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ABSTRACT

J ( J f l .35 f i5 /5 ( ) /2 I 534J514$( ) 2 . $)/OTH E JO URNAL OF PHARMACOLOGY AND EX PE RIM EN TAL TH ERAPEU TICSC opyrigh t (0 1 980 by Th e Am erican Soc iety for Pha rm acology and Exp er im enta l Th erapeutiC S

Es trog en -In duced A lte ra tion s in the A u tonom ic R esponses o fthe R abb it U rina ry B ladde r1

ROBERT M . LE V IN , FR ANCES S . SHO FER and ALAN J. W E IND iv is io n o f U ro log y , D e par tm en t o f Su rge ry , U n iv e rs ity o f Pe nn sy lva n ia Scho o l o f M ed ic ine an d th e P h ila de lph ia Ve te ran s A dm in is tra tionM ed ica l C en te rAccep ted fo r p ub lica tion Sep tem ber 1 2, 1 980

Lev in , R obert M ., F rances S . Sho fe r, and A lan J . W ein :E strog en -ind u ced a lte ra tio ns in th e au to nom ic resp onse s o fthe rab b it u rina ry B lad de r. J . P ha rm aco l. E xp . T he r. 2 1 5 : 6 14 -618 , 1 980 .The e ffec t o f e s tro gen adm in is tra tion on th e au to nom ic inn e r -vat ion o f th e ra bb it u rina ry b la dde r w as s tu d ied . Imm a tu refem a le w h ite ra bb its w e re in jec te d tw ice da ily w ith e s trog en(1 50 tg /kg ) fo r 4 consecu tiv e da ys . C on tro l a n im a ls re ce ive din je c tions o f veh ic le . T he re spon se o f b lad de r s trips to thefo llow in g agon is ts w ere de te rm in ed fo r co n tro l an d e s trog en -trea ted an im als : m e th oxam in e (a lpha adre ne rg ic ) ; is op ro te re -no l (be ta ad re ne rg ic ); b e th a ne cho l (m us ca r in ic ch o lin e rg ic );A TP (pu rine rg ic ); an d po ta ss ium ch lo ride (n o n re cep to r m e -d ia te d ). A dd ition a lly , th e ad rene rg ic a nd cho lin e rg ic re ce p to r

d e ns itie s w e re de te rm ined b y s ta ndard rad io liga nd b in d ingte chn iqu es . E s tro gen ind uced a m a rked in c re ase in th e re -spon se to a lpha ad re ne rg ic , m usca rin ic ch o lin e rg ic and pun-n e rg ic a gon is ts in the b la dde r b od y and m idb la d den . N o s ig -n if ica n t a lte ra tion s we re no ted in th e b la dder b ase . In add itio n ,n o a lte ra tio ns w ere no te d fo r iso p ro te ren o l on po tass ium ch lo -ride . T he rec ep to r d en s ities p a ra lle led c los e ly the ph ys io log ic a lre sp on se s . E s tro g en in d uced a s ig n ifica n t in c re ase in a lphaadre ne rg ic a nd m usca r in ic re ce p to rs in th e b la dder b od y andm idse c tio n , w herea s no change was no te d in th e rec ep to rd e ns ity o f th e b la dder ba se . E s tro gen d id no t a lte r the be tare ce p to r de n s ity . T he se find in g s ind ica te tha t the u r in a ry b lad -d e r resp ond s as a ta rge t o rg a n fo r es trog en -ind uced a lte ra tio nsin a u to n om ic in n e rv a tio n .

E strogen adm in is tration ha s been show n to a lte r the adre -ne rgic in ne rva tion of the u te ru s. M ille r and M arsh a ll (1 965)dem ons tra ted tha t hypogastric n erve s tim ulation in estrogen -trea ted rabb its p rodu ced u te rine con trac tion (a tpha adrenerg iceffec t), w hereas hypogas tn c s tim u la tion in prog es te rone-trea ted rab b its p roduced u te rine re laxa tion . A dditiona lly , estro -gen adm in is tration in rabb its has been show n to increase th edens ity of a lpha ad ren erg ic re cep tors in u teru s w ithout alte rin gth e be ta adrenerg ic recep to r density (R oberts e t a l., 1977) .

A lthough the u te rus is the pr imary ta rg et o rgan for es trogenand th e m ajority o f s tud ies on the pharm aco log ica l and phys i-o log ica l e ffec ts o f estrog en adm in istra tion have b een perfo rm edon the u te rus , c lin ica lly estrog en therap y and pregn an cy(Schreiter e t a t., 1976; Caine and R az , 1973) have been show nto a lter u rinary fun ction . T he fo llow in g stud ies w ere perfo rm edto de te rm ine if the au tonom ic innerva tion of the urinary b lad-der can be a lte red by estro gen adm in istra tion .

Me thodsEstrad io l (150 g ig/k g) w as adm in is te red to im m ature fem ale N ew

Z ea land W hite rab b its tw ic e daily fo r 4 co nse cu tive day s. C on tro lrab bits receiv ed in jection s o f vehic le. T his regim en has b ee n p re vi ou sl y

Received fo r pub lica tio n June 2 , 1980. T h is w ork w as sup ported in p art b y grants from the V ete rans A dm in is tration

and N a tiona l Ins titu te s o f H ea lth G ran t 1R O1-A M .265 08-O l.61 4

dem ons tra ted to pro duce m arked effec ts on the adrenerg ic in nerva tio nof the u te ru s (R oberts e t a l., 1977) .M uscle ba th stud ies . Urinary b ladd ers of 8 con tro l and 12 e s t rogenra bb its w e re rem ov ed un der l igh t sod ium pen toba rb ital anes the sia. T heb ladde rs w e re d issec ted fre e of fa t and se ro sa and separa ted in to threes egmen t s : body , m idb ladd er (at the leve l o f the ure teral o rifices) an dbase. S in ce th e sm ooth m usc le e lem ents o f th e b ladder ar e no t un i-f o rmly arranged in lay ers, it is possib le th at the leng th -tens io n re la tio n-ship s am ong th e bladd er section s m ay not b e equ iva lent . W ith th is inm ind , w e w ere care fu l to be co nsis ten t in the size of th e strip u tilizedand th e d irec tio n in w hich the s trips w ere m ounted . T he m usc le s tr ip sw ere m ounted in a m u sc le ba th co n ta in in g 30 m l of T yrodes so lu tioncon ta in ing 1 m g /m I of dextro se equ ilib rated w ith 95% 02=5% C O 2 at37#{176}C .O ne gram o f tension w as pla ced on each b ladder s trip . Isom etr iccon trac tile m easurem ent w as perfo rm ed as p rev iou sly described (Levisan d W ein , 197 9). D o se -re sp onse cu rv es w e re perfo rm ed by the sequen-tied addition of drug (in a cum ula tiv e m anner) a t 5 -m is in te rva ls . A TPp roduced a rap id increase in tens io n fo llow ed by a sho rt p la teau andrap id dec line in ten sion . T hus, to av o id the rap id tach yphy lax is , ATPw as adm inis tered at 3-mm in tervals.

P re lim in ary stu d ies con firm ed tha t the con tra ctile e ffect o f ep ineph-r ifle and m e thoxam in e (a lpha adrenergic) cou ld be com petitive ly in -h ib ited by ph en to lam in e, the re lax ation produced by ep inephrin e andisop roterenol could b e com peti tive ly an tagon ized by p rop ranolo l andthe con trac tile e ffec t ofb eth anecho lcould be com peti tive ly an tagonizedby at rop in e .

B indin g assay s. Th e ur ina ry bladd ers o f six con tro l and e igh tes trogen rabb its w ere rem ov ed under lig h t so d ium pen tobarb ita l a ries -thes ia. A fte r rap id ly rem oving the fa t depos its an d serosa , the b ladders

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F ig . 3 . E ffec t o f e s tro gen on the con tra c tile re sp onse to iso p ro te ren o l.T h e re spon se o f se quen tia l s tr ips iso la te d be tw een b lad der bod y andbase to is op ro te re no l w a s de te rm in ed a s de sc ribe d in M e th ods .E a ch va lue rep re sen ts th e m ean o f 8 to 1 0 de te rm in a tion s . V e rtic a lb rac ke ts in d ica te the SE .

