muscarinic response immediately following application of soman to cat bladder parasympathetic neurones. In order to

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1 Br. J. Phrmcol. (199), 99, Mcmilln Press Ltd, 199 Action of n irreversible cetylcholine esterse inhibitor, somn, on muscrinic hyperpolriztion in ct bldder prsympthetic gngli 'iichi Kummoto & Ptrici Shinnick-Gllgher Deprtment of Phrmcology nd Toxicology, University of Texs Medicl Brnch, Glveston, Texs 7755, U.S.A. 1 Intrcellulr recording techniques were used to exmine the ction of n irreversible cetylcholine esterse (ACh) inhibitor, somn, on the hyperpolriztions medited through muscrinic cholinoceptors in ct bldder prsympthetic neurones. 2 Somn (.1-1gM) depressed the mplitude nd prolonged the durtion of the muscrinic slow inhibitory postsynptic potentil (s-i.p.s.p.) elicited by pregnglionic tetnus (4 Hz for 1 s) in the presence of mecmylmine (2pum), phentolmine (1 pm) nd cffeine (1 mm), in dose-dependent mnner. The effect of somn on the mplitude of the s-i.p.s.p. ws prtilly reversible, while the effect on the durtion ws irreversible. 3 Somn hyperpolrized the membrne nd decresed input resistnce, but this effect could not ccount for somn-induced inhibition of the s-i.p.s.p. 4 Somn depressed the mplitude nd prolonged the durtion of muscrinic hyperpolriztion induced by pressure ppliction of cetylcholine (ACh) in the presence of mecmylmine, phentolmine nd cffeine. The time course of this effect prlleled tht on the synpticlly-evoked muscrinic s-i.p.s.p. S A reversible ACh inhibitor, pyridostigmine (1-1gM), lso depressed the mplitude nd prolonged the durtion of muscrinic hyperpolriztion induced by either pregnglionic stimultion or ACh pressure ppliction. These ctions were reversible, nd not ccompnied by significnt chnge in membrne potentil or input resistnce. 6 The inhibitory ction of somn (1 ym) on the muscrinic hyperpolriztion ws prevented by pyridostigmine (1OgM), but not by tropine (1 pm). 7 These results demonstrte tht somn prolongs not only the muscrinic hyperpolriztion, but lso inhibits its mplitude through postsynptic ction, probbly through ACh inhibition, in ct bldder prsympthetic neurones. Introduction It is known tht chronic tretment with irreversible orgnophosphorus cetylcholine esterse (ACh) inhibitors, DFP (diisopropylfluorophosphte; Ymd et l., 1983; Sivm et l., 1983) nd somn (pincolylmethylphosphonofluoridte; Churchill et l., 1984; As et l., 1987), reduces the number of muscrinic cetylcholine (ACh) receptors (see Russell & Overstreet, 1987, for review). This is ttributed to persistent cholinomimetic ctivity resulting from ACh inhibition (Klein et l., 1979). Such down-regultion of muscrinic receptors ws not observed fter cute dministrtion (Sivm et l., 1983; As et l., 1987). These results were obtined from binding studies of muscrinic ACh receptors. To our knowledge, there re few electrophysiologicl studies which exmine n effect of ACh inhibitors on muscrinic response. Recently, we found with intrcellulr recording techniques tht the ppliction of somn immeditely inhibited the membrne hyperpolriztion medited by the muscrinic receptors in ct bldder prsympthetic neurones (Kummoto & Shinnick-Gllgher, 1988). It is well known tht ACh inhibitors induce not only the persistence of ACh in the synptic cleft due to ACh inhibition, but lso other ctions (see Krczmr, 1984, for review), including blockde of the nicotinic ACh receptor-ion chnnel complex (Kub et l., 1974; Albuquerque et l., 1987). Therefore, the cute somn-induced depression of muscrinic hyperpolriztion my be due to n ction independent of ACh inhibition. This study ws undertken to exmine elec- ' Present ddress nd correspondence: Deprtment of Physiology, Sg Medicl School, Sg, 84-1, Jpn. trophysiologiclly the mechnism underlying the decrese in muscrinic response immeditely following ppliction of somn to ct bldder prsympthetic neurones. In order to determine the involvement of ACh inhibition in the ction of somn, the effect of reversible ACh inhibitor, pyridostigmine, on the muscrinic hyperpolriztion ws lso exmined. Methods Adult cts of either sex were used in ll experiments. The urinry bldder ws dissected from the ct under nesthesi induced by n intrperitonel injection of x-chlorlose (66mgkg-1) nd pentobrbitone (5.5mgkg-'). A single gnglion nd ccompnying pregnglionic nerve were visulized under microscope by inflting the urinry bldder with wrm sline injected through polyethylene ctheter inserted into the urethr. The gnglion nd pregnglionic nerve were dissected free of surrounding tissue. The isolted preprtion ws plced in physiologicl solution gssed with 95% 2-5% CO2 t room temperture nd remined vible for t lest two dys. Drug ppliction The gnglion with pregnglionic nerve ws trnsferred to n orgn bth of pproximtely.5 ml in volume nd continuously superfused t rte of bout 3 ml min 1 with preheted oxygented Krebs solution mintined t C t the

2 158. KUMAMOTO & P. SHINNICK-GALLAGHR Tble 1 Recovery of the mplitude nd the durtion of the muscrinic hyperpolriztion from somn (.1-1IM) tretment Amplitude (mv) Durtion (s) Recovery' (post-somn) Tretment time (min) Control (pre-somn) (.1 PM) (.1 PM) (l M) (68) (68) (6) (6) (16) (16) 5-1 (1PM) The muscrinic hyperpolriztion (s-i.p.s.p.) ws elicited by pregnglionic tetnus (4 Hz for 1 s) in the presence of mecmylmine (2Mm), phentolmine (1Mm) nd cffeine (1 mm). The stimulus strength used ws one t which mximl s-i.p.s.p. ws induced. ch vlue is expressed s men + s.e.men. The number in prentheses is the number of cells exmined. Somn,.1, 1 nd 1pM, superfusion with different tretment times hd comprble inhibitory effect on the s-i.p.s.p. (see Figure ic, nd Figures 2 nd 3 in Kummoto & Shinnick-Gllgher, 1988), wheres min tretment with.1 Mm somn hd lmost no effect on the mplitude of s-i.p.s.p. Mesurements during recovery were obtined.2-6 h fter somn tretment. recording site. Drugs were dissolved in the superfuste. The exchnge of the superfusing solution ws complete within 2.5min. Acetylcholine (ACh) ws pplied by pressure pulse (1-2 psi pressure; 3-5ms durtion; Picospritzer, Generl _. L- ~. ) (6) (4) 1- * \(11) (4) Somn concentrtion (>.M) b Control Somn (.1 FM) 28 min cq Control Somn (1 prm) 3 min L mv Figure 1 Somn depressed the muscrinic hyperpolriztions (si.p.s.ps) elicited by pregnglionic tetnus (4Hz for 1 s) in the presence of mecmylmine (2yM), phentolmine (1pM) nd cffeine (1 mm), in dose-dependent mnner. () Dose-response curve for the ction of somn. ch point shows the men of the % chnges in the mplitude of the s-i.p.s.p. Smin fter inititing somn superfusion. Verticl lines show s.e.men (t.1 ym somn the s.e.men ws within the size of the symbol). Corresponding durtions of s-i.p.s.p. t.1,.1 nd IlM somn were , nd % of control, respectively. Since somn (1pM) lmost completely bolished the s- i.p.s.p., it ws difficult to ssess ccurtely the prolongtion of the s-i.p.s.p. Vlues in prentheses re numbers of cells tested. The stimulus strength used ws one t which mximl s-i.p.s.p. ws elicited. (b) ffect of somn (.1 Mm) on the s-i.p.s.p. 28 min fter initition of tretment. The resting membrne potentil (RMP: -48 mv) remined constnt during somn superfusion. (c) Action of somn (1OMM) on the s-i.p.s.p. This effect ws ccompnied by membrne hyperpolriztion (7 mv); RMP = -68 mv. Stimulus strength used ws 5 V. Input resistnce (R1.) ws monitored by nelectronic potentils (downwrd deflection) resulting from injection of constnt current hyperpolrizing pulses (1ms durtion). The presynptic tetnus ws pplied t () shown under ech trce. Vlve) from low-resistnce micropipettes (filled with.1 mm ACh Cl) plced close to the neurone impled by the microelectrode. lectricl mesurements Neurones on the surfce of the gnglion were impled with glss microelectrodes (5-1 MC) filled with.5 M K2SO4. lectricl ctivity ws recorded by n Axoclmp-2A premplifier. Synptic responses were elicited by pplying rectngulr pulses, of.5 ms durtion nd vrious strengths, to the pregnglionic nerve held within smll polyethylene tube filled with the physiologicl solution nd contining Ag/AgCl wire. Dt were stored on video cssette, by use of Video Cssette Formt Instrumenttion Recorder (Model 42B, A. R. Vetter Co.), for subsequent nlysis. Figures were reproduced from pen recorder trcings. All dt re expressed s mens + s.e.men. Sttisticl significnce ws determined by Student's pired t test nd P vlues less thn.5 were considered significnt. In ll cses n refers to number of cells exmined. Solutions The norml Krebs solution hd the following composition (in mm): NCl 117, KCl 4.7, CCl2 2.5, MgCl2 1.2, NH2PO4 1.2, NHCO3 25. nd glucose Drugs used were mecmylmine HCl, tropine sulphte, cffeine, cetylcholine Cl (Sigm, St. Louis, MO, U.S.A.), phentolmine HCl (Summit, NJ, U.S.A.) nd pyridostigmine bromide (Hoffmn-L Roche, Nutley, NJ, U.S.A.). Somn ws obtined from the U.S. Army Medicl Reserch nd Development Commnd, Fort Detrick, Frederick, Mrylnd, U.S.A. Results In Krebs solution contining the nicotinic receptor ntgonist, mecmylmine (2 M), s well s phentolmine (1 M) nd cffeine (1 mm) which blocked -drenergic (Aksu et l., 1986b) nd purinergic (Aksu et l., 1984) slow hyperpolriztions, stimultion of the pregnglionic nerve trunk repetitively (4 Hz for Is) elicited slow muscrinic inhibitory postsynptic potentil (s-i.p.s.p.; Gllgher et l., 1982; Shinnick-Gllgher et l., 1987; Kummoto, 1989). The mplitude of the s-i.p.s.p. ws mv (n = 68; resting membrne potentil (RMP) = mv), nd its durtion ws s t mximl stimulus strengths (see Tble 1).

3 SOMAN INHIBITION OF MUSCARINIC INHIBITORY RSPONS 159 Action ofsomn on the s-i.p.s.p. elicited by the pregnglionic tetnus The ddition of the irreversible cetylcholine esterse (ACh) inhibitor, somn, to the superfuste depressed nd prolonged the muscrinic s-i.p.s.p. (Figures 1, 2, 3 nd 5; Kummoto & Shinnick-Gllgher, 1988). In generl, the higher the concentrtion of somn, the fster its onset of ction. The inhibitory effect of somn on the mplitude of s-i.p.s.p. ws observed more quickly with incresed concentrtions. A high concentrtion (1O pm) of somn rpidly depressed the s-i.p.s.p. within 3min fter initition of tretment (Figure ic; see Figures 2 nd 3 in Kummoto & Shinnick-Gllgher, 1988, for ctions of somn 1 nd.1 pm, respectively) nd this ws ccompnied by prolongtion of the s-i.p.s.p. In contrst, superfusion of low concentrtion (.1 M) of somn for 28 min hd lmost no effect on mplitude, but slowed the rising phse of the s-i.p.s.p. when compred to the control response (Figure lb). A comprison of the % chnge in the mplitude of the s- i.p.s.p. produced by somn (.1-1/pM) 5min fter the commencement of somn superfusion showed tht the ction of somn ws clerly dose-dependent (Figure l). After removl of somn from the superfusion solution, the mplitude of the s-i.p.s.p. prtilly recovered with slow time course, but the durtion remined prolonged (Tble 1). The mgnitude of the muscrinic s-i.p.s.p. ws dependent on the stimulus strength pplied to the pregnglionic nerve trunk (cf. Kummoto, 1989). The mplitude incresed with incresing stimulus strength nd ttined mximum vlue (Figure 2; n = 14). Somn (1 M) depressed this mximum response (Figure 2; n = 4), suggesting tht somn tretment elicited non-competitive decrese in the muscrinic synptic response. A, 15 c o N nco U B 2 25 V cmn C o d25v TRT~nTrnTffTr f32.5v Stimulus strength (V) b 27.5 V nntfth M e T,27.5 V 45 c32.5v mv Figure 2 ffect of somn (1 pm) on the muscrinic hyperpolriztion (s-i.p.s.p.) elicited by pregnglionic tetni (4Hz for is) of vrious stimulus strengths in the presence of mecmylmine (2 gm), phentolmine (1 pm) nd cffeine (1 mm). (A) Muscrinic s-i.p.s.ps re plotted ginst stimulus strengths of the pregnglionic tetni in control (; RMP = -53 mv), nd 1min (; RMP = -54 mv) fter the preprtion ws treted with somn for 1min (depressed the s-i.p.s.ps lmost completely). Curves were drwn by eye. (B nd C) Records, () to (c), in (B) nd (d) to (f), in (C) re smple trces of s-i.p.s.ps in control nd fter wshout of somn, respectively, t corresponding stimulus strengths shown in () to (f) of (A). The vlue next to ech record indictes the stimulus strength used to elicit the s-i.p.s.ps. Downwrd deflections re nelectronic potentils resulting from constnt current hyperpolrizing pulses (Oms durtion) s mesure of R1.. The pregnglionic tetnus ws pplied t () shown under ech trce. A > 2.2 o 15 Co 4 1 Somn r --- C o o o. o o - ) I oes( W. i 2 3 Time (min) B Pregnglionic tetnus Control Som n 7 m in "M Wsh C f f 4b 5 ACh puff ppliction d erryrrr2 m 1T T [LOMV * 27 min fl)ttifihjfl hhthh1httf Figure 3 ffect of somn (11pM) on muscrinic hyperpolriztions elicited by pregnglionic tetnus (4 Hz for 1 s; 5 V stimulus strength) nd by puff ppliction (5ms durtion; 1 psi pressure) of cetylcholine (ACh) in the presence of mecmylmine (2gM), phentolmine (1pM) nd cffeine (1 mm). (A) A muscrinic s-i.p.s.p. () induced by pregnglionic tetnus ws depressed by somn with time course prllel to tht for the muscrinic hyperpolriztion () elicited by puff ppliction of ACh. (B) Records () to (f) re exmples of muscrinic hyperpolriztions t times shown in () to (f) of (A), respectively. RMP: -56 mv in control; -65 mv during somn superfusion for 7min; -61 mv, 27 min fter wsh of somn. Rin ws monitored by nelectronic potentils (downwrd deflection) resulting from injection of constnt current hyperpolrizing pulses (12 ms durtion). The pregnglionic tetnus nd ACh puff were pplied t () nd (), respectively, shown under ech trce. Somn (1 pm) hyperpolrized the membrne ( mv, n = 13; P <.1; RMP = mv) nd decresed input resistnce ( %, n = 13, of control; P <.2) in the presence of