백서에서난소절제술후여성호르몬투여가방광에미치는영향 The Effects of Estrogen on Detrusor Contraction and the Expression of Muscarinic Receptors in Ovariectomized Rats Ho Young Chung, Jin Seok Chang, Se Hun Kim 1, Ki Hak Song, Young Seop Jang, Dong Seok Han From the Departments of Urology and 1 Physiology, Konyang University College of Medicine, Daejeon, Korea Purpose: The purpose of this study was to evaluate the effects of estrogen on detrusor contraction and the expression of muscarinic receptors in ovariectomized rats. Materials and Methods: 24 Sprague-Dawley female virgin rats (12 weeks old) were separated into three groups of 8 rats each. Group I served as a control group, group II was the ovariectomized only rats (Ovx group) and Group III was given estradiol benzoate (0.8mg/kg/day) subcutaneously for 7 consecutive days, beginning 1 week after ovariectomy (Ovx+E group). At the end of the experimental period, each rat was sacrificed and the urinary bladder was removed for contractile studies. The expressions of M 2 and M 3 receptors in the bladder epithelium and the muscle layer were investigated by performing immunofluorescent staining. Results: The Ovx group showed a significantly decreased bladder contractile function on the KCl and carbachol-induced contractile tests, whereas the Ovx+E group showed increased contractility (p<0.05). The Ovx+E group showed an increase of smooth muscle compared to the other groups. Ovariectomy induced a significant increase in the M 3 receptors density in the bladder body, as compared to the control group (p<0.05) but there was no significant difference between the Ovx group and the Ovx+E group. Conclusions: Bladder dysfunction of menopausal women is thought not to be related with the changes of muscarinic receptors. Our results suggest that the detrusor contractility of menopausal women might be improved after estrogen replacement therapy. (Korean J Urol 2008;49:1105-1111) Key Words: Ovariectomy, Estrogens, Muscarinic receptors, Urinary bladder, Muscle contraction 대한비뇨기과학회지제 49 권제 12 호 2008 건양대학교의과대학비뇨기과학교실, 1 생리학교실 정호영ㆍ장진석ㆍ김세훈 1 송기학ㆍ장영섭ㆍ한동석 접수일자 :2008년 7월 24일채택일자 :2008년 10월 10일 교신저자 : 한동석건양대학교의과대학비뇨기과학교실대전시서구가수원동 685 302-718 TEL: 042-600-9225 FAX: 042-542-3790 E-mail: hds@dreamwiz. com 이논문은 2006 년도건양대학교명곡학술연구비의지원에의하여이루어진것임. 서론폐경기여성에서여성호르몬의부족으로방광의기능장애가발생하는경향이있으며, 1 여성호르몬의변화가뇨절박, 빈뇨, 저활동성방광, 요도및골반근육의약화에주된영향을미치는것으로알려져있다. 2 이에대해여성호르몬 (estrogen) 은여성비뇨생식기의생리적유지에필수적이며, 3,4 여성호르몬대체치료가복압성요실금이있는폐경기 여성에게방광경부혈류량을증가시킬뿐만아니라, 5 뇨절박증상을의미있게감소시킨다고하였다. 4 또한, 동물실험에서난소절제에의한여성호르몬의결핍이점막의위축, 평활근 (smooth muscle) 세포의퇴화, 혈류량의감소및점막과평활근의저산소증을유발하여결국방광수축력을감소시키지만, 여성호르몬의대체치료가위와같은현상을되돌릴수있다고보고하고있으며, 6-8 방광의콜라겐대비평활근의비율을증가시킴으로써방광의수축력을증가시킨다고보고하고있다. 9 1105
1106 대한비뇨기과학회지 : 제 49 권제 12 호 2008 방광에는무스카린수용체아형중에서 M 2, M 3 가많으며, 방광의수축에중요한역할을한다고하였으나, 10 여성호르몬대체치료가이러한무스카린수용체에어떠한영향을미치는지에대해서는아직까지논란의여지가있다. 11,12 본연구에서는난소절제술을시행한처녀백서에게여성호르몬대체치료를시행하였을때방광의수축력의변화및무스카린수용체아형의분포변화를살펴보고자하였다. 재료및방법 1. 실험재료실험동물로는생후 12주된 230-270g의암컷처녀백서 (Sprague-Dawley) 24마리를사용하였다. 대조군, 난소절제술 (ovariectomized; Ovx) 군, 난소절제술후여성호르몬치료 (ovariectomized and estradiol treated; Ovx+E) 군으로각각 8 마리씩나누어실험하였으며, Ovx군과 Ovx+E군은 12주에난소절제술을각각시행하였다. 마취는 ketamine 100mg/kg 를사용하였고복강내투여후수술을시행하였다. 양측난소절제술은복와위 (prone position) 에서 0.5cm의양측 subcostal incision을통해시행하였다. 호르몬대체치료는 estradiol benzoate 20mg/10ml 제제를이용하였으며, 양측난소절제술후 1주일뒤부터 1주일간매일 0.1ml (0.8mg/kg) 를피하주사하였다. 2. 배뇨근수축실험 12주령의각군에속한쥐를난소절제술과여성호르몬치료시행후경추탈골로즉사시킨뒤에방광을적출하였다. 적출한방광을실온에서 100% 산소로포화된 HEPES 완충정상생리용액 (Nacl 140mM, KCl 4mM, CaCl 2 2mM, MgCl 2 1mM, HEPES 5mM, glucose 11mM, ph 7.4) 속에서 10배확대되는 stereoscope로관찰하면서주변결합조직을제거한후방광체부의가장넓은부분에서폭 2mm인고리모양의실험절편을제작하였다. 제작된실험절편을 100% 산소가공급되며, 37 o C가유지되는 HEPES 완충정상생리용액 (Nacl 140mM, KCl 4mM, CaCl 2 2mM, MgCl 2 1mM, HEPES 5mM, glucose 11mM, ph 7.4) 으로채워진용량 50ml의실험용기에옮긴후등장성장력변환기 (isometric force transducer) 에연결된아날로그기록기로방광수축을기록하였다. 고리모양의방광절편을 L모양의스테인레스 hook에걸어예비실험에서최대수축력을보인 2g 장력을준후 1시간동안안정화시킨뒤에수축실험을진행하였다. 안정화기간동안 30분경과후 HEPES 완충정상생리용액을 1회교환하였다. 1) 고농도의포타슘용액에의한수축반응 : 막전압의존성칼슘이온통로를직접적으로활성화시키는고농도포타슘 50mM 용액을투여하여각군사이에수축차이가있는지관찰하였다. 수축반응유발후 5분뒤 HEPES 완충정상생리용액을교환하여세척하였고, 5분간안정화기간을가진뒤에다시수축실험을시행하였다. 2) Carbachol에의한수축반응 : 방광의무스카린성수용체를자극하여수축을유발하기위해 carbachol을 1x10 7 부터 3x10 4 M까지투여하여각군의수축차이를비교하였다. 3x10 4 M까지투여한후 5분뒤에 HEPES 완충정상생리용액을교환하여세척하였고, 5분간안정화기간을가진뒤에다시수축실험을시행하였다. 3. 표본제작및면역형광염색 1) 표본제작 : 적출된방광의체부부분을 10% 중성포르말린에하루동안고정시키고흐르는물에수세하여고정제를제거하고탈수하였다. 투명화과정후에 paraffin에포매하였으며, 조직블럭은 slide glass에 4μm 두께로절편을제작하여부착시킨후 H-E 염색및면역형광염색을시행하였다. 2) 면역형광염색 : 제작된표본의탈파라핀화및함수화를위해 xylene I, II에각 10분씩반응시킨후 100%, 95%, 90%, 80%, 70% 알코올에순차적으로각각 5분씩반응시켰다. 항원을노출시키기위하여 1x sodium citrate buffer 용액에담가 3분간극초단파오븐 (microwave oven) 으로전처리하고, 0.01M (ph 7.4) PBS로 10분씩 3번세척후비특이적인반응을없애고자 10% 정상당나귀혈청 (normal donkey serum, Jackson immunoresearch Laboratories, lnc, USA) 으로 1시간동안 37 o C에서항온처리하였다. 