.44).10,13,15 The pKB value we obtained for BMY-378 in the dog intravesical ureter (6.78) was muchmaller than that previously reported for an �1D-AR-ediated response (rat aorta, pKB value 8.90),14 suggest-

ng that the �1D-AR subtype plays little role in intraves-cal ureteral contractions, at least in the dog. The pKB

alue we obtained for naftopidil (7.39) was greater thanhat obtained for BMY-7378 (6.78) in the dog intraves-cal ureter in the present study, and the values for these

drugs were very similar to those published for the dogrostate for contractions mediated by way of �1A-AR.13

ecause naftopidil has a threefold greater affinity for1A-AR than BMY-7378,13 it probably acted on �1A-R, as well as on �1D-AR, in the dog intravesical ureter

n the present study. Thus, our results suggest that stim-lation of the �1A-AR subtype is mainly responsible for

ponse to phenylephrine in isolated intravesical ureteralylephrine) (mol/L). (B) Graph of concentration-dependentan � SEM from 15 experiments.

resphen

ediating contractions in the dog intravesical ureter.

UROLOGY 74 (2), 2009

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Next, we examined the expression levels of the various1-AR mRNA subtypes in the dog intravesical ureter.he mRNA for all 3 �1-AR subtypes were expressed in

his tissue, with the rank order �1d-AR (72.68%),1a-AR (24.14%), and �1b-AR (3.18%). This is consis-

ent with a previous report showing that �1a- and �1d-Rs are more abundant than �1b-AR in the human distal

reter.8,9

Thus, our data have demonstrated that the �1A-ARubtype plays the predominant role in contractile func-ion, with the �1D-AR subtype participating much less,n the dog intravesical ureter. However, �1d-AR mRNAas the most abundant of the 3 �1-AR subtypes in this

igure 2. Antagonism by (A) prazosin, (B) silodosin, (C) nions of intravesical ureters isolated from dogs. Mean � S

Table 2. pKB values for �1-adrenoceptor antagonists

Drug pKB Value

Silodosin 9.45 � 0.14*†‡

Prazosin 8.16 � 0.08§¶

Naftopidil 7.39 � 0.19�

BMY-7378 6.78 � 0.20

* P � .05, silodosin vs prazosin.† P � .05, silodosin vs naftopidil.‡ P � .05, silodosin vs BMY-7378.§ P � .05, prazosin vs naftopidil.¶ P � .05, prazosin vs BMY-7378.� P � .05, naftopidil vs BMY-7378.

issue. It has been found in human embryonic kidney i

ROLOGY 74 (2), 2009

EK293 cells that although the �1A-AR subtype is ex-ressed both on the cell surface and intracellularly, the1B-AR subtype is expressed only on the cell surface and

he �1D-AR subtype is expressed only intracellularly.16

ur finding of little participation of �1D-ARs in ureteralontraction is most likely explained by �1D-ARs noteing expressed on the smooth muscle cell membrane inhis tissue, either. However, additional investigation iseeded to clarify the precise location of each �1-ARubtype in the dog intravesical ureteral smooth muscle.

Phenylephrine produced tonic contractions in isolatedog intravesical ureters in this study and in porcine

idil, and (D) BMY-7378 of phenylephrine-induced contrac-f 4-7 experiments.

igure 3. Relative expression levels of �1-adrenoceptorubtype mRNA in dog intravesical ureter. Values are meanrom 7 experiments.

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odel using anesthetized dogs, phenylephrine elevatedhe basal ureteral pressure without enhancing peristal-is.17 Moreover, intravenous administration of noradren-line or phenylephrine reduced the bolus volume and theuid flow of urine in anesthetized dogs.18 It seems likelyhat stimulation of �1A-AR is involved in the mainte-ance of ureteral tonus and resistance.In patients with urolithiasis, the �1A/1D-AR antagonist

amsulosin facilitates spontaneous stone passage and re-uces the time to expulsion of the ureteral stone,6,7

ndicating the close relationship of �1A-AR and/or1D-AR stimulation to ureteral contraction. Very re-ently, Sasaki et al.11 reported that the �1A-AR is theain participant in phenylephrine-induced ureteral con-

raction in the human isolated ureter. Our finding thathe �1A-AR antagonist silodosin was more effective thanhe selective �1D-AR antagonist BMY-7378 againsthenylephrine-induced contractions in the dog intraves-cal ureter makes it likely that no species difference existsetween dogs and humans in terms of the �1-AR subtypeie, �1A) participating in lower ureteral contractions.10,11