mecmylmine, phentolmine nd cffeine. Injection of current to return the membrne potentil to the control vlue did not ffect the somn-induced inhibition of the s-i.p.s.p. Mesurements of membrne potentil nd input resistnce obtined.45-6h fter wshout of somn were not significntly different from those of the control, lthough the somn-induced inhibition nd prolongtion of the s-i.p.s.p. persisted. Action ofsomn on the muscrinic hyperpolriztion elicited by puff ppliction of cetylcholine Somn lso depressed nd prolonged muscrinic hyperpolriztion elicited by puff ppliction of cetylcholine (ACh; Figures 3 nd 4). The mximl mplitude ( mv, n = 6; RMP = mV) nd durtion ( s) of the muscrinic hyperpolriztion elicited by ACh puff ppliction were, respectively, % nd %, of control 5min fter the initition of somn (1 M) superfusion. The effect of somn on the mplitude of the ACh hyperpolriztion ws not significntly different from its effect (52 + 3%; Figure l) on the synpticlly-evoked mximl muscrinic s- i.p.s.p. (P >.1). Furthermore, muscrinic hyperpolriztions elicited by ACh relesed from either the nerve terminl or micropipette locted ner the neurone were depressed by somn with similr time course (Figure 3; n = 4). The effect of somn on the reltionship between the durtion of the ACh pressure ppliction nd the mplitude of the muscrinic hyperpolriztion is plotted in Figure 4. The mplitude of the muscrinic hyperpolriztion incresed with

4 16. KUMAMOTO & P. SHINNICK-GALLAGHR A,, O cv~~~~~~ /.N c X A Mermbrne potentil (mv) -12-1() -8 ' -6 I i -4 > - c -5 X.L_. -5o ACh puff durtion (ms) Control d Somn 5 ms mm b e 1 Ms T f B -6 mv C o d -6 mv e -7 mv b -71 mv L Z_- c -91 V f -92 mv CL.-15 ụ _., -2 w 5e 11 mv 5s 5 ms c f Lmv i TTITM Figure 4 ffect of somn (1 M) on the muscrinic hyperpolriztions elicited by cetylcholine (ACh) puff ppliction (15 psi pressure) of vrious durtions in the presence of mecmylnmine (2pM), phentolmine (1 M) nd cffeine (1 mm). (A) The mplitude of the muscrinic hyperpolriztion is plotted ginst the durtion of the ACh pressure ppliction in control (; RMP =- 56 mv) nd 15 min fter inititing somn superfusion (RMP =-6mV; : responses mesured when the RMP ws returned to -56 mv by injecting cthodl current). Curves re drwn by eye. (B) Records () to (f) re smple trces of muscrinic hyperpolriztions elicited by ACh puff pplied with durtions corresponding to those shown in the grph. ACh puff ws pplied t () shown under ech trce. Downwrd deflections re nelectronic potentils elicited by constnt current hyperpolrizing pulses (1 ms durtion) s mesure of Rin Figure 5 xtrpolted reversl potentil of the muscrinic hyperpolriztion (s-i.p.s.p.) elicited by pregnglionic tetnus (4Hz for I s; 5 V stimulus strength) in the presence of mecmylmine (2M), phentolmine (1 JM) nd cffeine (1 mm) in control (RMP = - 5mV), nd 34min (RMP = -6 mv) fter n 8 min tretment with somn (1 pm) during which the s-i.p.s.p. ws lmost completely depressed. (A) The s-i.p.s.ps re plotted ginst membrne potentil in control () nd fter wsh of somn (). This cell hd more hyperpolrized reversl potentil of s-i.p.s.p. compred to the men vlue (-99.9 mv) obtined from mny cells, or the equilibrium potentil for K+ (-95.3 mv; Aksu et l., 1986) estimted from the reversl potentil of the fter-hyperpolriztion of the ction potentil. This my be due to the fct tht sites of synpse involved in the production of the s-i.p.s.p. exist in regions remote from the som into which the microelectrode ws impled. (B nd C), Records, () to (c), in (B) nd (d) to (f), in (C) re smple trces of s-i.p.s.ps in control nd fter wshout of somn, respectively, recorded t membrne potentils corresponding to those shown in () to (f) of (A). The vlue next to ech record indictes the membrne potentil t which s-i.p.s.p. ws mesured. The pregnglionic tetnus ws pplied t () shown under ech trce. longer ACh puff durtions nd sturted to stedy vlue (n = 19); somn (1 IM) depressed this mximum vlue (Figure 4; n = 3). This suggests tht somn tretment results in noncompetitive decrese of the muscrinic response. Somn does not ffect the extrpolted reversl potentil of the s-i.p.s.p. Muscrinic s-i.p.s.ps (mplitude: mv, n = 11; durtion: s; RMP = mv) decresed with membrne hyperpolriztion nd the extrpolted reversl potentil ws mv (n = 11; cf. Gllgher et l., 1982); this vlue ws not significntly different from tht, mv (n = 13), in neurones in which muscrinic s-i.p.s.ps ( mV; s; RMP = mV) hd been depressed nd prolonged by tretment with somn (1 JM; P >.1; Figure 5). The reltionship between the mplitude of the s-i.p.s.p. nd membrne potentil ws liner in control nd fter somn tretment, suggesting tht the effect of somn on the muscrinic hyperpolriztion ws not voltge-dependent. ffect of second ppliction ofsomn After wshout of somn, the mplitude of the depressed muscrinic s-i.p.s.p. prtilly reversed over long period of time (Tble 1). However, the prtilly-recovered s-i.p.s.p. (mplitude: mv, n = 5; durtion: i1.1 s; RMP = mV) ws not further inhibited by second ppliction of somn (mplitude: mv, n = 5; P >.1; not shown). An enhnced nicotinic depolriztion produced by pplying somn in norml Krebs solution (Kummoto & Shinnick-Gllgher, 1988) ws lso unffected by further ppliction of somn (not shown). Action ofpyridostigmine on the muscrinic hyperpolriztion The reversible ACh inhibitor, pyridostigmine, (Tylor, 1984) lso depressed nd prolonged the muscrinic s-i.p.s.p. elicited by pregnglionic tetnus, in dose-dependent mnner (Figure 6). This ction ws not ccompnied by significnt chnge in membrne potentil (from to mv, n = 12) or input resistnce (97 + 4% of control, n = 11) even when pyridostigmine ws pplied t high concentrtion (1pM). The ffinity of pyridostigmine t inhibiting the s-i.p.s.p. ws less thn tht of somn by fctor of over 1. Pyridostigmine (1 JM) lso depressed the mplitude ( mV, n = 4; RMP = mV) of the mximl muscrinic hyperpolriztion elicited by pressure

5 SOMAN INHIBITION OF MUSCARINIC INHIBITORY RSPONS c )._ Pyridostigmine concentrtion (>M) b Control Pyridostigmine (1 FM) Wsh 1 min 2 min Thr7fl 1Th1TPH~m1.5 2 c; 4- C) 4- co T~rlyrvjl [SmV Figure 6 Dose-response curves for the effect of pyridostigmine on the muscrinic hyperpolriztions observed in the presence of mecmylmine (2M), phentolmine (1pM) nd cffeine (1 mm). () Muscrinic hyperpolriztions (s-i.p.s.ps) were elicited with pregnglionic tetnus (4 Hz for 1 s). Vlues in prentheses re numbers of cells tested. ch point represents the men of the % chnges in the mplitude () nd durtion (-) of the s-i.p.s.p. 1min during tretment with pyridostigmine. The verticl lines show s.e.men unless it ws within the size of symbol. The dotted lines indicte 1%. The stimulus strength used ws one t which mximl s-i.p.s.p. ws elicited. (b) ffect of pyridostigmine (1pM) on muscrinic hyperpolriztions elicited by cetylcholine (ACh) pplied in pressure pulse (4 ms durtion; 18 psi pressure). Muscrinic hyperpolriztions were depressed nd prolonged 1min fter dding pyridostigmine to the superfusion solution (middle) compred to control (left). The muscrinic hyperpolriztion returned to control vlue 2min fter wshout of pyridostigmine (right). RMP (= -49mV) remined constnt throughout pyridostigmine superfusion. Downwrd deflections re nelectronic potentils resulting from constnt current hyperpolrizing pulses (1 ms durtion) s mesure of Ri. The puff of ACh ws pplied t () shown under ech trce. A 15 Pyridostigmine (1 FM) li~~~~~~~~.l B Control b Pyridostigmine (1 FtM) 1 min c Wsh 1 min Time (min) t TTmTfTTr Tr uqtm TT Wsh 2 min 3min 4min M mv Figure 7 Reversible effects of pyridostigmine (1pM) tretment on the muscrinic hyperpolriztion (s-i.p.s.p.) elicited by pregnglionic tetnus (4 Hz for 1 s) in the presence of mecmylmine (2 pm), phentolmine (1pM) nd cffeine (1mM). (A) Men mplitude (htched columns) nd durtion (open columns) of the s-i.p.s.ps obtined from five cells in control (RMP = mv), during nd fter pyridostigmine tretment. Verticl brs denote s.e.men. Dotted lines show the verged mplitude nd durtion of s-i.p.s.p. in control. (B) Smple trces of s-i.p.s.ps elicited by pregnglionic tetnus (5V stimulus strength) in control, 1min during, nd 1, 2, 3 nd 4min fter pyridostigmine tretment. RMP (= -5mV) remined constnt throughout pyridostigmine superfusion. Downwrd deflections re nelectronic potentils resulting from constnt current hyperpolrizing pulses (1ms durtion) s mesure of Ri.. The pregnglionic tetnus ws pplied t () shown under ech trce. ppliction of ACh by 52 ± 9% (P <.2) nd prolonged the durtion (5. +.) by % (P <.1; Figure 6b). The inhibitory effect of pyridostigmine on the muscrinic hyperpolriztion induced by pressure pulse ws not significntly different from tht, % (Figure 6), for synpticlly-evoked mximl muscrinic s-i.p.s.p. (P >.1). These dt indicte tht pyridostigmine cts on the postsynptic membrne. Figure 7 shows the time course of the ction of pyridostigmine (1pM) on the mplitude nd durtion of the muscrinic s-i.p.s.p. elicited by pregnglionic tetnus. The inhibitory effect on the mplitude nd the prolonged durtion of the s-i.p.s.p. observed with pyridostigmine tretment were lmost completely reversed 4 min fter wshout of pyridostigmine; this is consistent with the well-known reversible inhibitory ction of pyridostigmine on ACh (Tylor, 1984). The mplitude of the s-i.p.s.p. recovered fster from the effects of pyridostigmine thn the durtion. This my be due to the fct tht the durtion of s-i.p.s.p. is more sensitive to pyridostigmine thn the mplitude (see Figure 6). Protectionfrom somn effects by pyridostigmine Since both somn nd pyridostigmine depressed nd prolonged muscrinic hyperpolriztions, it ws possible tht these ctions of somn could be ltered by pretretment with pyridostigmine. When somn (1 pm) ws pplied in the presence of pyridostigmine (1 M), the mplitude of the s-i.p.s.p. ws depressed to % (n = 5) of control (in postpyridostigmine) 1min fter the initition of somn super- fusion (not shown); this depression ws significntly greter thn tht (6 + 3%, n = 9; 1min during somn superfusion) observed when somn (1 pm) ws pplied lone (P <.1). Furthermore, the mplitude of the s-i.p.s.p. recovered to 7 + 8% (n = 5) of control (in post-pyridostigmine) 2min fter wshout of somn when pyridostigmine ws still present (not shown). This mount of reversl ws lso greter thn the corresponding recovery (35 + 9%, n = 8) observed following somn ppliction which ws not preceded by pyridostigmine superfusion (P <.5). After wsh of.5-4h fter ll the tretments, the remining s-i.p.s.p. hd n mplitude nd durtion of mV (n = 8; RMP = mV) nd s, respectively. This s-i.p.s.p. ws prolonged compred to control (in pre-pyridostigmine; s, n = 68; Tble 1; P <.1), but hd n mplitude comprble to tht of control ( mv; Tble 1; P >.1). These results indicte tht pyridostigmine protects ginst the irreversible ctions of somn on the mplitude of the muscrinic hyperpolriztion. Does tropine inhibit the ction of somn? The muscrinic receptor ntgonist, tropine, blocks the muscrinic s-i.p.s.p. in ct bldder neurones (Gllgher et l., 1982; Kummoto, 1989). It ws possible tht the ction of somn ws direct, on the sme site s tropine nd thus, somn might depress the hyperpolriztion by blocking the muscrinic receptor. Superfusion of tropine (1 pm) completely blocked the s-i.p.s.p. within few minutes. When somn (1 gm) ws pplied for 1min to the tropine-treted preprtion nd then somn nd tropine were wshed out over 1 h, the

6 162. KUMAMOTO & P. SHINNICK-GALLAGHR muscrinic 'hyperpolriztion ws depressed ( mv, n = 12; RMP = mV) nd prolonged ( s). These vlues were not significntly different from the corresponding vlues (Tble 1; P >.1) observed fter tretment with only somn (1 ym). However, even in the presence of tropine, somn (1 Mm) hyperpolrized the membrne by mV (n = 3; RMP = mv). Discussion The present results demonstrte tht in ct bldder prsympthetic neurones, the irreversible cetylcholine esterse (ACh) inhibitor, somn, depresses the mplitude nd prolongs the durtion of the slow muscrinic inhibitory postsynptic potentil (s-i.p.s.p.), elicited by pregnglionic tetnus in dose-dependent mnner. The threshold concentrtion for prolonging the durtion of the s-i.p.s.p. ws lower thn the concentrtion required to depress its mplitude. The prolongtion of s-i.p.s.p. by somn is expected from its bility to inhibit ACh nd thus increse the lifetime of cetylcholine (ACh) in