그리고 1차항체인 rabbit M 2 monoclonal antibody (1:50, sigma-aldrich Corp., St. Louis, USA) 와 goat M3 polyclonal antibody (1:50, Santa Cruz Biotechnology, Inc., USA) 로 37 o C에서 30분간반응시킨뒤, 0.01M (ph 7.4) PBS로 10분씩 3번세척하였다. 2차항체로는 donkey anti-rabbit IgG-TR (1:50, Santa Cruz Biotechnology, Inc., USA), donkey anti-goat IgG-FITC (1:50, R&D immunoresearch Laboratories, lnc., USA) 를사용하여 37 o C의암실에서 30분동안반응시켰고, 0.1% Triton X-100 (Sigma- Aldrich Corp., St. Louis, USA) 이함유된 0.01M PBT로 30분씩 2회세척한후 PBS로 20분씩 15회세척하였다. 염색이끝난슬라이드는 mounting media (Dako, Carpinteria, USA) 를이용하여봉입하였다. 3) 영상분석 : 조직에서 M 2, M 3 의면역염색강도의평가는각군마다다섯장의슬라이드를분석하였으며, 각각의슬라이드마다점막상피층과근육층에서최소한 3군데이상
정호영외 : 여성호르몬치료시난소절제된백서방광의변화 1107 면역염색강도를측정하여평균값을구하였고, 이는공초점레이저주사현미경 (Zeiss LSM, Jena, Germany) 과영상분석장치를이용하여측정하였다. 4. 연구결과의분석수축실험시각각의약물에의한최초 2-3회의수축반응평균값을구하여각군사이의차이를비교하였고통계적분석은 SPSS (version 13.0) 를사용하였고 Kruskal-Wallis test 와 Mann-Whitney U test를이용하여분석하였으며 p값이 0.05 미만인경우에만통계학적으로의미있는것으로정하였다. 결과막전압의존성칼슘이온통로를직접적으로활성화시키는고농도포타슘 50mM 용액을투여하여각군의수축반응을비교한결과대조군이 1.61±0.16g이었고 Ovx군은 0.78±0.13g 으로가장작은수축반응을나타냈으며 Ovx+E군이 1.96± 0.12g으로가장큰수축반응을나타냈다. Ovx군은대조군과 Ovx+E군에비하여통계학적으로의미가있는낮은수축반응을보였으며 (p<0.05), 대조군과 Ovx+E군은통계학적으로의미있는수축반응의차이는보이지않았다 (p> 0.05) (Fig. 1). 방광의무스카린성수용체를자극하여수축을유발하기 Fig. 1. KCl 50mM induced contractile response of the rat bladder. The ovariectomized (Ovx) group showed a decreased contractile response to KCl 50mM compared with the control group and the Ovx+estradiol treated (Ovx+E) group. Each bar is the mean±se. *: significantly different from the control group and the Ovx+E group (p<0.05). Fig. 2. Carbachol (Cch) induced contractile responses of the rat bladder. The ovariectomized (Ovx) group showed decreased contractile responses to Cch, as compared with the control group and the Ovx+estradiol treated (Ovx+E) group. Values are plotted as means±se. *: significantly different from the control group and the Ovx+E group (p<0.05). Fig. 3. Representative photomicrographs showing staining of the control group (A), the Ovx group (B) and the Ovx+E group (C). The connective tissue in the Ovx group was markedly increased (white arrow) more than that in the control group and the Ovx+E group. Smooth muscle hypertrophy was observed in the Ovx+E group (black arrow) (H&E stain, original magnification, x40).
1108 대한비뇨기과학회지 : 제 49 권제 12 호 2008 위해 carbachol을 1x10 7 부터 3x10 4 M까지투여하여각군의수축차이를비교하였을때모든군에서 1x10 5 M 농도투여시수축반응이시작되었고, 그이후농도의존적인배뇨근수축이유발됨을확인하였다. 3x10 4 M 농도에서각군의수축반응을비교한결과대조군이 2.33±0.09g이었고 Ovx군은 1.17±0.22g으로가장작은수축반응을나타냈으며 Ovx+E군이 2.74±0.17g으로가장큰수축반응을나타냈다. Ovx군은대조군과 Ovx+E군에비하여통계학적으로의미가있는낮은수축반응을보였으며 (p<0.05), 대조군과 Ovx +E군은통계학적으로의미있는수축반응의차이는보이지않았다 (p>0.05) (Fig. 2). 방광의결합조직대비평활근의비율에대하여형태학적정성 (quality) 분석결과 Ovx+E군이대조군과 Ovx군에비해방광평활근이증가해있었다 (Fig. 3). 무스카린수용체아형 (M 2, M 3) 을면역형광염색하여분석한결과 M 2 수용체는 Ovx+E군이대조군및 Ovx군에비해점막상피층과근육층에서그발현이의미있게증가되어나타났으며 (p<0.05), Ovx군과대조군사이에는통계학적 Table 1. Comparisons of the M 2 receptor expression among the three groups Group Epithelial layer Density of M 2 receptor Muscle layer Control 82.2±3.7 55.4±2.1 Ovx 94.2±4.1 56.2±1.5 Ovx+E 113.2±6.0* 63.6±2.3* Ovx: ovariectomized group, Ovx+E: ovariectomized and estradiol treated group. Each value represents the mean±se, *: Ovx+E vs. Ovx (p<0.05), : Ovx+E vs. control (p<0.05) Table 2. Comparisons of the M 3 receptor expression among the three groups Group Density of M 3 receptor Epithelial layer Muscle layer Control 121.6±8.2 62.2±1.3 Ovx 156.0±2.4* 70.4±3.4* Ovx+E 164.8±7.3 76.0±1.5 Ovx: ovariectomized group, Ovx+E: ovariectomized and estradiol treated group. Each value represents the mean±se. *: Ovx vs. control (p<0.05), : Ovx+E vs. control (p<0.05) Fig. 4. Comparisons of the M 3 and M 2 receptor expressions among the three groups on the immunofluorescent study. The expressions of M 3 (green) and M 2 (red) were showed in the control group (A, B), the Ovx group (D, E) and the Ovx+E group (G, H). Merged images of the M 3 and M 2 were showed in the control group (C), the Ovx group (F) and the Ovx+E group (I). The M 3 receptor expressed in the epithelial area (arrow), and the M 2 receptor had a similar pattern. The M 3 receptor was more strongly expressed in the Ovx+E and Ovx groups than that in the control group. The M 2 receptor was more strongly expressed in the Ovx+E group than that in the Ovx group and the control group (original magnification, x400).
정호영외 : 여성호르몬치료시난소절제된백서방광의변화 1109 으로의미있는발현의차이는없었다 (Table 1)(Fig. 4). M 3 수용체는 Ovx+E군이대조군에비해점막상피층과근육층에서그발현이의미있게증가되어나타났다 (p< 0.05). Ovx군역시대조군에비해점막상피층과근육층에서통계학적으로의미있는발현증가가있었다 (p<0.05) (Table 2) (Fig. 4). 고찰양측난소절제술을시행받은쥐는통상적으로폐경기여성의동물모델로사용되고있으나 13,14 방광에관련된연구는부족한상태이다. 폐경기이후많은여성들이배뇨장애를가지고있으며이러한환자에게여성호르몬대체치료를시행하는것에대해서는논란이많다. 5,7,14 양측난소절제술후여성호르몬대체치료를시행받은경우 carbachol에의한방광의수축반응이양측난소절제술만시행받은경우보다감소하였다는보고가있으나, 14 많은다른연구들이양측난소절제술후여성호르몬대체치료가방광의수축반응을증가시킨다고보고하였다. 1,6-8 본연구결과에서양측난소절제술후여성호르몬대체치료가고농도의포타슘용액과 carbachol에의한방광의수축반응을증가시켰으며, 양측난소절제술은위와같은약물에대한방광의수축반응을감소시켰다. 여성호르몬의결핍이방광의구조에미치는영향에대해 Aikawa 등 7 은양측난소절제술후여성호르몬대체치료가방광평활근의비대를유발한다고보고하였고 Susset 등 15 은사후부검에서 50세여성이남성과젊은여성에비해방광의콜라겐이증가하였으며이러한구조적변화가방광의수축력저하, 순응도의감소, 콜린성자극에대한감각의변화를유발하여여성에게서배뇨근불안정을야기한다고하였다. 여성호르몬이방광의구조적변화를유발하는기전에대해 Lin 등 16 은여성호르몬이방광평활근내의혈관신생을유발하여방광내의혈류를증가시키며방광평활근의비대를초래한다고보고하였고, Blacher 등 17 은여성호르몬의대체치료가섬유모세포의증식을막고혈관과심장의콜라겐침착을막는다고하였으며, 방광에서도혈관, 심장과유사한기전으로콜라겐침착을억제할것으로추측한다. 이번연구에서방광의평활근과결합조직의정량적인분석은시행하지않았지만형태학적인정성분석을시행한결과에서다른정량분석을시행한연구결과와 7,18 유사하게양측난소절제술후여성호르몬대체치료를시행받은군이다른군에비해방광평활근이증가되었고, 이와같은결과는여성호르몬대체치료가방광의수축반응을증가시킨다는결과를뒷받침해주며, 폐경기여성의저활동성방광 에대해여성호르몬대체치료를시행하는근거가될수있을것으로생각한다. 콜린성신경계는배뇨에있어일차적인역할을하며콜린성무스카린수용체는방광전체에분포하지만방광체부가기저부에비해그밀도가더높다. 19 방광평활근에서무스카린수용체아형중 M 2, M 3 가많으며 10 M 2 가 M 3 에비해그밀도가 3-10배더높으나, 20 M 3 가아세틸콜린에의한방광수축의주된역할을한다. 21 M 3 수용체를자극하면 pertussis toxin-insensitive G protein을통해 phospholipase C를활성화시켜 phosphatidylinositol을가수분해하여세포내의칼슘을유동화 (mobilization) 시켜수축을유도하며, M 3 선천성억제쥐는비선택적무스카린작용제인 carbachol에대해방광수축이전혀없거나매우약하게나타난다고한다. 22 M 2 는억제성 G 단백을통하여 adenylate cyclase를억제하여베타아드레날린성작용에의해방광을이완시키는 cyclic AMP가생성되는것을막아방광수축을유도하지만, M 2 선천성억제쥐는 M 3 선천성억제쥐와는달리비선택적무스카린작용제인 carbachol에대해대조군에비해약간감소한수축반응을보인다고한다. 23 양측난소절제술후여성호르몬대체치료가방광의무스카린수용체의발현에어떠한영향을미치는지에대해서는아직까지논란의여지가있다. Levin 등 11 은 4일간여성호르몬치료를시행한결과암컷토끼의방광에서무스카린수용체의밀도가증가하였다고보고였으나, Shapiro 12 는암컷토끼에서양측난소절제술후 3주간여성호르몬대체치료를시행한결과대조군과양측난소절제술만시행한군에비해방광의무스카린수용체의밀도가감소하였다고보고하였으며, Matsumoto 등 24 은암컷쥐에서 12주간여성호르몬치료를시행한결과방광의 M 3 수용체 mrnas가대조군에비해감소하였다고보고하였다. 본연구에서는양측난소절제술후 7일간여성호르몬대체치료를시행하였고여성호르몬대체치료를시행받은군이나머지두군에비하여 M 3, M 2 수용체의밀도가증가하였으며단기간여성호르몬치료를시행한 Levin 등 11 의실험결과와유사하게나타났다. 본연구는 12주령의처녀백서를대상으로하였고 Diep 과 Constantinou 25 는본연구처럼양측난소절제술시행후 1주일뒤부터여성호르몬대체치료를시행한결과젊은쥐는성숙한쥐에비해난소절제술후외부에서주입하는여성호르몬에대한방광수축력의회복이떨어진다고보고하였으며, 젊은쥐는여성호르몬대체치료를한군이대조군보다도수축력이낮게나타났다. 그러나본연구결과는젊은쥐를사용하였음에도불구하고위의연구결과와다르게 OVx+E군의수축력이대조군보다도증가한것으로나
1110 대한비뇨기과학회지 : 제 49 권제 12 호 2008 타났다. Palea와 Angel 14 은본연구보다더짧은 5일간여성호르몬대체치료를시행하였으며결과는본연구와다르게 OVx+E군이 OVx군에비해수축력이감소한것으로나타났다. 본실험이기존의연구와유사하게실험하였음에도불구하고 14,25 결과가다다르게나타나는것은여러원인이있겠지만그이외의다른연구와 1,6-8,11 유사하게결과가나타난것을고려한다면실험이잘못된것은아니라고생각한다. 이번실험결과만으로폐경기여성에게서여성호르몬대체치료와 M 2, M 3 수용체변화와의관계에대해결론내리기는어려우며본연구에서난소절제술이방광의 M 3 수용체발현을증가시키는것으로나타났으나반대의결과를보고한연구도있고, 24 난소절제술이나여성호르몬대체치료가방광의무스카린수용체변화와무관하다는보고가있으므로 26 이에대해좀더많은연구가필요하리라생각한다. 결 여성호르몬대체치료의효과에대해결론을내리기는어렵지만폐경기여성에게서나타나는방광의기능장애는다른연구결과와비교해볼때 M 2, M 3 수용체의변화와별다른관계가없을것으로생각하며, 폐경기여성에게서나타나는방광의기능장애에대해시행되는여성호르몬대체치료는방광의수축반응을증가시켜저활동성방광증상개선에도움이될것이라고생각한다. 그러나폐경기여성의방광의기능장애에대한여성호르몬대체치료의결과에대해아직까지논란의여지가있는부분이많이있으므로좀더많은연구와실험이필요하리라생각한다. 론 REFERENCES 1. Keane DP, O'Sullivan S. Urinary incontinence: anatomy, physiology and pathophysiology. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:207-26 2. Lin AD, Levin R, Kogan B, Whitbeck C, Chichester P, Sokol R, et al. Estrogen induced functional hypertrophy and increased force generation of the female rabbit bladder. Neurourol Urodyn 2006;25:473-9 3. Simunic V, Banovic I, Ciglar S, Jeren L, Pavicic Baldani D, Sprem M. Local estrogen treatment in patients with urogenital symptoms. Int J Gynaecol Obstet 2003;82:187-97 4. Zullo MA, Plotti F, Calcagno M, Palaia I, Muzii L, Manci N, et al. Vaginal estrogen