ONCLUSIONShe results of our study have shown that the �1A-ARlays the major role in mediating intravesical (lower)reteral contractions in dogs, as it does in humans.

eferences1. Pak CY. Kidney stones. Lancet. 1998;351:1797-1801.2. Carstensen HE, Hansen JS. Stones in the ureter. Acta Chir Scand

Suppl. 1973;433:66.3. Morita T, Ando M, Kihara K, et al. Function and distribution of

autonomic receptors in canine ureteral smooth muscle. NeurourolUrodyn. 1994;13:315-321.

4. Healy KA, Ogan K. Nonsurgical management of urolithiasis: an

overview of expulsive therapy. J Endourol. 2005;19:759-767.

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5. Küpeli B, Irkilata L, Gürocak S, et al. Does tamsulosin enhancelower ureteral stone clearance with or without shock wave litho-tripsy? Urology. 2004;64:1111-1115.

6. Ukhal M, Malomuzh O, Strashny V. Administration of doxazosinfor speedy elimination of stones from lower part of ureter. Presentedat the XIV Congress of the European Association of Urology,Stockholm, Sweden, 1999.

7. Parsons JK, Hergan LA, Sakamoto K, et al. Efficacy of �-blockersfor the treatment of ureteral stones. J Urol. 2007;177:983-987.

8. Itoh Y, Kojima Y, Yasui T, et al. Examination of alpha 1 adreno-ceptor subtypes in the human ureter. Int J Urol. 2007;14:749-753.

9. Sigala S, Dellabella M, Milanese G, et al. Evidence for the presenceof �1-adrenoceptor subtypes in the human ureter. Neurourol Uro-dyn. 2005;24:142-148.

0. Tomiyama Y, Kobayashi K, Tadachi M, et al. Expressions andmechanical functions of alpha(1)-adrenoceptor subtypes in ham-ster ureter. Eur J Pharmacol. 2007;573:201-205.

1. Sasaki S, Itoh Y, Kojima Y, et al. Expression level and role inureteral contraction of alpha1-adrenoceptor subtypes in humanureter. J Urol. 2008;179(suppl):559.

2. Hernández M, Prieto D, Simonsen U, et al. Noradrenaline modu-lates smooth muscle activity of the isolated intravesical ureter ofthe pig through different types of adrenoceptors. Br J Pharmacol.1992;107:924-931.

3. Tatemichi S, Tomiyama Y, Maruyama I, et al. Uroselectivity inmale dogs of silodosin (KMD-3213), a novel drug for the obstruc-tive component of benign prostatic hyperplasia. Neurourol Urodyn.2006;25:792-799.

4. Goetz AS, King HK, Ward SD, et al. BMY 7378 is a selectiveantagonist of the D subtype of alpha 1-adrenoceptors. Eur J Phar-macol. 1995;272:R5-R6.

5. Murata S, Taniguchi T, Takahashi M, et al. Tissue selectivity ofKMD-3213, an �1-adrenoceptor antagonist, in human prostate andvasculature. J Urol. 2000;164:578-583.

6. Chalothorn D, Mccune DF, Edelmann SE, et al. Differences in thecellular localization and agonist-mediated internalization proper-ties of the �1A-AR subtypes. Mol Pharmacol. 2002;61:1008-1016.

7. Tomiyama Y, Murakami M, Akiyama K, et al. Modification ofureteral motility and promotion of urine flow around an intrau-reteral obstruction by CL-316243, phenylephrine, and furosemidein dogs. Neurourol Urodyn. 2002;21:251-257.

8. Morita T, Wada I, Suzuki T, et al. Characterization of �-adreno-ceptor subtypes involved in regulation of ureteral fluid transport.

Tohoku J Exp Med. 1987;152:111-118.

UROLOGY 74 (2), 2009