the synptic cleft. Somn lso induced membrne hyperpolriztion ssocited with n incresed input conductnce. The observed chnges in these membrne properties could not explin the somn-induced inhibition of s-i.p.s.p. Somn-induced membrne hyperpolriztion Membrne potentil chnges induced by somn seem to depend on the preprtions used. In rt superior cervicl (Yrowsky et l., 1984) or bullfrog sympthetic (Heppner & Fiekers, 1988) gngli, somn produced membrne depolriztion, ccompnied by decrese in input resistnce; the former ction ws different from tht in ct bldder prsympthetic neurone. The somn-induced hyperpolriztion in bldder neurones ws not due to n excessive concentrtion of ACh in the synptic cleft resulting from n inhibition of ACh ctivity, becuse the hyperpolriztion ws not blocked by tropine. Furthermore, the reversible ACh inhibitor, pyridostigmine, did not hyperpolrize the membrne. It is lso unlikely tht this ction of somn ws due to relese of endogenous (nordrenergic or purinergic) neurotrnsmitters known to hyperpolrize these neurones (cf. Aksu et l., 1984; 1986b; Kummoto, 1989) becuse this ction of somn ws observed in the presence of phentolmine nd cffeine. Hyperpolriztion following somn superfusion ws lso observed in drug-free Krebs solutions (Kummoto & Shinnick- Gllgher, 1988). Although these dt do not rule out the possibility tht some unknown substnce my be relesed by somn, they do suggest tht the membrne hyperpolriztion my be due to direct ction of somn. Somn-induced depression ofmuscrinic response In the superior cervicl gnglion (Yrowsky et l., 1984) or phrenic nerve-hemidiphrgm (Andersen et l., 1987) of rt, it hs been suggested tht somn hs presynptic ction. It is unlikely tht the inhibition of the muscrinic s-i.p.s.p. observed with somn is due to decrese in ACh relese from nerve terminls, since somn lso depressed the muscrinic hyperpolriztion elicited by exogenous ppliction of ACh. Furthermore, the depolriztion medited by nicotinic receptors in ct bldder neurones ws not depressed, but enhnced by somn (Kummoto & Shinnick-Gllgher, 1988). These dt indicte tht somn cts on the postsynptic membrne to inhibit the muscrinic hyperpolriztion. In ct bldder prsympthetic neurones, the muscrinic s-i.p.s.p. is due to ctivtion of K chnnel (Gllgher et l., 1982; Shinnick-Gllgher et l., 1987). Somn my ffect the distribution of ions cross the membrne nd thus depress the muscrinic response. However, the extrpolted reversl potentil for the muscrinic s-i.p.s.p. ws unchnged lthough the s-i.p.s.p. ws depressed. Somn my reduce the number of muscrinic receptors cutely, s hs been suggested with chronic somn tretment (Churchill et l., 1984; As et l., 1987). Alterntively, if somn incresed the dissocition constnt for ACh binding to the muscrinic receptor, the muscrinic response would be depressed. The somn-induced inhibition of the mximl mplitude of the muscrinic s-i.p.s.p. elicited by pregnglionic stimultion or ACh ppliction, i.e., non-competitive inhibition of muscrinic response, suggested tht the number of muscrinic receptors my be ffected by somn. Alterntively, this observtion my be due to decrese in the number of functionl muscrinic receptors even if the number of receptors themselves is not chnged. As et l. (1987) demonstrted tht cute exposure to somn reduced the mximl vlue of the contrction induced by muscrinic ction of ACh without chnging the number of muscrinic receptors in bronchil smooth muscle. It hs been suggested tht somn my interct directly with sites on the nicotinic ACh receptor mcromolecule (Albuquerque et l., 1987). Therefore, somn my reduce the muscrinic response by cting t site other thn t the ACh ctive centre. This trget site is probbly not the site where tropine cts on the muscrinic receptor, becuse tropine filed to inhibit the effect of somn in bldder neurones. If somn blocks the open stte of K chnnel involved in the muscrinic response, tretment with somn would result in decrese in the number of functionl muscrinic receptors. Clsiclly, gents ssocited with non-competitive ntgonism of responses induce n open chnnel block of the receptorchnnel complex which is voltge-dependent (cf. Ascher et l., 1979). This is probbly not the mechnism of ction of somn, becuse the effect of somn on the muscrinic s-i.p.s.p. ws not voltge-dependent. Is the effect of somn due to ACh inhibition? The effect of somn my be due to persistent cholinomimetic ctivity resulting from ACh inhibition. Consistent with this hypothesis is our finding tht the reversible ACh inhibitor, pyridostigmine, lso depressed the mplitude of the muscrinic hyperpolriztion; this effect reversed on wshing, result clerly different from the prtilly reversible ction of somn on the mplitude of the muscrinic hyperpolriztion. Thus, inhibition of muscrinic response cn be observed not only with somn, but lso with other ACh inhibitors. It is known tht the ction of n irreversible inhibitor on ACh cn be prevented by reversible inhibitor (Hrris et l., 1978; Vn Meter et l., 1978; Ymd et l., 1983; McGee & Brezenoff, 1987). The irreversible ction of somn on bldder neurones ws lso prevented by pretretment with pyridostigmine. These results suggest tht the effects of somn my be due to inhibition of ACh ctivity, resulting in n ccumultion of ACh in the synptic cleft nd n excessive excittion of muscrinic receptors. It hs been suggested tht this extreme stimultion of muscrinic receptors results in downregultion or loss of muscrinic receptors (Klein et l., 1979). Feigenbum & l-fkhny (1984) hve observed decrese in the number of binding sites for muscrinic receptors following 3min preincubtion of neuroblstom cells with muscrinic gonist. Furthermore, down-regultion of muscrinic receptors hs been shown for chronic tretment of orgnophosphte ACh inhibitors such s DFP (Ymd et l., 1983) nd somn (Churchill et l., 1984). The loss of muscrinic receptors produced by chronic DFP tretment ws ntgonized by the muscrinic receptor ntgonist, tropine (Ymd et l., 1983), suggesting tht excessive stimultion of receptors ws the custive fctor. In ddition, somninduced depression of synptic trnsmission in superior cervicl gngli ws shown to be inhibited by tropine pretretment (Yrowsky et l., 1984). In contrst, the somninduced inhibition of the muscrinic hyperpolriztion in

7 SOMAN INHIBITION OF MUSCARINIC INHIBITORY RSPONS 163 bldder neurones ws not suppressed by pretretment with tropine. Thus, the inhibition of ACh my not necessrily ccount for this ction of somn. However, becuse ACh ctivity ws still inhibited during wshout of tropine, it is possible tht the persistent cholinergic ctivity could still led to n cute down-regultion of muscrinic receptors. Recovery from somn ction After wshout of somn, the mplitude of the muscrinic hyperpolriztion prtilly recovered, lthough the durtion remined prolonged. This finding is consistent with observtions of spontneous recovery of gnglionic (Lukomsky et l., 198) or neuromusculr (Vn Dongen et l., 1988) trnsmission following tretment with irreversible ACh inhibitors. Recovery from somn tretment my occur through n unknown mechnism independent of de novo synthesis of ACh. When somn ws pplied to bldder neurones second time, the prtilly-recovered muscrinic response ws not depressed further. The lck of n effect of second ppliction of orgnophosphorus ACh inhibitors hs lso been demonstrted for G seizure elicited by DFP in rbbits (Vn Meter et l., 1978) nd for inhibition of neuromusculr trnsmission by somn in rts (Vn Dongen et l., 1988). These dt lso suggest tht the somn-induced depression of the muscrinic hyperpolriztion in bldder neurones is due to inhibition of ACh. In conclusion, somn not only prolongs the muscrinic hyperpolriztion, but lso depresses its mplitude in noncompetitive mnner; both effects re probbly due to inhibition of ACh. A direct ction of somn to hyperpolrize the neuronl membrne my not result from ACh inhibition. This work ws supported by DAMD C-633 to P.S.G. References AAS, P., VITBRG, T. & FONNUM, F. (1987). Acute nd sub-cute inhltion of n orgnophosphte induce ltertion of cholinergic muscrinic receptors. Biochem. Phrmcol., 36, AKASU, T., SHINNICK-GALLAGHR, P. & GALLAGHR, J.P. (1984). 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