Br. J. Pharmaol. (1994), 111, 283-287 17" Mamillan Press Ltd, 1994 xtraellular panuronium affets sodium urrent in hik embryo sensory neurones gidio Maestrone, *Valeria Magnelli, tmario Nobile & "tcesare Usai Centro Studio della Funzione Neuromusolare, Ospedale Civile di Sondrio, via Stelvio 25, 23100 Sondrio, taly; *stituto di Anatomia e Fisiologia Umana, Universita' di Torino, C. so Raffaello 30, 10125 Torino, taly and tlstituto di Cibernetia e Biofisia, C.N.R., via Dodeaneso 33, 16146 Genova, taly 1 The ation of panuronium on transmembrane sodium ondutane was investigated in dorsal root ganglion neurones of hik embryos. The Na+ urrent was measured by use of the path-lamp tehnique in whole-ell onfiguration. 2 xternally perfused panuronium (50 1AM to 1 mm) reversibly inhibited the urrent by a fast mehanism of ation. nhibition was onentration-dependent (with a half-effetive dose of 170 1AM) but not voltage-dependent. 3 The ativation and inativation kinetis of the Na+ urrent were estimated in panuronium and in ontrol solution by fitting experimental data with a Hodgkin-Huxley theoretial model. 4 The ativation time onstant Tm, at negative membrane voltages, was larger in the presene of panuronium than in the ontrol. n ontrast, the inativation time onstant Th was smaller during drug perfusion at membrane voltages <-10 mv. The steady-state inativation h,, was not affeted by panuronium. 5 These results suggest that panuronium may redue sodium hannels in both the resting and open states. the sodium urrent by interating with the Keywords: Panuronium; musle relaxants; sodium hannel; dorsal root ganglion; path-lamp; hik embryo; sensory neurones ntrodution Panuronium bromide, C35H6OBr2N204, is a bis-quaternary ammonium ompound with the steroidal hemial struture shown in Figure 1 (Savage et al., 1971). Panuronium is a non-depolarizing neuromusular bloking agent developed more than twenty years ago (Baird & Reid, 1967; Bukett et al., 1967). Panuronium is still widely administered in linial environments to provide musle relaxation both during surgial operations and in ritially ill subjets in ntensive Care Units. To patients needing longterm mehanial ventilation, huge doses (1 mg kg-' body weight, twenty times as large as the average dose administered to surgial patients) are given for days, sometimes for weeks (Agoston et al., 1990). 'Like other non-depolarizing musle relaxants, panuronium is known to inhibit neuromusular transmission by ompeting with aetylholine for binding sites on niotini reeptors (Bowman, 1990). Moreover, Yeh & Narahashi (1977) showed that additional mehanisms of ation an be onsidered. These authors observed that internally perfused panuronium inhibited the sodium urrent of voltagelamped squid axons. This ompound was quite ineffetive if applied to the extraellular site of the axon and did not exhibit any notable ation on potassium membrane ondutane. Sine the interation of the drug with sodium ondutane in ell membranes is a pharmaologially and linially important phenomenon, it seemed appropriate to hek on this interation: (a) in a different neuronal preparation, i.e. the dorsal root ganglion (DRG) sensory neurones of hik embryos; (b) under different experimental onditions, perfusing panuronium from the external side of the ell membrane; () by employing a different eletrophysiologial tehnique (the path-lamp tehnique in whole-ell onfiguration). ' Author for orrespondene. Methods The experiments were performed on primary ulture neurones from DRG of 10-day old hik embryos, with the approval of the ad ho Committees on Animal Researh at our institutions. Sensory neurones were grown aording to the proedure desribed elsewhere (Barde et al., 1980; Carbone & Lux, 1984). xperiments were performed from 4 h to 4 days after plating. The external and internal solutions were hosen suh as to eliminate potassium and alium urrents. The omposition of the external solution was as follows (mm): NaC 120, KC 3, CaCl2 2, MgCl2 2, gluose 20, HPS 10 and CdCl2 1. The eletrode filling solution ontained (mm): CsCl 120, TAC 20, GTA 10, HPS 10 and MgCl2 2. Solutions were adjusted to ph 7.3 with the addition of NaOH (external solution) or CsOH (internal solution), and the osmolarity was adjusted to 300 mosm kg-' by adding mannitol. 0N CH3COO CH H C3 CH3COO CH3 H 2Br Chemial struture of panuronium bromide: 1,l'-(3a,17p- Figure 1 dihydroxy-2-5a - androstan - 20,16f - ylene) bis[l - methylpiperidinium] dibromide diaetate. Moleular weight 732.7. Perentage omposition: C 57.39, H 8.25, Br 21.81, N 3.82, 0 8.73. Panuronium dissolves in 30 parts hloroform, one part water at 20'C.
284. MASTRON et al. Panuronium bromide for linial use (Pavulon, from N.V. Organon, Oss, Holland) was applied by means of a perfusion system working at a flow-rate of 1 ml min-'. This system allowed a full hange of solution in less than 0.5 s via a blunt glass pipette held lose to the ell with a miromanipulator. Control experiments were performed with pure panuronium bromide, supplied by Sigma Co., St. Louis, MO, U.S.A., but no differene was ever observed. The experimental setup has been desribed previously (Nobile et al., 1990). Sodium urrent measurements were made at room temperature (20 to 220C) by using the pathlamp tehnique in whole-ell onfiguration (Hamill et al., 1981). Membrane urrents were evoked by voltage steps of 10 mv, 40 ms long, from -40 to 60 mv, at a holding potential (VH) of -60 mv or -80 mv, and reorded by use of an L/M PC-7 path-lamp amplifier (List Medial letronis, Darmstad, Germany). Speial are was exerised in ompensating for aess resistane to improve the auray of voltage ontrol and the response time-onstant of the amplifier. Large eletrodes with low series resistane (2 to 3 MC) were used. The iterative inrease in the positive-feedbak seriesresistane ompensation iruit and an aurate tuning of apaitane ompensation allowed us to reah a good ompromise with the tendeny of the amplifier to osillate at about 75 to 80% ompensation. Finally, measurements where a time-dependent hange in the aess resistane was suspeted, were disarded. Passive urrent omponents were orreted by using the P/4 stimulation protool from a VH of - 120 mv. xperimental data were digitized at 16 bit by employing a digital audio proessor (SONY PCM 701 S), and stored in a video tape reorder (SONY SL HF 100). Data were retrieved from the reorder, filtered at 3 khz with an 8-pole Bessel filter, and then aquired with a personal omputer (BM PC AT) for further analyses. The eletrophysiologial data-proessing software, VCAN V-i, was kindly provided by Dr J. Dempster, Dept. of Physiology and Pharmaology, University of Strathlyde, Glasgow, Sotland. Results The time-ourse of a family of whole-ell urrents, under ontrol onditions and during perfusion with 150 tm panuronium, is shown in Figure 2. Currents were evoked in a voltage-lamped neurone held at -60 mv VH by step depolarizations from -30 to 10 mv. The external solution ontained Na' (120 mm), Ca2+ (2 mm), and Cd2+ (1 mm), and the internal solution ontained Cs' (120 mm) and TA (20 mm). A fast transient inward urrent was present at eah applied depolarizing potential, with a maximum peak amplitude between -10 and 0 mv. By ontrast, a low, longlasting, residual outward urrent was observed at membrane depolarizations more positive than -20 mv. n the absene of Ca2+ and K+ urrents, the fast transient disappeared in the presene of 3ytM tetrodotoxin (TTX) in the bath, or when the external sodium was replaed with holine. These effets, together with the fast ativation and inativation rates and with the voltage range of exitation, indiated that the inward urrent was arried by Na+ ions. The Na+ urrent was inhibited by panuronium at eah applied potential, and the peak amplitude was redued to about 60% of its previous value. nterestingly, the residual outward urrent was affeted by the presene of panuronium, as well. At VH = - 60 mv, the presene of the Na+ hannel ultraslow inativation proess must be taken into aount (Carbone & Lux, 1986; Ruben et al., 1992). For this reason, the same experiments were performed on ells held at VH = - 80 mv: the perentages of Na+ urrent inhibition due to panuronium ation turned out to be quite omparable in both ases. Therefore, the -60 mv value of VH was preferred in the following experiments, as the preparation showed a better stability and a longer life. The urrent-voltage relationships in ontrol onditions, in VH = -60 mv Control 0.2 na 2 ms Panuronium 10 mv 0 mv -10 mv -20 mv -30 mv Figure 2 Whole-ell urrents evoked by depolarizing steps to indiated values in ontrol onditions and during perfusion with 150 im panuronium (VH = -60 mv). After removal of Ca2l and K+, a transient inward Na+ urrent, showing a maximum between -10 and 0 mv, is evoked. A low, long-lasting outward urrent is still present. the presene of 250 gm panuronium, and after wash-out of the drug are shown in Figure 3. pal, values were measured by using 10 mv depolarizing steps from -40 to 60 mv, at VH = -60 mv, and averaged over seven neurones. The urrent reovery was omplete after wash-out with ontrol solution. n Figure 4, the ratios between Na' urrent in panuronium and in ontrol onditions are plotted versus depolarization. The Na' urrent inhibition by panuronium is not voltage-dependent exept for very large depolarizations, where the outward urrent prevails. The inhibitory effet of panuronium on the Na' urrent was onentration-dependent. Conentrations between 50 f1m and 1 mm were tested: in every ase, the observed inhibition of the Na' urrent amplitude was ompletely reversible. Similar results were obtained in 65 DRG neurones. The dose-response relation is shown in Figure 5. The ratio of the peak urrent with and without panuronium (/.), was measured at six different onentrations by using depolarizing voltage pulses to -10 mv from a holding potential of
PANCURONUM AFFCTS Na CURRNT N DRG NURONS 285 1.0-0.8 - wx 01 x o 0.6-0.4-0.2-0.0 - -40-20 0 20 40 60 Figure 3 Current-voltage relationship in ontrol onditions (0), during the perfusion of 250 4M panuronium (V), and after washout with ontrol solution (V); 10 mv depolarizing steps from -40 mv to 60 mv (VH = -60 mv) were used. Data were averaged over seven neurones. Mean values ± s.e.mean are shown. '*7* 10-4 10-3 Conentration (M) Figure 5 Panuronium dose-response urve. Depolarizing pulses to - 10 mv (VH = -60 mv) were used to measure the ratio of the peak urrent with and without panuronium. Values of /.m. were averaged over ten ells, and the s.e.means are shown. The experimental points were best approximated with the equation shown in the text. The half-effetive dose was 170 M. 100-1.0-80 - 0.8-0 0.6- - C to 0.4-4 1 4 1 4 0 4 0 4 1 - - 0 -o._._._o 60 40- -L- -L- -A- - 0.2-20 -40-20 0 20 40 60 Figure 4 Voltage-dependene of Na+ urrent inhibition. Peak urrent ratios in panuronium and in the ontrol have been alulated from the same data as in Figure 3. Mean values ± s.e.mean are shown. 0 t (s) Figure 6 Time-ourse of Na+ urrent inhibition and reovery. Currents were eliited by repetitive (period = 6 s) depolarizing pulses to -20 mv, 40 ms long. At t = 18 s, panuronium 500 pm (0) or 150 JM (A) was applied. After the steady-state of drug ation was reahed, the ell was washed with drug-free solution (t = 36 s). T = 22 C. -60 mv. At a 50 tim onentration, the ratio /ma, was lose to one, while at 1 mm the urrent was fully bloked. xperimental data were fitted aording to the equation: = 1 - [Cn/(Cn + K1n)] where C is the panuronium onentration, K4 is the onentration value produing a half-inhibition of urrent and n is the Hill oeffiient. The best fit gave K1 = 170 jim and n = 2.1, suggesting that two panuronium moleules must bind to a site in order to inhibit the Na+ urrent. The time-dependene of Na+ urrent inhibition and reovery at different panuronium onentrations are shown in Figure 6: depolarizing voltage pulses of 40 ms duration from VH = - 60 mv to -20 mv, were employed. Pulses were repeated every 6 s, and panuronium was perfused during stimulation. At 500 glm panuronium, the steady-state inhibition value was reahed very rapidly (< 6 s, for 95% inhibition), at the first pulse after perfusion. The lak of blok aumulation with repetitive stimuli suggests that inhibition is not use-dependent at this frequeny and onentration. Similar results were obtained at higher onentrations. At onentrations lower than 500 pm, the inhibition time-ourse
286. MASTRON et al. was relatively slower (about 12 s to 60% steady-state blok, at 150 pm). An almost omplete reovery was then reahed in a slightly longer time after wash-out (t15 s), at eah tested onentration. The ativation and inativation kinetis of the Na' urrent were estimated, in the absene and in the presene of panuronium, by fitting experimental data with a simplified Hodgkin-Huxley model: Na = maxil- exp( - t/tm)]3 x [her- (ho,- )exp(- t/th)] where Tm and Th are the ativation and inativation time onstants respetively, and h., is the steady-state inativation. The resulting values of Tm and Th were voltage-dependent, with larger values at more negative membrane potentials. The voltage dependene of Tm is shown in Figure 7a, in ontrol onditions, during drug perfusion and after reovery. The ativation time onstant in the tested voltage range (-30 to 0 mv) was signifiantly larger in the presene of panuronium than in ontrol or reovery onditions. The differene beame negligible at positive membrane potentials. The voltage dependene of Th is shown in Figure 7b. Unlike Tm, Th showed a signifiant redution during perfusion with the drug at membrane potentials more negative than -10mV. a 2.0-1.8-1.0 0.8-0.6-0.4-0.2-0.0-100 -80-60 -40-20 VP (mv) