neuromuscular junctions

Transcription

1 Br. J. Phrmcol. (1992), 15, (D Mcmilln Press Ltd, 1992 The effects of vesmicol on trins of endplte currents nd on foclly recorded nerve terminl currents t mmmlin neuromusculr junctions Kren E. Pemerton, 'Chris Prior & In G. Mrshll Deprtment of Physiology nd Phrmcology, University of Strthclyde, Glsgow, GI 1XW 1 The effects of vesmicol, n inhiitor of vesiculr cetylcholine (ACh) storge, were studied on trins of endplte currents (e.p.cs) in the cut rt hemidiphrgm nerve-muscle preprtion nd on trins of foclly recorded nerve terminl current wveforms in the mouse tringulris sterni nerve-muscle preprtion. 2 In the rt,.1 nd 1,M (-)-vesmicol produced n enhncement of the rundown of e.p.c. mplitudes during trins of high frequency (5 Hz) nerve stimultion. However, 1 M (+)-vesmicol hd no effect on the rundown of e.p.c. mplitudes. 3 In the mouse, high concentrtions of (-)-vesmicol (1-1OOM) produced concentrtion- nd stimultion-dependent decrese in the mplitude of the second negtive-going deflection of foclly recorded nerve terminl current wveforms. 4 At mm, (-)-vesmicol produced stimultion-independent decrese in the mplitude of the first negtive-going deflection of the nerve terminl current wveforms, n increse in signl dely nd evidence of nerve conduction filure. These ll indicte locl nesthetic-like lock of nodl N+-chnnels. 5 In contrst to its effects on trins of e.p.cs, the effects of vesmicol on the nerve terminl current wveforms were not stereoselective, the (+ )-isomer eing equipotent with the (-)-isomer. 6 Low concentrtions of the N+-chnnel locking toxin, tetrodotoxin (15-6nM), produced similr chnges in the foclly recorded nerve terminl current wveforms to those seen with vesmicol. 7 It is concluded tht the stereoselective rundown of e.p.c. mplitudes produced y (-)-vesmicol is due to n effect, either direct or indirect, on ACh moiliztion within motor nerve terminls. Furthermore, in mmmlin species, the inhiitory effects of vesmicol on nodl N+-chnnels which re seen t high concentrtions do not contriute to the principl neuromusculr effects of the compound. Keywords: Rt hemidiphrgm; mouse tringulris sterni; neuromusculr junction; motor nerve ending; endplte currents; perineurl wveform; cetylcholine moiliztion; sodium currents Introduction Following the initil proposl (Mrshll, 197) nd susequent confirmtion (Anderson et l., 1983) tht vesmicol (trns-2-[4-phenylpiperidino] cyclohexnol; AH5183) inhiits the trnsport of cetylcholine (ACh) into synptic vesicles the compound hs ecome widely used in mmmlin preprtions s prejunctionlly-ctive tool for studying the role of vesiculr ACh in trnsmitter relese (see Mrshll & Prsons, 1987, for review). However, t high concentrtions, vesmicol hs een reported to exhiit rnge of phrmcologicl ctions in ddition to the effects ttriutle to lock of ACh storge. These include postjunctionl receptor nd ion chnnel lock (Enomoto, 1988); nticholinesterse ctivity (Vn der Kloot, 1986); locl nesthetic ctivity (Mrshll, 197; Estrell et l., 1987); twitch ugmenttion (Mrshll, 197; Estrell et l., 1987); nd effects t the sympthetic neuroeffector junction (Mrshll, 197; Wnnn et l., 1991). Mny of these 'side-effects' do not exhiit the stereospecificity seen for the iochemicl nd phrmcologicl ctions of the compound on synptic vesicles nd neuromusculr trnsmission. Hence, lthough the (-)-isomer of vesmicol is 2 times more potent t inhiiting the synptic vesicle trnsport of ACh thn the (+)-isomer (Bhr & Prsons, 1986), the two isomers re equipotent for the locl nesthetic nd twitch ugmenttion effects (Estrell et l., 1987) nd for lock of l1-drenoceptors (Wnnn et l., 1991). A consequence of the non-cholinergic side-effects of vesmicol is the prolem of unequivoclly linking vesmicol-induced chnges in neuromusculr trnsmission to n effect on the ' Author for correspondence. vesiculr pckging of ACh. Thus, Girod et l. (1991) hve shown tht in the Torpedo electroplx the successive diminution of synptic potentil mplitudes during high frequency trins of