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1980 E strogen E ffe c ts on U r in a ry B ladder 615were sep arated in to th ree sec tions : bo dy , m id (a t the leve l o f theurete ra l o rif ice s) and ba se. The tissues w ere e ither u sed im m ediate ly orf rozen an d s to red under liqu id n itrog en . R ecep to r b ind in g assays fo ra lpha adrenerg ic , be ta adrenergic and m uscarin ic -cho linerg ic w ereperfo rm ed as prev io usly described (L ev in an d Wein , 1979; Levis e t a t.,1980) . The rad io ligands u tilized w ere: [3H }dihydro ergocryp tine ([3H ]-DH E; 0 .3 -6 0 nm ); [3H ]d ihydro a lp reno lo l ([3H ID H A ; 0.1-20 nm ) and[3H ]qu inuc id iny l benz ila te ([3H ]QN B ; 0 .03-3 0 nm ).

P ro tein con cen trations w ere de te rm ined by th e m ethod of Lowry etat . (1 951 ). S tat istical sig nif icance w as dete rm ined by an aly sis o f van -an ce .

Mater ials. [ 3H ]DHA (48 .6 C i/m m ol), [3H ]D H E (38 .8 C i/m m ol) an d[ 3H IQNB (25 .4 C i/m m ol) w ere o b tain ed from New Eng land N u cle ar,Bo ston , M A . O ther reagen ts w ere ob ta ined from genera l com m erc ia lsources.

Resu l tsAdren erg ic resp onses to es trogen adm in is tra tion . F ig -

u re 1 dem on stra tes the effec t o f estrogen adm in istra tion on the

E PIN EP HR HE (S M)F ig . 1 . E ffe c t o f e s trog en on th e con tra c tile re spon se to ep in e ph rin e .T he re spon se o f s trips o f u rin a ry b lad de r to e p ine phrine w as de te r-m ine d as d es crib ed in Me thod s . Each po in t rep re sen ts th e m ean o f1 2 se pa ra te d e te rm ina tio ns . Ve rtica l b ra cke ts in d ic a te the S .E . Sig -n ifica n tly d iffe re n t from th e re spon se o f the con tro l t issu e (P < 0 .05 ) .

F ig . 2 . E ffec t o f e s tro ge n o n th e co n trac t ile re sp onse to m e tho xam ine .Th e re sp on se o f s eq ue n tia l s trips iso la ted b e tw een b ladd er b od y andbase to m e tho xam ine w as de te rm in ed a s de sc ribe d in M e th ods . E a ch va lu e rep res en ts th e m ean o f 8 to 1 0 de te rm in a tion s . V e rtica lb rac ke ts ind ica te th e S E . S ign ific an tly d if fe ren t from th e re sp on se o fthe con tro l t iss ue (P < 0 .0 5 ) .

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CE CE CEF ig . 6 . E ffec t o f e s tro gen on th e con tra c tile re sp on se to KC I. T here spon se o f se quen tia l s tr ips iso la te d be tw een b la dde r b ody and baseto KC I (1 5 0 mM ) was de te rm in ed as de sc ribe d in M e th od s . E a chva lu e rep re sen ts th e m ean o f fo u r to e igh t de te rm in a tion s . V e rtica lb rac ke ts ind ic a te the S E .