therapy and overactive bladder symptoms in postmenopausal patients after a tension-free vaginal tape procedure: a randomized clinical trial. Menopause 2005;12:421-7 5.Tsai E, Yang C, Chen H, Wu C, Lee J. Bladder neck circulation by Doppler ultrasonography in postmenopausal women with urinary stress incontinence. Obstet Gynecol 2001;98:52-6 6. Zhu Q, Ritchie J, Marouf N, Dion SB, Resnick NM, Elbadawi A, et al. Role of ovarian hormones in the pathogenesis of impaired detrusor contractility: evidence in ovariectomized rodents. J Urol 2001;166:1136-41 7. Aikawa K, Sugino T, Matsumoto S, Chichester P, Whitbeck C, Levin RM. The effect of ovariectomy and estradiol on rabbit bladder smooth muscle contraction and morphology. J Urol 2003;170:634-7 8. Parekh MH, Chichester P, Lobel RW, Aikawa K, Levin RM. Effects of castration on female rabbit bladder physiology and morphology. Urology 2004;64:1048-51 9. Fleischmann N, Christ G, Sclafani T, Melman A. The effect of ovariectomy and long-term estrogen replacement on bladder structure and function in the rat. J Urol 2002;168:1265-8 10. Andersson KE. Advances in the pharmacological control of the bladder. Exp Physiol 1999;84:195-213 11. Levin RM, Shofer FS, Wein AJ. Estrogen-induced alterations in the autonomic responses of the rabbit urinary bladder. J Pharmacol Exp Ther 1980;215:614-8 12. Shapiro E. Effect of estrogens on the weight and muscarinic cholinergic receptor density of the rabbit bladder and urethra. J Urol 1986;135:1084-7 13. Longhurst PA, Kauer J, Leggett RE, Levin RM. The influence of ovariectomy and estradiol replacement on urinary bladder function in rats. J Urol 1992;148:915-9 14. Palea S, Angel I. The effect of ovariectomy on the contractile response of the rat isolated detrusor muscle and urethra. Life Sci 1997;61:PL21-6 15. Susset JG, Servot-Viguier D, Lamy F, Madernas P, Black R. Collagen in 155 human bladders. Invest Urol 1978;16:204-6 16. Lin AD, Mannikarottu A, Kogan BA, Whitbeck C, Chichester P, Leggett RE, et al. Estrogen induces angiogenesis of the female rabbit bladder. J Endocrinol 2006;190:241-6 17.Blacher J, Dabire H, Promies JP, Safar ME, Stimpel M. Long-term cardiovascular effects of high "osteoprotective" dose levels of 17-beta estradiol in spontaneously hypertensive rats. Cardiovasc Drugs Ther 2000;14:303-7 18. Juan YS, Mannikarottu A, Kogan BA, Leggett RE, Whitbeck C, Chichester P, et al. The effect of low-dose estrogen therapy on ovariectomized female rabbit bladder. Urology 2008;71: 1209-13 19. Longhurst PA, Uvelius B. Pharmacological techniques for the in vitro study of the urinary bladder. J Pharmacol Toxicol Methods 2001;45:91-108 20. Wang P, Luthin GR, Ruggieri MR. Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J Pharmacol Exp Ther 1995;273:959-66
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