motor nerve stimultion is consequence of the ility of vesmicol to lock N+-chnnels, nd hence to prevent invsion of the nerve terminls y ction potentils, rther thn result of its ility to inhiit vesiculr ACh storge. In contrst, results from experiments in our own lortory hve een interpreted in terms of the enhnced rundown of endplte current (e.p.c.) mplitudes during high frequency trins of nerve stimultion in mmmlin preprtions eing reflection of n effect, direct or indirect, of vesmicol on trnsmitter moiliztion (Prior et l., 1989; Bowmn et l., 199). However, it is lso possile tht the vesmicol-induced rundown in e.p.c. mplitudes could e due to postjunctionl ion chnnel lock s demonstrted for trimetphn (Gi & Mrshll, 1984) or to n incresed relese during the first few e.p.cs of the trin s result of lock of nerve terminl K + -chnnels similr to tht seen with 4- minopyridine (Lundh, 1978; Molgo et l., 1979). Indeed, it hs een suggested tht the twitch ugmenttion effect of ( +)- nd (-)-vesmicol seen t low rtes of nerve stimultion (1 Hz) could e due, in prt, to lock of nerve terminl K+chnnels (Estrell et l., 1987). To determine the role of the ion chnnel locking ctivities of vesmicol in its neuromusculr effects t high frequencies of nerve stimultion t mmmlin neuromusculr junctions, we hve studied the concentrtion-dependence nd stereoselectivity of vesmicol on trins of e.p.cs recorded from cut rt hemidiphrgm motor endpltes nd on perineurlly recorded nerve terminl current wveforms recorded from motor nerve endings in the mouse tringulris nerve-muscle preprtion.

2 114 K.E. PEMBERTON et l. Methods Endplte current trins The cut rt hemidiphrgm preprtion used hs een descried extensively elsewhere (Brstd & Lilliheil, 1968; Glvinovic, 1979; Gi & Mrshll, 1984). Briefly, hemidiphrgms were removed, long with 1-2mm of their phrenic nerve supply, from mle Sprgue-Dwley rts (15-2 g) nd pinned to the Sylgrd se of 5 ml tissue th. Preprtions were continully superfused (5-1mlmin-1) t 32C with Kres-Hensleit solution of the following composition (mm): NCl 118, KCl 5, CCl2 2.5, NHCO3 25, MgSO4 1 nd glucose 11; gssed with 95% 2:5% CO2 to ph 7.4. The phrenic nerve ws stimulted t 5 Hz vi silver wire electrodes with fifty pulses of 5-lps durtion nd suprmximl voltge. Trins of e.p.cs were recorded from endpltes voltge-clmped t -5 mv y conventionl two microelectrode voltge clmp recording technique (Axon Instruments, Axoclmp 2A). Intrcellulr microelectrodes were filled with either 3 M KCI (voltge recording, resistnces 5-1 MQ) or.6 M K2SO4 (current pssing, resistnces 2-5MQ). In ech fire, following minimum of 3min rest with no nerve stimultion, four trins of e.p.cs, ech seprted y 2s rest, were recorded in either the sence of vesmicol or in the presence of either.1 or 1 UM (-)-vesmicol or 1 UM (+ )-vesmicol. For ech trin, e.p.c. rundown ws clculted s the verge loss of mplitude of the lst ten e.p.c. in the trin s percentge of the mplitude of the first e.p.c. Vlues for ech of the four seprtely recorded trins were verged to give men vlue for ech fire studied. Nerve terminl current wveforms Experiments were performed on the left tringulris sterni nerve-muscle preprtion (McArdle et l., 1981) isolted from 2-25 g mle Bl C mice. The entire dissection ws performed under continuous superfusion with physiologicl slt solution of the following composition (mm): NCl 154, KCI 5, MgCI2 1.2, HEPES 5, CCl2 2.5 nd glucose 11; uffered to ph 7.4 nd continully gssed with 1% 2. Following isoltion, preprtions were mounted in 5ml tissue th nd superfused with physiologicl solution t rte of 5-1ml min t 32C. The intercostl nerves were stimulted vi suction electrode with pulses of 5-1ps durtion nd suprmximl voltge. Muscle movement in response to nerve stimultion ws olished y dding 1 UM vecuronium romide to the thing medium. Presynptic wveforms were recorded from the terminl region of intercostl nerves with glss microelectrodes plced inside the perineurl sheth (Mllrt, 1985). Extrcellulr microelectrodes were filled with 2 M NCl nd hd resistnces of 5-15 MC. Signls were monitored using stndrd high impednce unity gin electrometer (World Precision Instruments, model 75). For ech perineurl implement, single recording of 3 wveforms evoked t 5 Hz ws mde in the sence nd in the presence of one of (-)-vesmicol (1, 1, 1, 1pM), (+)- vesmicol (1pM) or tetrodotoxin (15, 3, 6nM). Ech concentrtion of vesmicol ws perfused through the tissue th for 5min (5-1mlmin-1) efore mking ny recording. For experiments with tetrodotoxin, the preprtions were exposed to ech concentrtion of the toxin for 5, 1 nd 15min efore recording single trin of 3 wveforms evoked t 5 Hz. Anlysis nd sttistics Trins of e.p.cs nd nerve terminl current wveforms were recorded (d.c. - S khz) on FM tpe for susequent nlysis. Signls were digitized t 25 khz (Dt Trnsltion, DT281A lortory interfce) nd nlysed with suite of purposedesigned progrmmes (Dempster, 1988) running on n IBMcomptile microcomputer (Vnill, SX-386). All numericl dt re expressed s men + s.e.men of 3-9 implements (mouse tringulris sterni), or 8-14 implements (rt hemidiphrgm). For the nerve terminl current wveforms, ech implement ws mde in different muscle preprtion nd for the endplte current trins no more thn three fires were smpled in ny single muscle preprtion. Sttisticl testing ws performed with either pired Student's t test (nerve terminl current wveforms), n unpired Student's t test (endplte current trins) or, where pproprite, one smple Student's t test on dt normlized to control vlue of 1%. In ll sttisticl tests significnce ws set s P <.5. Results Effects of vesmicol on trins of endplte currents In the sence of vesmicol, the mplitudes of successive e.p.cs in trins of currents diminished y round 3% until plteu level ws reched. In the presence of.1 or 1 gum (-)- vesmicol, the iochemiclly ctive isomer, there ws sttisticlly significnt increse of pproximtely 5% in the rundown in e.p.c. mplitudes during the trin compred to tht seen in the sence of the drug (Tle 1). Neither concentrtion of (-)-vesmicol hd ny sttisticlly significnt effect on the mplitude or the decy time constnt of the first e.p.c. in the trin (Tle 1). In contrst to the effects of (-)-vesmicol, 1,M of (+)-vesmicol, the iochemiclly inctive isomer, hd no significnt effect on the rundown of e.p.c. mplitudes nd, like the ctive isomer, hd no effect on the mplitude nd time course of the first e.p.c. of the trin (Tle 1). Effects of vesmicol on nerve terminl current wveforms In the sence of vesmicol, high frequency nerve stimultion (3 pulses t 5 Hz) produced consistent nd chrcteristic chnge in the shpe of the nerve terminl current wveform (Figures 1 nd 2). As stimultion progressed, the mplitude of the first negtive-going deflection (the component of the wveform normlly ttriuted to the inwrd nodl N+-current) grdully incresed, so tht y the 3th wveform, it ws significntly incresed to % (n = 9) of its initil mplitude (P <.5, pired Student's t test). At the sme time the mplitude of the second negtive-going deflection (the component of the wveform normlly ttriuted to the outwrd nerve terminl K+-current) grdully decresed to % (n = 9) of its initil vlue (P <.5, pired Student's t test). At concentrtion of 1 UM, (-)-vesmicol hd no clerly discernile effect on the nerve terminl current wveforms. However, higher concentrtions of the compound produced mrked chnges in the mplitude of oth the first nd second negtive-going components of the wveforms (Figure 1). At concentrtions of 1pM nd ove, (-)-vesmicol produced concentrtion-dependent decrese in the mplitude of the first negtive-going component of the initil nerve terminl Tle 1 Effect of vesmicol on endplte current (e.p.c.) trin prmeters t 5 Hz Control (-)-Vesmicol.1 gm (-)-Vesmicol 1 AM (+)-Vesmicol 1 JiM n e.p.c.1 (na) T, e.p.c. rundown (Ms) (%) * * All dt re men + s.e.men verged over the numer of cells shown in n. E.p.c., nd r1 re, respectively, the pek mplitude nd time constnt of decy of the first e.p.c. in the stimultion period. *Significntly different from control (P <.5, unpired Student's t test).