61 6 Lev ine ta l . V ol. 21 5

F ig . 4 . E ffec t o f e s tro gen on the con tra c tile re spon se to b e th anecho l.T he re spon se o f se quen tia l s tr ip s is o la te d be tw een b la dde r b ody andba se to u re cho line w as de te rm ined as de sc r ib ed in Me thod s . Eachva lu e rep re sen ts th e m ean o f 8 to 1 2 de te rm in a tion s . V e rtica l b ra cke tsin d ic a te the S E . S ig n ifica n tly d iffe re n t from th e re spon se o f thecon tro l tiss ue (P < 0 .05 ).respon se of the urinary b ladd er to ep ineph rin e . E p in ep hrine isa po ten t stim ula to r o fbo th a tpha an d be ta ad renerg ic recep to rs.In the co n tro l an im als , ep inephn in e adm in is tration produced adose-dependen t re laxa tion in the b ladder body , d ose-d ep enden tcon trac tio n in the b ladder b ase and a b iphasic response in them idsec tion consis tin g of an in itia l re laxa tio n (be ta effec t) fo l-low ed by a m ild con trac tion (a tpha effect).

T he estrog en -treated an im als respond ed as fo llow s: ep in ep h-rine produced a b iphas ic resp onse in the b ladder b ody cons ist-in g of an in itia l re laxa tio n fo llow ed by con traction ; dose-depen-den t con trac tio n of the b ladd er base and a b iphas ic response ofth e b ladder m idsec tion cons isting of a s ligh t re laxa tion fo llow edby a subs tan tia l con trac tion . In genera l, a ll sections of thees trogen-trea ted urinary b lad der dem ons tra ted a subs tan tia llygreater a lpha adrenerg ic resp onse th an the b ladd er o f con tro lanimals.

The respon se of th e b lad der strips to m ethoxam ine (a tphaag on ist) and isopro teren o l (be ta agon is t) a re shown in figu res2 and 3 , respec tiv ely . M eth oxam ine w as sig n ifican tly m o reef fec tive in s tim u la ting con trac tion in b ladders (b ody and m id-sec tio n) ob ta ined from es trogen-trea ted an im als than in blad-ders from contro l rabb its. E s trogen adm in is tra tion d id no t a lte rthe sensitiv ity of any tissue to m ethoxam ine , i.e., the ED ,o w asu nchanged by estrogen trea tm ent. T he response of th e b lad derstr ips to isopro teren o l w as the sam e for the sm oo th m uscles trips o b ta in ed from bo th co n tro l and es trogen-trea ted rabb its.

T he resp onse of th e b ladder strips to be thanecho l is shownin figure 4 . In bo th con tro l and estrog en -treated an im als , theb lad der body respond ed to be thanecho l to a grea te r ex ten t th and id the b lad der base . A dd itiona lly , the b ladd er s tr ip s (body andm id sec tio n) iso la ted from the estrogen -trea ted rab b its re -sp onded to a sign ifican tly grea te r degree th an d id the con tro lstrips.

ATP is a proposed neurohum ora l transm itte r in the urinaryb ladder (B urn stock , 1979); the effec t o fes trogen th erap y on the

AT P (mM)F ig . 5 . E ffec t o f es trog en on th e con trac tile resp onse to ATP . T heresponse o f sequen tia l s tr ips iso la ted be tw een b lad de r b ody and ba seto ATP was de te rm ined a s de sc ribe d in Me thod s . Each va lue re p -res en ts th e m ean o f 8 to 1 2 de te rm ina tio ns . V e rtica l b rack e ts ind ic a tethe S E . S ig n ifica n tly d iffe re n t from th e re sponse o f the con tro l tissu e(P < 0 .05 ) .

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F ig . 7 . E ffe c t o f es tro g en on th e adre ne rg ic a nd cho lin -e rg ic rece p to r de ns itie s o f the u rin a ry b lad de r b ody , m ida nd base . B ind ing expe rim en ts w ere pe rfo rm ed as d e-ta ile d in M eth od s. T he con ce n tra tio ns u t iliz ed w ere : 3 0nM [3H ]D HE , 1 0 nM [3H ]D HA and 1 0 nm [3H ]Q N B fo r thede te rm ina tio n o f a lp ha , be ta and m usca r in ic rece p to rs ,re sp ect ive ly . Ea ch va lue rep resen ts th e m ean o f s ix toe igh t d e te rm ina tio ns . V e rtica l b rac ke ts in d ic a te th e S E .C S ig n if ica n tly d iffe ren t from the recep to r de ns ity o f th econ tro l tis su e (P < 0 .0 5 ).I

1980 Estro g en E ffe c ts o n U rina ry B lad der 617

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respon se o f the b lad der to ATP is presen ted in figu re 5 . T hem agnitu de of the respon se o f the b ladder body and m idsec tionto ATP was su bstan tia lly increased by es trogen adm in istra tion .

In ord er to de te rm ine if es trogen-ind uced a lte ra tion s in m us-cle m ass con tribu te to the enhanced response to pharm aco log -ica l stim ula tio n , the response of the m u sc le s trips to 1 50 m MKC1 w as de te rm in ed . T he resu lts (fig . 6 ) dem on stra te tha tes trogen adm in is tration pro duced no ch ange in the respon se toKC1 .

In ord er to de term in e if the a lterations in the response of th eb lad der to au to nom ic ag on ists m igh t be due to a lte red recep to rd istribu tions , w e de te rm ined the a tpha an d be ta adrenergic an dmuscar in ic cho linerg ic recep to r densities in the b ladder body ,m id sec tio n and base . T he resu lts a re show n in figure 7.

Th e a tpha recep to r dens ities ([3H JD HE b in d ing sites) in theb lad der body and m idsection w ere sub stan tially grea te r in th eestrog en -trea ted an im als than in con tro l an im als. T here w ereno s ign ifican t d iffe rences in be ta recep to r d en sities in any ofth e tissues stud ied . T he m u scarin ic recep to r dens ities in theb lad der body and midsection w ere sign ifican tly grea ter in thees trogen rabb its than in con tro l an im als. T here w ere no s ig n ifi-can t d iffe rences in any recep to r concen tra tions in the b ladd erbase .

S catchard ana lys is o f [3H JDH E (a tpha) and [3H ]Q N B (m us-ca.r in ic) bind ing to the b ladder b ody iso la ted from both con tro lan d estrogen- trea ted rabb its dem onstra ted th at the Kd w asunchanged for bo th rad io lig an ds (d a ta no t show n).

Discuss ionThe presen t stud ies dem ons trate tha t the urinary b ladd er

can be a ta rge t o rgan for a lte ra tion s in es trogen leve ls . In th esam e m anner as has been show n for the u te rus and ov id uc t(H ow e and B lack , 1973 ; R oberts et a l., 1977; M ille r and M ar-sha ll, 1965 ), estro gen adm in is tration produced a m arked in -c rease in the response o f th e urinary b ladder to a tpha adrener-g ic stim ula tion . A ddition ally , es trogen adm in istra tion s ign ifi-can tly increased th e resp onse of b ladd er to bo th be th an echo land A TP, w hereas there w as no change to iso pro te reno l.W hether these a lterations are d ue to a d irec t e ffec t o f estrog enon th e b ladder, o r an ind irec t e ffect, po ss ib ly th rough there lease of a seco nd agen t, is n o t known .

R ecep to r b ind ing stud ies dem ons trated tha t th e observedalte ra tio ns in the respon se of the b ladder to m ethoxam ine andbe th an echo l w ere d ue prim arily to a m ark ed increase in th ea lpha and m uscar in ic recep to r dens ities (respec tive ly ) o f th eb ladder .

During the perfo rm ance of these stud ies, it appeared tha t th eb lad ders from the es trogen-trea ted rabb its w ere la rger than th eb lad ders from th e con tro l an im als . O ur resu lts sh ow ing tha tes trogen pro duced no change in the respon se of the b lad derstrips to KC 1 ind ica te th a t changes in m u scu la r con ten t o f th estrips cou ld no t accoun t fo r th e ph arm aco lo g ica l a lte ra tion sobserved . A dditio na lly , a lte ra tion s in m u scu la tu re co u ld no tacco un t fo r either: 1 ) the m ark ed change in the response of th eb ladder to ep in ep hrine (tha t is, the change from be ta to a tphaad rene rgic re sponse ) o r 2 ) th e increase in ad ren erg i#{232} and cho-linergic recep to r d en sities s ince these dens ities a re calcu la tedper m illig ram of tissue pro te in .

T h e find ing tha t estrogen adm in is tra tion indu ced a subs tan -tia l inc rease in the respon se of th e b ladder body and m id sec tio nto A TP prov ides ind irec t ev id ence tha t A TP m ay b e ac ting asa neurohum ora l transm itte r. S im ila rities be tw een the effec t o fATP and effects o f adren erg ic an d cho linerg ic agon ists a re : th eresponse of the b ladder body to all agen ts is subs tan tia llydif ferent f rom th e response of th e b ladder b ase . E strog en ad-m in istra tion a lte rs the response of the b ladder body and m id-sec tions to adrenerg ic (a tpha) agen ts , cho linerg ic (m u scarm nic)agen ts and A TP.

In conc lus ion , es trogen adm in is tration to im m ature fem alerabb its p roduced a sign ifican t increase in the response of b lad-der s tr ip s to a tpha ad renerg ic , m uscarin ic and pur in erg ic s tim -u la tion . T he increased adrenerg ic and cho linerg ic response ism os t like ly a resu lt o f the increased a lpha and m uscarin icrecep to r dens ities.

AcknowledgmentsW e w ou ld like to thank M s. S uzan Be rg an d M rs . S heila Levin for the ir

exce llen t te chn ica l ass ista nc e.

Ref eren ce sBURNS TOCK , G .: Pa st and cu rren t evid ence for th e pur ine rgic ne rve hypoth esis.

In Ph ys io log ical and Regu la to ry Func tion s of A d eno sin e and A den ine N uc leo-tides , ed . b y H . P . B aer and G . I. D rum m ond , p p. 3 -32 , R av en Press , N ewY or k, 19 79 .

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618 Lev ine ta l . Vo l . 21 5CA I NE , M . AND RA Z , S .: T he R ole o f F em a le H orm one s in S tress Incon tin ence.

P resen ted a t the 16 th C ongre ss o f So ciete In terna tiona le d U ro log ie, Am ste r-dam , 1 973 .

H O W E , G . R . A ND BLACK , D . L .: A utonom ic nervou s system and ov idu ct fun ctionin the rab b it. J . R ep rod . F ert il. 33 : 425-430 , 1973 .

LEV IN , R . M ., SHOFER , F . S . AND W E IN , A . J .: Cholin erg ic, adrene rgic andpur ine rgic respon se of sequen tial strips of rabb it urinary bladd er. J. P harm acol.Exp . The r. 2 12 : 5 36-54 0 , 1980 .

LEV I N , R . M . AND W E IN , A . J .: D istribu tio n and fun ctio n o f ad rene rgic recepto rsin th e u rin ary bladd er o f th e rabb it. M ol. P harm acol. 16: 4 41-448 , 1 979 .

LOWRY , 0 . H ., ROSEBROUGH , N . J., FARR , A . L . AN D RA ND ALL , R . J.: P ro te inm easurem ent w i th th e Fo lin pheno l Reag en t. J. B io l. C hem . 1 93 : 265 -2 75 ,1 9 5 1 .

MILLER , M . D . AN D M AR SHA LL , J . M .: U te rin e response to nerve stim ulatio n;

re la t ion to h orm on al s ta tu s and ca techo lam in es . A m . J. P hy sio l. 2 09 : 859-865,1965 .

ROBERTS , J. M ., I N SEL , P . A ., GOLDFIEN , R . D . A ND G OL DFIE N, A .: a a dr en or e-c ep to r s but no t fi adrenorecepto rs in cre ase in rabb it u terus w ith o es trogen .Nature (L end .) 2 70 : 624-625 , 1977 .

S cH axrrxa , F ., FucH s, P . A ND STO CK AM P, K .: E strogenic sen sit iv ity o f alpharecepto rs in the u reth ral m usculatu re. U ro l. m t. 31: 13-18, 1976 .

Sen d reprin t requ es ts to : D r. R Ober t M . Lev in , D iv isio n o f U ro logy, H ospitalof the U niv ers ity of P ennsy lv an ia , 30 10 Ravdin C our tya rd B ldg., P hilade lph ia,PA 19104 .