Effects of LY274614, a competitive NMDA receptor antagonist, on the micturition reflex in the urethane-anaesthetized rat

Transcription

1 Br. J. Phrmcol. (1993), 11, Br. J. Phrmcol. (I993), 11, '." Mcmilln Press Ltd, 1993 Effects of LY274614, competitive NMDA receptor ntgonist, on the micturition reflex in the urethne-nesthetized rt M. Yoshiym, 1J.R. Roppolo, *K.B. Thor & W.C. de Grot Deprtment of Phrmcology, School of Medicine, University of Pittsburgh, Pittsburgh PA 15261, U.S.A. nd *Division of CNS/GI/GU Reserch, Lilly Reserch Lbortories, Eli Lilly nd Compny, Indinpolis IN 46285, U.S.A. 1 The effects of 3 competitive N-methyl-D-sprtte (NMDA) receptor ntgonists, LY274614, LY nd LY235723, on the micturition reflex nd externl urethrl sphincter EMG ctivity, were exmined either under isovolumetric conditions or during continuous filling cystometry in urethnenesthetized (1.2 g kg-', s.c.) rts. 2 Intrvenous dministrtion of LY (3-3 mg kg-') inhibited in dose-dependent fshion both bldder nd sphincter ctivity in the intct rts. In ddition, the volume threshold for inducing micturition ws incresed nd voided volume ws decresed. 3 Intrthecl dministrtion of LY (.6-3 fg) similrly inhibited bldder nd sphincter ctivity during cystometry in intct rts. 4 In chronic spinl cord (T6-T8) trnsected rts LY (.1-3 mg kg-, i.v.) did not lter bldder ctivity under isovolumetric conditions but decresed the mplitude of micturition contrctions nd sphincter EMG ctivity during cystometry t dose of 1-3 mg kg-'. 5 The inhibitory effects of i.v. dministrtion of LY274614, on bldder nd sphincter ctivity induced by infusion of chemicl irritnt (.1% cetic cid) or sline, were similr; except tht slightly lrger dose ws needed to inhibit sphincter ctivity during cetic cid infusion. 6 Pek mplitude of micturition contrctions recovered to 5% of control 3 h following i.v. (3mg kg-') or i.t. (6 jg) dministrtion of LY Two other chemiclly relted NMDA ntgonists, LY nd LY produced similr but less potent effects thn LY when given i.v. 8 These dt indicte tht glutmtergic trnsmitter mechnisms t the level of the spinl cord re importnt in modulting bldder ctivity in the intct niml, but tht these mechnisms do not contribute to bldder reflexes in the chronic spinl rt. These mechnisms my, however, contribute to sphincter ctivity in both intct or chronic spinl rts. Keywords: NMDA receptor ntgonist; rt bldder; EMG; externl urethrl sphincter; glutmte; micturition reflex Introduction Reflex micturition is produced by contrction of the smooth muscle of the urinry bldder nd reciprocl relxtion of bldder neck, urethr, nd the externl urethrl sphincter. However, in rts (Kruse et l., 1993) nd to lesser degree in dog (Nishizw et l., 1985) the strited muscle of the externl urethrl sphincter contrcts intermittently in high (4-6 Hz) frequency bursts during voiding. This enhnced nd pulstile sphincter ctivity my id in expulsion of urine in these nimls, while continuous contrction of sphincter prevents urine relese. In mny species these responses re medited by spinobulbospinl reflex pthwy tht psses through rely centres in the lumboscrl spinl cord nd the rostrl brin stem (the pontine micturition centre) (de Grot et l., 1992). The reflex pthwy is ctivted by distension of the urinry bldder nd subsequent firing in tension receptor fferent fibres tht pss through the pelvic nerve to the lumboscrl spinl cord. Phrmcologicl studies hve implicted number of neurotrnsmitters in the centrl nervous control of micturition (de Grot et l., 1992). Among the puttive excittory trnsmitters, glutmic cid hs ttrcted the most ttention. Microinjection, of glutmic cid or relted excittory mino cids, into the pontine micturition centre or djcent pontine re, fcilitted micturition in the ct (Mllory et l., 1991) nd rt (Lumb & Morrison, 1987). Wheres, systemic dministrtion of MK-81, n N-methyl-D-sprtte (NMDA) glutmte receptor ntgonist, inhibited bldder nd sphincter reflexes in nesthetized rts (Mggi et l., 199; ' AuthoT for correspondence. Yoshiym et l., 1991,b; 1993). MK-81 lso blocked the bldder contrctions elicited by electricl stimultion of the pontine micturition centre (Suzuki et l., 1991) nd depressed the expression of the immedite erly gene, c-fos, in spinl neurones induced by chemicl irrittion of the bldder mucos (Birder & de Grot, 1992). Lignd binding studies hve reveled tht NMDA receptors re present t vrious loctions long the micturition reflex pthwy; including res in proximity to the lumboscrl prsympthetic nucleus, the urethrl sphincter motor nucleus nd the pontine micturition centre (Jnsen et l., 199; Shw et l., 1991). The present study ws undertken to evlute further the role of NMDA receptors in the micturition reflex pthwy. We hve exmined the chnges in bldder nd sphincter ctivity induced by LY274614, potent competitive NMDA receptor ntgonist (Ornstein et l., 1991), which enters the centrl nervous system following systemic dministrtion. Two other competitive NMDA receptor ntgonists (LY nd LY235723) were lso exmined in few experiments. Unlike the non-competitive ntgonist, MK-81, which blocks the ction chnnel of the NMDA receptor (Wong et l., 1986), LY cts t the glutmte binding site on the NMDA receptor (Ornstein et l., 1991). In ddition, LY is likely to hve more selective ction thn MK-81 since, in high concentrtions, MK-81 is known to block nicotinic cetylcholine receptor chnnels (Amdor & Dni, 1991). The ltter effect could lter gnglionic trnsmission in the peripherl utonomic pthwys to the bldder, s well s trnsmission t the neuromusculr junction, in the strited sphincter muscle nd thereby lter micturition reflexes by peripherl non-glutmtergic mechnism. The