3 NEUROMUSCULAR EFFECTS OF VESAMICOL c 2 mv 2 ms d /85 "' /79 Figure 1 Representtive exmples of nerve terminl current wveforms recorded from single implement in the sence (, ) nd presence (c, d) of 1pUM (-)-vesmicol. Figure shows the first (, c) nd lst (, d) wveforms from 6 s period of 5 Hz nerve stimultion. The numers in (), (c) nd (d) give the mplitude of the two negtivegoing components of the wveform s percentge of their mplitude in (). current wveform of the trin of stimuli. Thus 1/AM (-)- vesmicol produced slight, ut not sttisticlly significnt, decrese in the mplitude of the first negtive-going component of the initil wveform in the trin of stimuli, which remined constnt throughout the 6s stimultion period (Figure 2), while 1 mm (-)-vesmicol produced sttisticlly significnt decrese in the mplitude of the first negtive-going component of the initil wveform in the trin of stimuli to % of control (n = 4, P <.5, one smple Student's t test). The effects of (-)-vesmicol on the second negtivegoing component of the nerve terminl current wveforms were more pronounced thn the effects on the first negtivegoing component nd were seen t lower concentrtions. Thus 1-1/AM (-)-vesmicol produced concentrtiondependent decrese in the mplitude of the second negtivegoing component of the initil wveform of the trin of stimuli (Figure 2). In ddition, 1/uM (-)-vesmicol enhnced the stimultion-induced depression of the second negtive-going component seen s stimultion progressed (Figure 2). None of the effects of vesmicol on nerve terminl current wveforms were stereoselective (Figure 3). Thus, s for 1/UM (-)-vesmicol, 1/AM (+)-vesmicol produced smll, sttisticlly non-significnt, decrese in the mplitude of the first negtive-going component of ll wveforms in the trin of stimuli (Figure 4) nd sttisticlly significnt decrese in the mplitude of the second negtive-going component of the initil wveform (Figure 4). Additionlly, s for 1/UM (-)- vesmicol, the stimultion-induced decline in the mplitude of the second negtive-going component of the wveform ws enhnced y 1/uM (+)-vesmicol (Figure 4). These effects of 1juM (+)-vesmicol were of similr mgnitude to those seen for 1/AM (-)-vesmicol. In the presence of high concentrtions of (-)-vesmicol (1/AM nd 1 mm) there ws concentrtion-dependent, stimultion-induced, increse in signl dely, i.e. the time etween the stimulus rtefct nd the strt of the nerve terminl current wveform. The stimultion-induced increse in signl dely ws rpid, eing seen within the first 5-1 wveforms (Figure 5). Susequent to the increse in signl dely, s stimultion progressed in the presence of the highest concentrtion of (-)-vesmicol (1 mm), the size of the nerve terminl current wveform fluctuted mrkedly nd, occsionlly, no '49-8- c Time (s) Figure 2 Effect of stimultion t 5Hz on the mplitude of the first () nd second () negtive-going components of nerve terminl current wveforms recorded in the sence of (-)-vesmicol (@) nd in the presence of 1 (e) nd 1pM (*) (-)vesmicol. For ech implement, individul mplitude vlues were expressed s percentge of the mplitude of the pproprite negtive-going component in the first control wveform. Ech plotted point is the men of dt from the sme six implements; s.e.men shown y verticl rs. (-)- Vesmicol, 1pM, hd no effect on the first negtive-going component of the wveform nd therefore dt for this concentrtion hs een omitted from () for clrity. wveform ws detected ssocited with stimulus rtefct. The vrile wveform size, nd occsionl sence of signl, suggest nerve conduction filure in some, or ll, of the nerve fires responsile for generting the signl. f87 ThImt Figure 3 Representtive exmples of nerve terminl current wveforms recorded from single implement in the sence (, ) nd presence (c, d) of 1puM (+)-vesmicol. Figure shows the first (, c) nd lst (, d) wveforms from 6 s period of 5 Hz nerve stimultion. The numers in (), (c) nd (d) give the mplitude of the two negtivegoing components of the wveform s percentge of their mplitude in (). d 11 78

4 116 K.E. PEMBERTON et l C.) S 8- T -g-4p--f T - 1- C8) "'8- me~~~~ m ' C.) I' d~~~~~~~~~ Time (s) Figure 4 Effect of stimultion t 5 Hz on the mplitude of the first () nd second () negtive-going components of nerve terminl current wveforms recorded in the sence () nd presence (*) of 1 #M (+)-vesmicol. For ech implement, individul mplitude vlues were expressed s percentge of the mplitude of the pproprite negtive-going component in the first control wveform. Ech plotted point is the men of dt from the sme three implements; s.e.men shown y verticl rs. Effects of tetrodotoxin on nerve terminl current wveforms As 1 mm (-)-vesmicol ws shown to produce mrked effects on oth negtive-going components of the nerve terminl ,- 4 8 Time (ms) Figure 5 Effects of (-)-vesmicol on nerve terminl current wveform signl dely. Grph shows signl dely for first seven signls recorded t 5Hz in the sence () nd presence of 1uM (*) or 1 mm (V) (-)-vesmicol. For ech implement, individul signl dely vlues were expressed s percentge of the signl dely of the first control wveform. Ech point is the men of dt from 4-8 different implements; verticl rs show s.e.men. Error rs for control points lie within the plotted symols Time (s) Figure 6 Effect of stimultion t 5 Hz on the mplitude of the first () nd second () negtive-going components of nerve terminl current wveforms recorded in the sence of tetrodotoxin () nd following 1min incution in 15 (e) or 3nM (*) of the toxin. For ech implement, individul mplitude vlues were expressed s percentge of the mplitude of the pproprite negtive-going component in the first control wveform. Ech plotted point is the men of dt from the sme three implements; s.e.men shown y verticl rs. Tetrodotoxin, 15nm, hd no effect on the first negtive-going component of the wveform nd therefore dt for this concentrtion hve een omitted from () for clrity. current wveforms, it is not cler whether its effects on the second negtive-going component t lower concentrtions re due to lock of nerve terminl K+-chnnels or, lterntively, consequence of n undetectle effect on the first negtive-going component, i.e. the nodl N + -chnnels. Accordingly, we tested the effects of the specific N'-chnnel locking toxin, tetrodotoxin (15-6nM), under conditions identicl to those used for vesmicol. The results otined with tetrodotoxin were essentilly similr to those seen for 1-1,UM (-)-vesmicol. Hence, tetrodotoxin produced concentrtion-dependent depression of the second negtivegoing component of the nerve terminl current wveforms t concentrtions tht produced only very smll, nonsignificnt, effect on the mplitude of the first negtive-going component (Figure 6). Unlike vesmicol, tetrodotoxin did not enhnce the stimultion-induced depression in the mplitude of the second negtive-going component of the nerve terminl current wveforms (Figure 6). Discussion As reported previously, vesmicol, n inhiitor of vesiculr cetylcholine uptke, produced n enhnced rundown of evoked synptic responses during high frequency repetitive stimultion (Suszkiw & Mnlis, 1987; Prior et l., 1989; Bowmn et l., 199; Girod et l., 1991). The compound lso produced chnges in prejunctionl current wveforms tht re similr to chnges produced y tetrodotoxin nd re consistent with the previously reported locl nesthetic ctivity of vesmicol (Mrshll, 197; Estrell et l., 1987). Girod et l. (1991) hve demonstrted in the Torpedo electroplx tht the

5 NEUROMUSCULAR EFFECTS OF VESAMICOL 117 effects of vesmicol on evoked synptic responses cn e demonstrted only t high concentrtions of the drug nd re not demonstrle when the synptic responses re evoked y focl stimultion of relese in tetrodotoxin-locked preprtions. They conclude tht the effects of vesmicol in the Torpedo re consequence of the locl nesthetic ctivity of the compound. Their experiments point to the difference etween the ctions of vesmicol on mmmlin, reptilin nd mphiin neuromusculr junctions on the one hnd, nd t the Torpedo electroplx on the other hnd. Girod et l. (1991) suggest tht the locl nesthetic ctivity of vesmicol should e tken into ccount when ssessing the mechnism of ction of the compound during high frequency trins of stimultion. As our own interprettion of the enhnced rundown of endplte responses during high frequency trins of stimuli in the presence of vesmicol nd other drugs (Prior et l., 1989; Bowmn et l., 199) hs een in terms of gents ffecting trnsmitter moiliztion from the depot to the redily relesle store, we hve now re-ssessed the ctions of vesmicol y studying its concentrtion-dependent nd stereospecific effects on the rundown of e.p.c. mplitudes nd nerve terminl current wveforms. The effect of the iochemiclly-ctive isomer of vesmicol on e.p.c. rundown ws oserved t very low concentrtions. Thus O.1 pm (-)-vesmicol produced virtul mximl effect on e.p.c. rundown. This concentrtion of (-)-vesmicol is close to the IC5 of 5nm for the inhiitory effects of the gent on cetylcholine trnsport into isolted synptic vesicles (Bhr & Prsons, 1986). Additionlly, the iochemiclly inctive (+)-isomer of vesmicol ws devoid of ctivity on e.p.c. rundown t concentrtion of 1 m. Thus, the effects of the vesmicol stereoisomers on the rundown of e.p.c. mplitudes during high frequency nerve stimultion re consistent with effects medited through the sme, or similr, mechnisms to those involved in inhiition of vesiculr cetylcholine trnsport. However, the lck of n effect of vesmicol on the mplitude of the first e.p.c. of the trin is in greement with our previous oservtion tht, t low rtes of nerve stimultion, (-)-vesmicol does not ffect evoked quntl relese in the rt (Serl et l., 1991) nd suggests tht the compound hs no effect on the trnsmitter relese mechnism per se. Furthermore, the lck of n effect of vesmicol on the time constnt of decy of the first e.p.c. of the trin suggests tht t this concentrtion the compound does not ffect the cetylcholine receptor-ssocited ion chnnel. This elimintes the possiility tht the e.p.c. rundown is consequence of usedependent endplte ion chnnel lock. In contrst to its effects on e.p.cs, the effects of vesmicol on nerve terminl current wveforms were oserved t much higher concentrtions thn those used to demonstrte e.p.c. rundown. Thus, no effects on nerve terminl current wveforms were mesurle t 1 gm (-)-vesmicol, which produced mximl effect on e.p.c. rundown. The lowest concentrtion used t which effects on nerve terminl current wveforms were detected ws 1pM. This concentrtion produced smll, ut significnt, reduction of the second negtive-going deflection of the wveform, ut mm ws required to demonstrte significnt effect on the first negtive-going deflection of the wveform, normlly ssocited with the nodl N+-current. Additionlly, unlike the effects on neuromusculr trnsmission, the effects of vesmicol, s reported previously for extrcellulrly recorded gross nerve ction potentils (Estrell et l., 1989) were not stereospecific. We thus conclude tht the stereospecific enhnced rundown of e.p.c. mplitudes seen t low concentrtions of vesmicol, in the rt hemidiphrgm, is unlikely to e relted to the locl nesthetic ctivity of the compound. Nevertheless, our results with vesmicol on the nerve terminl wveforms do provide more informtion on the locl nesthetic ctivity of vesmicol which my e pplicle in species like Torpedo where the locl nesthetic ctivity of the compound is clerly of importnce. Thus, the effect of 1pM vesmicol ws primrily on the second negtive-going component of the nerve terminl wveform, i.e. the wveform normlly ssocited with the nerve terminl K+-current. On this sis, it could e rgued tht vesmicol hs specific effect on K+-chnnels similr to tht of the minopyridines. However, diminution of the second negtive-going component of the wveforms produced y the specific K+-chnnel locker 3,4- diminopyridine, similr in degree to tht produced y 1UM (-)-vesmicol, produces mrked increse in e.p.p. quntl content (Hrvey & Rown, 199; Brg et l., 1991). In smll numer of experiments performed on the Mg2 +- prlysed, voltge-clmped (-55 mv) mouse tringulris sterni nerve-muscle preprtion we oserved no detectle effect of lo1um (-)-vesmicol on e.p.c. quntl content mesured from the men e.p.c. mplitude (1 Hz) divided y the men m.e.p.c. mplitude: control, ; 1pM (-)-vesmicol, (n = 6, P >.5, pired Student's t test). This is in greement with our previous oservtions in the snke (Serl et l., 199) nd rt (Serl et l., 1991) tht (-)-vesmicol hs no effect on e.p.c. quntl content when smll numers of qunt re eing relesed. One noticele difference etween the effects of vesmicol nd 3,4-diminopyridine on the nerve terminl current wveforms is tht, unlike vesmicol, even t very high concentrtions, 3,4-diminopyridine hs no effect on the first negtive-going component of the wveform (Brg et l., 1991). However, low concentrtions of the N+-chnnel selective toxin p-conotoxin decrese the mplitude of oth negtive-going components of the nerve terminl current wveform (M.F.M. Brg, personl communiction). Therefore, we investigted whether the effect of vesmicol on the second negtive-going component of the wveform could e n effect secondry to prime effect on the first negtivegoing component, the N+-current. We were le to demonstrte tht tetrodotoxin, specific N+-chnnel locker, produced effects similr to those of vesmicol, i.e. mrked reduction in the mplitude of the second negtive-going component of the wveform t concentrtions producing only slight, non-significnt, reduction in the mplitude of the first negtive-going component. The ility of low concentrtions of N+-chnnel lockers selectively to ffect the second negtive-going component of the nerve terminl current wveforms presumly indictes tht even very smll, undetectle, chnge in the nodl N+-current is sufficient to impir mrkedly the spred of the depolrizing wve into the nerve terminl nd the susequent ctivtion of the nerve terminl voltge-dependent K+-chnnels. Alterntively it is possile tht low concentrtions of N'-chnnel lockers selectively ffect N+-chnnels in the nerve terminl memrne itself. The existence of such N+-chnnels in the nerve terminl memrne hs een postulted (Konishi & Sers, 1984) nd it is possile tht these chnnels could ply n importnt, s yet unidentified, role in determining the mgnitude of the depolriztion reching the nerve ending. This could explin the lrge effects oserved on the nerve terminl K+-current in the sence of significnt effects on the nodl N + -current. In summry, the oserved effects of vesmicol on nerve terminl current wveforms, plus the vesmicol-induced, stimultion-dependent, increse in signl dely time, leds us to suggest tht it is use-dependent lock of the nodl N+ - chnnels which underlies ll of the oserved effects of vesmicol on the nerve terminl current wveforms. The ility of vesmicol, ut not tetrodotoxin, to enhnce the stimultioninduced depression of the mplitude of the second negtivegoing component of the nerve terminl current wveforms presumly reflects differences in the use-dependence of the N+-chnnel lock y the two gents. We therefore conclude tht the use-dependent lock of N+-chnnels t high concentrtions is the primry cuse of the locl nesthetic ctivity of oth (-)- nd (+ )-vesmicol. Additionlly, the results suggest tht lock of cetylcholine moiliztion, nd not the locl nesthetic-like effect, underlies the stereospecific enhnced rundown of e.p.c. mplitudes seen t high frequencies of stimultion with low concentrtions of (-)-vesmicol.

6 118 K.E. PEMBERTON et l. This work is supported y postgrdute reserch studentship from the MRC (K.E.P.) nd project grnts from The Wellcome Trust (I.G.M. nd C.P.). We re grteful to Dr S.M. Prsons, University of Cliforni t Snt Brr for the supply of (+)- nd (-)-vesmicol. References ANDERSON, D.C., KING, S.C. & PARSONS, S.M. (1983). Phrmcologicl chrcteriztion of cetylcholine trnsport in purified Torpedo electric orgn synptic vesicles. Mol. Phrmcol., 24, BAHR, B.A. & PARSONS, S.M. (1986). Demonstrtion of receptor in Torpedo synptic vesicles for the cetylcholine storge locker L- trns-2-(4-phenylpiperidino) cyclohexnol. Proc. Ntl. Acd. Sci. U.S.A., 83, BARSTAD, J.A.B. & LILLIHEIL, G. (1968). Trnsverslly cut diphrgm preprtion from the rt. Arch. Int. Phrmcodynm. Ther., 175, BOWMAN, W.C., PRIOR, C. & MARSHALL, I.G. (199). Presynptic receptors in the neuromusculr junction. Ann. N.Y. Acd. Sci., 65, BRAGA, M.F.M., HARVEY, A.L. & ROWAN, E.G. (1991). Effects of tcrine, velncrine (HP29), suroncrine (HP128), nd 3,4- diminopyridine on skeletl neuromusculr trnsmission in vitro. Br. J. Phrmcol., 12, DEMPSTER, J. (1988). Computer nlysis of electrophysiologicl signls. In Microcomputers in Physiology: Prcticl Approch. ed. Frzer, P.J. pp Oxford: IRL Press. ENOMOTO, K. (1988). Post- nd presynptic effects of vesmicol (AH5183) on the frog neuromusculr junction. Eur. J. Phrmcol., 147, ESTRELLA, D., GREEN, K.L., PRIOR, C., DEMPSTER, J., HALLIWELL, R.F., JACOBS, R.S., PARSONS, S.M., PARSONS, R.L. & MARSHALL, I.G. (1987). A further study of the neuromusculr effects of vesmicol (AH5183) nd of its enntiomer specificity. Br. J. Phrmcol., 93, GIBB, A.J. & MARSHALL, I.G. (1984). Pre- nd post-junctionl effects of tuocurrine nd other nicotinic ntgonists during repetitive stimultion in the rt. J. Physiol., 351, GIROD, R., LOCTIN, F. & DUNANT, Y. (1991). Locl nesthetic ctivity of vesmicol in the electric orgn of Torpedo. Eur. J. Phrmcol., 195, 1-1. GLAVINOVIC, M.I. (1979). Voltge clmping of unprlysed cut rt diphrgm for study of trnsmitter relese. J. Physiol., 29, HARVEY, A.L. & ROWAN, E.G. (199). Effects of tcrine, minopyridines, nd physostigmine on cetylcholinesterse, cetylcholine relese nd potssium currents. In Alzheimer's Disese. ed. We thnk Dr E.G. Rown nd Ms M.F.M. Brg for dvice on the perineurl recording technique nd for helpful discussion of the results nd J. Dempster for computer progrmming. Wurtmn, R.J., Corkin, S., Growdon, J.H. & Ritter-Wlker, E. pp New York: Rven Press. KONISHI, T. & SEARS, T.A. (1984). Electricl ctivity of mouse motor nerve terminls. Proc. R. Soc. B, 222, LUNDH, H. (1978). Effects of 4-minopyridine on neuromusculr trnsmission. Brin. Res., 153, MALLART, A. (1985). Electric current flow inside perineurl sheths of mouse motor nerves. J. Physiol., 368, MARSHALL, I.G. (197). Studies on the locking ction of 244-phenylpiperidino) cyclohexnol (AH5183). Br. J. Phrmcol., 38, MARSHALL, I.G. & PARSONS, S.M. (1987). The vesiculr cetylcholine trnsporter system. Trends Neurosci., 1, McARDLE, JJ., ANGAUT-PETIT, D., MALLART, A., BOURNAUD, R., FAILLE, L. & BRIGANT, J.L. (1981). Advntges of the tringulris sterni muscle of the mouse for investigtions of synptic phenomen. J. Neurosci. Meth., 4, MOLGO, J., LEMEIGNAN, M. & LECHAT, P. (1979). Anlysis of the ction of 4-minopyridine during repetitive stimultion t the neuromusculr junction. Eur. J. Phrmcol., 53, PRIOR, C., SEARL, T. & MARSHALL, I.G. (1989). The effects of 1- vesmicol on trnsmitter relese from rt motor nerve terminls t high frequencies of nerve stimultion. Br. J. Phrmcol., 98, 826P. SEARL, T., PRIOR, C. & MARSHALL, I.G. (199). The effects of 1- vesmicol, n inhiitor of vesiculr cetylcholine uptke, on two popultions of miniture endplte currents t the snke neuromusculr junction. Neuroscience, 35, SEARL, T., PRIOR, C. & MARSHALL, I.G. (1991). Acetylcholine recycling nd relese t rt motor nerve terminls studied using (- vesmicol nd troxypyrrolium. J. Physiol., (in press). SUSZKIW, J.B. & MANALIS, R.S. (1987). Acetylcholine moiliztion: effects of vesiculr ACh uptke locker, AH5183, on ACh relese in rt rin synptosomes, Torpedo electroplx nd frog neuromusculr junction. In Cellulr nd Moleculr Bsis of Cholinergic Function. ed. Dowdll, M.J. & Hwthorne, J.N. pp Ellis Horwood, Chichester. VAN DER KLOOT, W. (1986). 2-(4-phenylpiperidino) cyclohexnol (AH 5183) decreses quntl size t the frog neuromusculr junction. Pfliugers Arch., 46, WANNAN, G., PRIOR, C. & MARSHALL, I.G. (1991). Alphdrenoceptor locking properties of vesmicol. Eur. J. Phrmcol., 21, (Received August 2, 1991 Revised Septemer 24, 1991 Accepted Septemer 26, 1991)