2 78 M. YOSHIYAMA et l. present results with LY confirm nd extend the findings with MK-81; indicting tht glutmtergic trnsmission t NMDA receptors hs n importnt fcilittory influence on bldder nd sphincter reflexes in the nesthetized rt. This influence must occur, t lest in prt, t the level of the lumboscrl spinl cord, since intrtheclly dministered LY mimicked the effect of systemic dministrtion. A preliminry ccount of this work hs been presented in n bstrct (Yoshiym et l., 1992). Methods Animl preprtion Experiments were performed on 79 femle urethnenesthetized (1.2 g kg-', s.c.) Sprgue-Dwley rts weighing 17 to 31 g (men 24 g). The trche ws cnnulted with polyethylene tube (PE-25) to fcilitte respirtion; nd cnnule (PE-5) were plced in the externl jugulr vein or femorl vein for intrvenous drug dministrtion. A trnsurethrl bldder ctheter connected to pressure trnsducer ws used to record the bldder pressure isovolumetriclly with the urethrl outlet ligted or to record pressure during cystometry when the bldder ws filled with constnt infusion of physiologicl sline or cetic cid (.1%) nd llowed to empty round the ctheter. To evlute voiding efficiency (% of bldder volume voided) sline ws infused into the bldder (.4 ml min-') until the pek of voiding bldder contrction; then the infusion ws stopped nd the sline voided from the bldder ws collected nd mesured to determine the volume voided. The bldder ws then emptied, the residul volume mesured nd cystometrogrm repeted. The procedure ws repeted minimum of three times for ech drug dose nd control. Continuous cystometry ws performed by constnt infusion (.21 ml min-') of sline into the bldder to elicit repetitive voidings, which llowed rpid collection of dt for lrge number of voiding cycles (Mggi et l., 1986). PE-9 nd PE-5 cnnule were used for isovolumetric recording nd constnt infusion cystometry (continuous nd single), respectively. For isovolumetric recording, the ureters were tied distlly, cut nd the proximl ends cnnulted (PE-1) nd drined externlly. The procedure prevented the bldder from filling with urine during the experiment. Eleven rts were spinlized under hlothne nesthesi. After T6-T8 lminectomy, the dur nd spinl cord were cut with scissors, nd sterile sponge (Gelfom, The Upjohn Compny) ws plced between the cut ends. The bldders of spinl rts were expressed mnully two or three times dily, nd perigenitl stimultion with cotton swb ws performed to encourge reflex bldder emptying (Mllory et l., 1989). The experiments on spinlized rts were performed 2 to 3 weeks postspinliztion. In 19 rts nesthetized with urethne (1.2 g kg- ', s.c.), n intrthecl (i.t.) ctheter ws inserted ccording to the technique of Yksh & Rudy, The occipitl crest of the skull ws exposed nd the tlnto-occipitl membrne ws incised t the midline using the tip of 19 guge needle s cutting edge. A ctheter (PE-1) ws inserted through the slit nd pssed cudlly to the L6-level of the spinl cord. The volume of fluid within the ctheter ws kept constnt t 6 Al. Cumultive dose-response curves were obtined by dministering the drugs in 6 yl injections followed by 6 ;LI flush with rtificil CSF t 15 min intervls. At the end of the experiment lminectomy ws performed to verify the loction of the ctheter tip. In 23 experiments (5 intct, 5 chronic spinl, 8 intct with i.t. cnnule, nd in 5 intct nimls with cetic cid infusion 12 * * o 1 8 %I. 8 \ 6 o4 2 * Dose (mg kg-', i.v.) Figure 2 Log dose-response curves showing the effects of incresing doses of LY (.1-3mg kg', i.v.) on the mplitude of reflex bldder contrctions under isovolumetric conditions in the urethnenesthetized intct (-) nd chronic spinl (A) rts. Absciss scle: the cumultive dose of LY (mg kg-', i.v.) plotted on log scle. Ordinte scle: contrction mplitude s % of control. Men ± s.e.men is plotted for ech point. Dt in the intct rt (n = 5) shows dose-dependent significnt decrese from control. Dose-response curve in the chronic spinl rt (n = 5) shows smll but significnt increse t doses of.3 nd 1 mg kg-'. Individul doses re compred to control by pired t test (*P <.5, **P<.O1). As comprison of dose-response curves between the intct nd chronic spinl rts t ech dose, significnt differences were seen t.3-3 mg kg-' (.3-1 mg kg-', P<.5; 3 mg kg-', P<.1; 1-3mgkg-', P<.1, unpired t test). m L b m ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~. m _ - * -E. _ Il in Control 3 mg kg-' 1 mg kg- Figure 1 The effects of incresing doses of LY on rhythmic reflex bldder contrctions under isovolumetric conditions in the urethne-nesthetized intct () nd chronic spinl rts (b). Doses represent totl cumultive doses. Note tht the mplitude of bldder contrctions is reduced by incresing doses of LY (3-1 mg kg- ', i.v.) in the intct rt while smll increse is seen in the chronic spinl rt. The spinl cord ws sectioned t the level of T6-T8, 3 weeks prior to the experiment.

3 EFFECTS OF LY ON THE MICTURITION REFLEX 79 insted of sline), fine (76 gm) wire EMG electrodes were plced percutneously in the externl urethrel sphincter to record the electricl ctivity of the strited muscle. A 3 guge needle with hooked EMG electrode positioned t the tip ws inserted into sphincter pproximtely 5-1 mm lterl to the urethr nd then withdrwn leving the EMG wires embedded in the muscle (Kruse et l., 199). The EMG ctivity obtined ws pssed through rtemeter nd recorded on chrt recorder. The pek firing rte during ech micturition contrction ws mesured. Acetic cid (.1%) infusion into the bldder Acetic cid infused into the bldder ws used s model for bldder irrittion (Birder & de Grot, 1992). In 12 experiments,.1% cetic cid (ph = 3.5) ws infused into the bldder, fter recording control bldder contrctions induced by norml sline infusion. Control bldder ctivity during chemicl irrittion ws nlyzed h fter beginning cetic cid infusion, to llow time for the bldder contrctions to stbilize. Evlution nd sttisticl nlysis The effects of cumultive doses of LY274614, LY or LY injected t pproximtely 15 min intervls on the mplitude, durtion nd frequency of reflex bldder contrctions were recorded under isovolumetric conditions with the urethr closed or with it open to llow the bldder to empty. The intervl between two voiding cycles, termed the intercontrction intervl (Mggi et l., 1986), ws lso mesured during continuous cystometry. During single cystometry, ech dose of LY ws given 2 min prior to the first test. Two to four records were obtined fter ech dose. The effects of the drug on volume threshold (VT) to induce micturition nd the volume of fluid relesed (voided volume; Vv) during ech voiding reflex ws mesured. Bsed on this vlue, voiding efficiency (%) (VE = 1 VV(VT)-') could be estimted. All vlues re expressed s men ± s.e.men. Repeted mesures nlysis of vrince (ANOVA) nd Student's t test were used when pproprite for sttisticl dt nlysis. For ll sttisticl tests, P <.5 ws considered significnt. The ED5 vlues were clculted from the dose-response curves. Drugs Drugs used in these studies included urethne (ethyl crbmte, Sigm), hlothne (Ayerst Lb. Inc.), LY ((± )-dechydro-6-(phosphonomethyl)-3-isoquinolinecrboxylic cid), LY ((± )-dechydro-6-(2h-tetrzol-5-ylmethyl)- 3-isoquinolinecrboxylic cid) nd LY ([cis-(-)]-4- (2H-tetrzol-5-ylmethyl)-2-piperidinecrboxylic cid) (Lilly Res. Lb.). LY ws dissolved in few drops of IN sodium hydroxide nd physiologicl sline for i.v. dministrtion nd in rtificil CSF (Merlis, 194; Feldberg & Fleischhuer, 196) for i.t. injection. LY nd LY were dissolved in physiologicl sline for i.v. dministrtion. Drugs doses were clculted for the slts of ech compound. Results The effects of LY on bldder reflexes were studied in four different preprtions: (1) nimls with n intct 1-~co 3 42 JMJWRJ 1~ cn j)c( l w. b 3 1 In AEA A -o i i I I t I UI krraa ( fl U) J JA i &I L L Control 3 mg kg-' 3 mg kg1 1 min Figure 3 The effects of incresing doses of LY on continuous filling (.21 ml min-') cystometry nd EMG of the externl urethrl sphincter muscle in urethne-nesthetized intct () nd chronic spinl (b) rts. Doses represent totl cumultive doses. Note tht the mplitude of micturition contrctions nd sphincter EMG ctivity re reduced by incresing doses of LY (3-3mg kg-', i.v.) both in the intct nd chronic spinl rts. Some bldder nd sphincter EMG ctivity in the chronic spinl rt remined even t the lrgest dose 3 mg kg- ' while those in the intct rt re completely bolished t this dose. Dt in the intct rt t 3 mg kg-' show bldder inhibition with higher bseline intrvesicl pressure. The reduction in mplitude of micturition contrctions in the chronic spinl niml is probbly due to inhibition of externl urethrl sphincter ctivity (see Discussion). EUS = externl urethrl sphincter.

4 8 M. YOSHIYAMA et l. neurxis (intct) nd (2) chronic spinl nimls in which the i.v. route of dministrtion ws used, (3) intct nimls in which drugs were dministered intrtheclly (i.t.) nd (4) intct rts during bldder irrittion produced by infusion of.1% cetic cid into the bldder. Six prmeters of bldder ctivity were mesured: pek mplitude of intrvesicl pressure, durtion nd frequency of bldder contrctions, intercontrction intervl (intervl between voids during continuous cystometry), volume threshold for inducing micturition (VT or bldder cpcity) nd voided volume (Vv). Bsed on VT nd Vv,, voiding efficiency s percentge of bldder volume (VE = 1VV (VT)-') ws estimted. In some experiments the externl urethrl sphincter EMG ctivity ws lso recorded while recording micturition reflexes. Intct rts Bldder ctivity recorded under constnt volume conditions (n = 5) consisted of rhythmic contrctions occurring t pek mplitudes of mmhg (men = 33 mmhg), frequencies of min-' (men =.69 min-') nd durtions of s (men = 38 s). The mplitude of bldder contrctions ws decresed or inhibited completely following the dministrtion of incresing doses of LY (Figure l). As shown in the dose-response curve in Figure 2, significnt decrese in mplitude of bldder contrctions ws seen t doses between 3-3 mg kg-' with n ED5 of 2.3 mg kg-'. LY in doses of.1-3 mg kg-' did not ffect either frequency or durtion of bldder contrctions. In 12 nimls, urinry bldder contrctions recorded during continuous trnsurethrl infusion (.21 ml min-') of sline exhibited pek pressures rnging from mmhg (men = 5 mmhg), durtion of s (men = 68 s) nd intercontrction intervl of 1-32 s (men = 118 s). The mplitude of micturition contrctions ws reduced in dosedependent mnner following the dministrtion of incresing doses of LY (3-3mg kg-', i.v.; Figures 3 nd 4) with n ED5, of 12.2 mg kg-'. Although chnges in intercontrction intervl nd durtion of micturition contrctions were seen in some experiments, these chnges were not consistent or sttisticlly significnt (n = 12). In 3 of 12 nimls, 3 mg kg-' or higher doses did not completely bolish the rhythmic bldder ctivity but these doses did reduce mplitude to 48% of control (rnge: 26-86% of control). In 5 of 9 nimls following complete inhibition of rhythmic bldder contrctions, bseline bldder pressure ws higher thn control levels (rnge: 1-23 mmhg, bove control bseline men = 16 mmhg) (Figure 3). Sphincter EMG recorded simultneously with bldder reflex ctivity ws lso reduced in dose-dependent fshion by LY (.1-3 mg kg', n = 5) with n ED5, of 3.5 mg kg' (Figures 3 nd 4b). O 12 o1 O 8 d E < C- b L- cj ol -C,._e QL cn Dose (mg kg-1, i.v.) Figure 4 Log dose-response curves showing the effects of incresing doses of LY (.1-3 mg kg-', i.v.) on the mplitude of micturition contrctions () nd sphincter EMG (b), during continuous filling (.21 ml min') cystometry in the urethne-nesthetized intct () nd chronic spinl (A) rts. Absciss scle: the cumultive dose of LY (mg kg-', i.v.) plotted on log scle. Ordinte scle: contrction mplitude () or pek firing of EMG in spikes s-' (b) s % of control. Men ± s.e.men is plotted for ech point. Dose-response curves for mplitude of micturition contrctions or sphincter EMG both in the intct (n = 12, cystometrogrms; n = 5, EMG) nd chronic spinl (n = 6, cystometrogrms; n = 5, EMG) rts show significnt decrese. Individul doses re compred to control by pired t test (*P<.5, **P<.1, ***P<.1). No significnt difference between the pirs of dose-response curves ws reveled by two-wy repeted mesures ANOVA in either () or (b). 1'.: Control VT.3 ml ^ m3mgk91 VT=O5m lso rdcdyy264m mcutovt= 14uml - Sti q X Xliz infubn (O.J4 mlr n-) { Figure S The effects of incresing doses of LY during single filling (.4 ml min-') cystometry in the urethne-nesthetized intct rt. Doses represent totl cumultive doses. Note tht the intervl from the strt of sline infusion to the point where micturition occurs ws prolonged by incresing doses of LY (3-3 mg kg-', i.v.). Amplitude of micturition contrctions ws lso reduced by LY M = micturition. VT = volume threshold.

5 EFFECTS OF LY ON THE MICTURITION REFLEX 81 U L. 4- c * *I w loor 8[ 6[ 4 T T T 2 2 E. U '- I [ 15 [ u L 3 Dose (mg kg-1, i.v.) Figure 7 The depressnt effect of incresing doses of LY (.3-3 mg kg-', i.v.) on the efficiency of voiding during single filling (.4mlmin-') cystometry in the urethne-nesthetized intct rts (n = 5). Voiding efficiency (VE, %) is defined s 1 Vv (VT) - Absciss scle: the dose of LY (mgkg-', i.v.). Ordinte scle: voiding efficiency (%). Men ± s.e.men is plotted for ech column. Individul doses re compred to control by pired t test (*P<.5, **P<.1). C = control. 2 1O 8 6 C r.3 T 4 * 21[ 1 3 * 3 Dose (mg kg-', i.v.) Flgur 6 () The effect of LY on the mplitude of micturition contrction during single filling (.4 ml min-') cystometry in the urethne-nesthetized intct rts (n = 5). LY (.3-3 mg kg- ', i.v.) decreses the mplitude in dose-dependent mnner similr to the result during continuous filling (.21 ml min-') cystometry (Figure 4). There is no sttisticl difference t ech dose between continuous nd single cystometry. (b) LY increses VT (volume threshold) in dose-dependent mnner. (c) LY decreses Vv (voided volume) in dose-dependent mnner. Men ± s.e.men is indicted for ech column. Individul doses re compred to control by pired t test (*P<.5, **P<.1, ***P<.1). In 5 nimls, urinry bldder ctivity ws recorded during cystometry performed using slower rte of trnsurethrl infusion (.4 ml min') which more closely pproximted the rte of urine formtion (Sillen, 198). The bldder ws emptied t the end of ech cystometrogrm. The pek mplitudes of micturition contrctions rnged from 26-4 mmhg (men = 33 mmhg) nd durtions between s (men = 34 s). These prmeters were not significntly different from the mesurements obtined during continuous cystometry. LY produced dose-dependent decrese in the mplitude of micturition contrctions (Figures 5 nd 6) similr to the effect during continuous cystometry (Figure 4). Figure 5 shows tht with incresing doses of LY the volume of sline (bldder cpcity or volume threshold) to produce micturition contrction ws incresed 1.5 nd 3 times control levels with 3 nd 3 mg kg-' of LY274614, respectively. In ddition, s the bldder begn to fill, smll bldder contrctions which did not produce void were seen prior to 3 micturition (Figure 5). Control VT nd Vv were ml (men =.41 ml) nd ml (men =.31 ml), respectively, with VE of 74%. Incresing doses of LY incresed VT (Figure 6b) nd reduced Vv (Figure 6c). VE ws reduced to 11% of control t the mximl dose of LY (Figure 7). Chronic spinl rts In 5 chronic spinl nimls, bldder ctivity recorded under constnt volume conditions consisted of rhythmic contrctions occurring t pek mplitudes of 1-23 mmhg (men = 16 mmhg), frequencies of min ' (men =.96 min-') nd durtions of s (men = 37 s). As shown in Figure lb, LY274614, t doses of.3 nd 1 mg kg- 'in this preprtion slightly but significntly incresed the mplitude of bldder contrctions (Figure 2), while the effects of lrger doses were not significntly different from control. Doses of LY (.1-3mg kg-' i.v.) hd no effect on frequency or durtion of bldder contrctions. In 6 chronic spinl nimls, micturition contrctions recorded during continuous trnsurethrl infusion (.21mlmin-') of sline exhibited pek pressures rnging from 1-42 mmhg (men = 18 mmhg), durtions of s (men = 31 s) nd intercontrction intervls of 8-42 s (men = 18 s). Incresing doses of LY (.1-3 mg kg-', i.v.) reduced the mplitude of the micturition contrctions (Figure 3b). A significnt decrese ws seen t doses of 1 nd 3 mg kg-' of LY (Figure 4) with n ED5 of 18.5 mg kg-'. Durtions nd intercontrction intervls were not ffected by LY Following the 3 mg kg-' dose of LY bldder ctivity ws depressed for the reminder of the experiment (2-8 h). In 5 nimls in which sphincter EMG ws recorded during cystometry, LY significntly decresed sphincter ctivity with n ED5 of 4.7 mg kg-' (Figures 3b nd 4b). Intct rts: i.t. dministrtion of LY In 13 nimls with n intrthecl ctheter, voiding reflexes induced by continuous infusion of sline (.21 ml min-') hd pek intrvesicl pressures of mmhg (men = 58 mmhg), intercontrction intervls of s (men = 16 s) nd durtions of s (men = 92 s). An i.t. dministrtion of LY (.6-3 fig) produced

6 82 M. YOSHIYAMA et l. dose-relted reduction in micturition contrction mplitude (EDm of.11 tg) (Figures 8 nd 9). In 7 nimls (greter thn 5% of nimls tested) bldder ctivity ws bolished t dose of 121g of LY In 9 nimls fter complete inhibition, the bseline bldder pressure mrkedly incresed (2 mmhg; rnge: mmhg) nd sline constntly leked from the externl urethrl orifice (overflow incontinence). In 7 nimls, bldder ctivity ws mesured 5-18 h following complete inhibition with doses rnging from ltg. During this period, 5 of 7 nimls showed prtil recovery of bldder ctivity (35-141% of beginning control, men = 71%). In 4 of these nimls, second dose of LY lso bolished the bldder ctivity. The effects of LY on durtion nd intercontrction intervl were quite vrible nd lthough few doses showed sttisticlly significnt decrese, the effects were not dose-dependent. Sphincter EMG recorded simultneously with bldder reflex ctivity ws lso reduced in dose-dependent fshion by LY (.6-3 Lg, n = 8) (Figures 8 nd 9b). A sttisticlly significnt reduction in the EMG ctivity ws noted t dose of.12 jg (64.% of control) with n EDm of.16 fig. Time course of LY effects on mplitude of micturition contrctions mesured during continuous cystometry The durrtion of bldder inhibition produced by LY ws studied following both i.v. nd i.t. dministrtion of the drug. During continuous cystometry, single dose (3mgkg-', i.v., n=5 or 6 xg i.t., n=5) of LY ws given to ech niml nd time course of recovery recorded for up to 1 h following drug dministrtion. Figure 1 shows the time course (up to 6 h) of recovery. Some recovery ws seen t 2.5 to 3 h following LY In 2 nimls following i.v. nd in 3 nimls following i.t. dministrtion, the mplitude of the micturition contrctions hd not recovered to control levels for up to 1 h following dministrtion. Influence of bldder irrittion with cetic cid (.1%) on the effects of L Y (i.v.) Since drugs tht suppress the micturition reflex might be used cliniclly to diminish bldder hyperctivity induced by neurologicl disorders, series of experiments ws conducted to exmine the effect of LY on bldder hyperctivity induced by chemicl irrittion. In 12 nimls, urinry bldder ctivity ws recorded during continuous trnsurethrl infusion (.21 ml min-'). Control micturition contrctions induced by sline infusion occurred t pek mplitudes of mmhg, intercontrction intervls of s nd durtions of s (Tble 1). Following control period of 3 min with sline infusion, the infusion solution ws swit- 1 co 8 C ' * 4 4 Ē 2 E -W 1 c o 8 Co 1 Ī 4 X 2 Cn) ly.6.6 Dose (,ug, i.t.) I,I 6 6 Figure 9 Log dose-response curves showing the effects of incresing doses of LY (i.t.) on the mplitude of micturition contrctions () nd sphincter EMG (b) during continuous filling (.21 ml min -') cystometry in the urethne-nesthetized intct rts (n = 13, cystometrogrms; n = 6, sphincter EMG). Absciss scle: the cumultive dose of LY (jig) plotted on log scle. Ordinte scle: contrction of micturition mplitude () or pek firing of EMG in spikes s-i (b) s % control. Men ± s.e.men is plotted for ech point. Individul doses re compred to control by pired t test (: P<.5, P: P<.1, y: P<-1)- ched to.1% cetic cid, which hd little effect on the mplitude (32-69 mmhg) or durtion (25-56 s) of micturition contrctions but significntly decresed intercontrction intervl (9-83 s) (Tble 1). In these nimls the mplitude of micturition contrctions (n = 12) nd sphincter EMG ctivity (n = 5) were reduced following the dministrtion of incresing doses of LY (.1-3 mg kg- ' i.v.: Figure 11). The ED5s for these effects were 24.3 mg kg-' nd 16.6mg kg-', respectively. Although reduction in mplitude of micturition contrctions seemed to require lrger dose of LY in the cetic cid-infused bldder (Figure ll), this effect ws,cd_ 3 x, E I -]J 251 ~CD W Co I Control.12 pg 18 pg 1 min Figure 8 The effects of incresing doses of LY dministered by i.t. injection on continuous filling (.21 ml min') cystometry nd sphincter EMG in the urethne-nesthetized intct rt. Doses represent totl cumultive doses. Drug injections were mde subdurlly t the L6-S, level of spinl cord. Note the decrese in the mplitude of the micturition contrctions nd the sphincter EMG ctivity following LY ( jig). EUS = externl urethrl sphincter.

7 EFFECTS OF LY ON THE MICTURITION REFLEX 83 Tble 1 The effects of cetic cid (.1%) continuous filling infusion (.21 ml min ') to the bldder Continuous filling cystometrogrm Sline Acetic cid Amplitude (mmhg) 53 ± 5 52 ± 3 Intercontrction intervl* (s) 95 ± 2 27 ± 6 Durtion (s) 41 ± 6 4 ± 3 *Significnt difference between 'sline' nd 'cetic cid' (P<.1, pired t test). Only the intercontrction intervl ws chnged by cetic cid continuous infusion to the bldder but not the mplitude nd durtion of micturition contrctions. All vlues re expressed s men ± s.e.men (n= 12). not sttisticlly significnt (two-wy repeted mesures ANOVA) from sline infusion. Reduction in sphincter EMG did, however, require lrger dose of LY in the cetic cid infused bldder s compred to sline-infused preprtions (Figure 1 Ib). A significnt reduction in sphincter EMG ws only detected t dose of 3 mg kg-' (23.2% of control); nd significnt difference from the control experiment with sline infusion occurred t 1 mg kg-' (two-wy repeted mesures ANOVA followed by unpired t test, P <.5). A significnt decrese in durtion of micturition contrctions ws seen t 1 nd 3 mg kg' for cetic cidinfused bldder. Effects of LY nd L Y on continuous filling cystometry Two other competitive NMDA receptor ntgonists were studied on micturition reflexes. LY is relted chemiclly to LY274614, while LY is chemiclly distinct. Both compounds (1-1 mg kg-', i.v. of LY233536, n = 5 or LY235723, n = 6) decresed the mplitude of micturition contrctions in dose-dependent mnner when studied with continuous cystometry. Micturition contrctions were completely inhibited by LY or LY t 1mgkg-' with ED5s of 18 or 2 mg kg-', respectively. A significnt decrese in mplitude occurred t doses of 1mgkg-' nd lrger of LY235723, nd 3 mg kg- nd lrger of LY These drugs did not chnge durtions or intercontrction intervls with the exception of n increse of these intervls by LY t doses of I mg kg- (P <.5, pired t test) nd 3 mg kg' (P <.1, pired t test). Discussion The results of the present study indicte tht LY274614, LY nd LY235723, competitive NMDA receptor ntgonists tht enter the centrl nervous system (CNS) following systemic dministrtion, cn inhibit bldder nd externl urethrl sphincter reflexes in the urethne-nesthetized rt. The site of these inhibitory effects is, t lest in prt, t the level of the lumboscrl spinl cord nd dependent on n intct descending pthwy from suprspinl sites. These dt support the conclusions tht emerged from previous studies with the non-competitive NMDA receptor ntgonist, MK-81, tht CNS glutmtergic mechnisms involving NMDA receptors re importnt in modulting bldder nd sphincter ctivities. Although the block of the micturition reflex produced by MK-81 is t the ctionic chnnel ssocited with the NMDA receptor complex (Foster & Fgg, 1987) while LY274614, LY nd LY block t the recognition site for NMDA, the effects of the drugs re qulittively similr in most respects. Both LY nd MK-81 inhibit bldder nd sphincter reflexes when dministered either intr- 14 & 12 I- 8 1oo ' 4 E 2 1- I- t **I Time (h) Figure 1 Grph showing the time course of the effect of LY (3 mg kg-,' i.v.,, n = 6 nd 6 fig i.t., A, n = 6) on the mplitude of micturition contrctions during continuous filling (.21 ml min-') cystometry in the urethne-nesthetized intct rts. Absciss scle: time scle (h) since LY ws dministered. Ordinte scle: contrction mplitude s % of control. Men ± s.e.men is plotted for ech point. Individul points re compred to control by pired t test (*P<.5, **P<.1, ***P<.1). A comprison between the i.v. nd i.t. dministrtion t ech period, showed no significnt difference up to 6 h. Note tht some recovery from LY ws seen 2.5 to 3 h fter injection but complete recovery ws not seen in ll nimls even 6 h fter LY = F 6 ~4- X 2 E 6 \ b n -b_ t, ~ ~~~~~~~~ 2 co X,* Ql 1 3 Dose (mg kg-' i.v.) Figure 11 Log dose-response curves showing the effects of incresing doses of LY (i.v.) on the mplitude of micturition contrction () nd sphincter EMG (b) during continuous filling (.21 ml min-') cystometry with either sline () or.1% cetic cid () in intct rts. Absciss scle: the cumultive dose of LY (mg kg-', i.v.) plotted on log scle. Ordinte scle: contrction mplitude () or rtemeter output of EMG in spikes s- I s % control (b). Men ± s.e.men is plotted for ech point. Dose-response curves both with sline (n = 12, cystometrogrms; n = 5, sphincter EMG) nd cetic cid (n = 12, cystometrogrms; n = 5, sphincter EMG) infusion show sttisticlly significnt decrese in mplitude nd sphincter EMG pek firing. Individul doses re compred to control by pired t test (*P<.1, **P<.1). There is no significnt difference between pirs of dose-response curves with sline or cetic cid infusion in () lthough significnt difference is seen in (b) t dose of 1mg kg-' (two-wy repeted mesured ANOVA followed by unpired t test, P<.5). venously (i.v.) or intrtheclly (i.t.). MK-81 is however more potent (ED5 =.36mg kg-') thn LY (ED5 = 12.2 mg kg-') on i.v. dministrtion but LY is more potent on i.t. dministrtion (ED5 of MK-81 = 17 tsg; of LY =.11 pg) (Yoshiym et l., 1993). LY hs slightly

8 84 M. YOSHIYAMA et l. longer time for onset thn MK-81 (4-6 min compred to 1-2 min). These differences re consistent with the poorer penetrtion of LY cross the blood-brin brrier nd the greter ffinity of LY for its binding to the NMDA receptor complex (personl communictions with Drs Ornstein & Schoepp), in comprison to MK-81. Similr differences in effective doses of MK-81 nd LY were seen by other investigtors exmining the effects of these NMDA ntgonists on pin pthwys (Elliott et l., 1991) nd on neurotoxic effects of excittory mino cids nd mphetmine (Fuller et l., 1992). One other qulittive difference between these two drugs ws the elevted bldder tone following complete inhibition of bldder contrctions with LY274614, seen when bldder reflexes were studied during continuous cystometry. The bseline bldder pressure ws high (verge 2mmHg) with LY (i.v. nd i.t.), but this ws rrely seen with MK-81. Overflow incontinence occurred with both drugs following inhibition of bldder contrctions but only with LY ws n elevted resting pressure observed. The mgnitude of the pressure recorded in the bldder during continuous infusion cystometry depends on two fctors, the contrctile force of the bldder nd the pressure t which the strited nd smooth muscle sphincters relx (outlet resistnce). Since both LY nd MK-81 inhibit micturition contrctions, the increse in bseline pressure seen following LY is probbly due to some residul urethrl resistnce reducing flow through the urethr. The exct mechnism of this high resting tone is not known but my represent some differentil ction of the two drugs on either urethrl smooth or strited muscle sphincter ctivity. Although the mechnism of this elevted bldder pressure ws not investigted in the present study one might speculte from in vitro dt, which shows tht MK-81 blocks nicotinic chnnels in cultured strited muscle cells (Amdor & Dni, 1991), tht MK-81 my produce more effective relxtion of externl urethrl sphincter thn LY An lterntive explntion would be tht MK-81 is more effective in producing relxtion of the smooth muscle of the urethr. The site of ction of LY within the CNS ppers to be similr to tht proposed for MK-81 (Yoshiym et l., 1993). Since n i.t. dministrtion of LY or MK-81 t the level of L6-S1 spinl cord inhibited bldder nd sphincter ctivity, spinl site of ction is likely. However, the bsence of n effect of LY or MK-81 (Yoshiym et l., 1993) on bldder contrctions in chronic spinl nimls studied under isovolumetric conditions would suggest tht n intct descending pthwy from the pontine micturition centre is necessry for the ction of the drugs. However, decrese in sphincter ctivity with LY or MK-81 ws seen both in nimls with n intct spinl cord nd in chronic spinl cord trnsected nimls; suggesting tht NMDA receptor mechnisms re importnt for both the spinl nd suprspinl control of externl urethrl sphincter ctivity. It should be noted, however, tht LY did prtilly reduce the mplitude of the micturition contrctions in chronic spinl nimls when using continuous cystometry with the urethrl outlet open. This effect is probbly produced by decresed outlet resistnce rther thn decrese in bldder contrctility since with continuous cystometry, bldder pressure begins to decrese s soon s the urethrl outlet relxes nd voiding begins. Furthermore, LY274614, even in very lrge doses (>3mgkg-'), produced no decrese in reflex bldder contrctions in group of nimls studied with closed bldder outlets (ligted) nd t constnt bldder volume. In these nimls, chnges in outlet resistnce could not modify bldder pressure since ligture seprtes the bldder from the urethr. It is, therefore, concluded tht reflex bldder contrctions re not reduced by LY in chronic spinl nimls. The lck of effect on bldder contrctions in chronic spinl nimls with both LY nd MK-81 would suggest tht neurl elements in the spinl bldder reflex pthwy including bldder fferent neurones, interneurones nd pregnglionic efferent neurones do not utilize glutmtergic NMDA receptor mechnism. On the other hnd i.t. dministrtion of very smll doses of LY to nimls with n intct spinl cord inhibited bldder ctivity, indicting tht the descending limb of the spinobulbospinl micturition reflex pthwy (de Grot et l., 1992) my utilize glutmte s trnsmitter nd must be intct for LY to be effective. However, since the drugs do depress sphincter ctivity in chronic spinl nimls, NMDA receptor-relted glutmtergic mechnisms must be involved in the spinl reflex pthwy controlling sphincter function. In intct nimls, the sphincter ctivity is suppressed t lower dose (EDj, = 3.5 mg kg-') thn bldder ctivity (ED_o= 12.2mg kg-'), which suggests tht the drug ffects bldder nd sphincter by different mechnisms. NMDA receptors in the spinl cord re prominent in the vicinity of the motoneurones which control sphincter, nd round the centrl cnl nd in the intermediolterl gry (Jnsen et l., 199; Shw et l., 1991). The ltter regions of the cord contin bldder pregnglionic neurones or interneurones which re involved in lower urinry trct function (de Grot et l., 1992), nd thus re likely to be one site of ction of the NMDA ntgonists. Although the present study suggests spinl site of ction for LY274614, n effect on suprspinl pthwys is lso possible. A suprspinl site of ction is supported by receptor binding studies which show tht NMDA receptors re present in the brinstem ner the pontine micturition centre (Monghm & Cotmn, 1985). Further support for suprspinl site of ction is provided by studies from this nd other lbortories which exmined the effects of neuromodultors t the level of the pontine micturition centre (Lumb & Morrison, 1987; Willette et l., 1988; Mllory et l., 1991). In these studies glutmte ws n effective gent in modulting micturition reflexes producing reduction in VT when injected directly into the pontine micturition centre. LY274614, much like MK-81 (Yoshiym et l., 1993), incresed the VT nd decresed the Vv when given i.v. nd tested using cystometry. The EDm for LY in these experiments is bout 3 times tht for MK-81: suggesting tht LY is less potent thn MK-81. The durtion of ction of LY following either i.v. or i.t. ws long, requiring pproximtely 4 h for 5% recovery. These results re consistent with those reported by other lbortories which hve evluted different phrmcologicl effects of LY (Schoepp et l., 1991; Fuller et l., 1992). We therefore were ble to use, in the mjority of our studies, cumultive doses of LY given t 15 min intervls with complete dose-response curves usully requiring less thn 9 min. This pproch could be used since there ws no evidence for rpid tolernce development to repeted i.v. dministrtion of LY Following prtil or totl recovery from LY274614, second dose of the drug produced the sme mgnitude of response s seen with the first dose. The effects of LY on bldder hyperctivity due to bldder irrittion ws lso exmined in the present study. Bldder irrittion ws produced by substituting.1% cetic cid for norml sline during continuous cystometry. In the irritted bldder model, LY reduced the mplitude of micturition contrctions nd sphincter EMG ctivity with the dose-response curve for sphincter EMG showing smll shift to the right. The typicl effect of LY in reducing bldder nd sphincter ctivity occurred t slightly higher dose for sphincter EMG ctivity when the bldder ws irritted with cetic cid. The mjor effect produced by cetic cid ws to decrese the intercontrction intervl (increse in frequency), nd this effect of irrittion ws not reduced by LY This ltter result would be in greement with our conclusions tht neither LY nor MK-81 (Yoshiym et l., 1993) hve ny effect on the fferent limb of the micturition reflex or on nociceptive fferents tht modulte the reflex. These findings re lso consistent with the effects of MK-81 on incresed expression) of the immedite erly gene, c-fos, in spinl neurones induced by stimultion of bldder fferents. MK-81

9 EFFECTS OF LY ON THE MICTURITION REFLEX 85 in doses tht blocked the micturition reflex did not lter c-fos expression elicited by non-noxious distension of the bldder of the rt (Birder & de Grot, 1993). Lrge doses of MK-81 did reduce by 5% the incresed c-fos expression induced by chemicl irrittion of the bldder (Birder & de Grot, 1992). However, this- effect ws remrkbly reduced in spinl trnsected nimls, suggesting tht the effect of MK-81 ws dependent upon the integrity of suprspinl pthwys nd ws not medited by direct ctions on spinl nociceptive mechnisms. Although the phrmcology nd possible sites of ction of LY were the primry focus of this study, two dditionl competitive NMDA receptor ntgonists were exmined for their effects on micturition reflexes. LY is chemiclly similr to LY except tht the phosphonic cid moiety is replced with tetrzole group, while LY is the (-)- isomer of LY23353, 4-tetrzolyllkyl substituted piperidine- 2-crboxylic cids with competitive NMDA ntgonist ctivity (Schoepp et l., 199; Ornstein et l., 1991; 1992,b; Zimmermn et l., 1992). Both LY nd LY produced effects, qulittively similr to those of LY nd MK- 81, but were less potent in reducing micturition bldder contrctions. The reduced potency of these compounds is in greement with lower NMDA receptor ffinity in vitro nd lower in vivo nd in vitro ntgonist ctivity reported by others (Ornstein et l., 1991). The fct tht vriety of compounds which possess high ffinity for NMDA receptors in vitro produce profound effects on micturition reflexes, provides strong support for the ide tht glutmtergic mechnisms re importnt in modulting reflex micturition. In summry, these experiments suggest tht glutmic cid is n importnt neurotrnsmitter in the micturition reflex pthwy of the rt. This substnce cts vi NMDA receptors in the lumboscrl spinl cord when descending pthwys from the brinstem to the cord re intct. On the other hnd, in chronic prplegic rts when the descending pthwys re eliminted, NMDA receptor medited glutmtergic trnsmission is not essentil for the genertion of spinl micturition reflexes. The inhibitory effects of LY on externl urethrl sphincter, however, still occurred fter chronic spinliztion; indicting tht glutmte is trnsmitter in the spinl reflex pthwys controlling the urethrl sphincter. LY is long cting NMDA ntgonist nd my be potentilly useful in the tretment of neurogenic bldder hyperctivity. It hs the dvntge over MK-81 of hving fewer phencyclidine-like side effects (Rsmussen, 1991; Rsmussen et l., 1991). 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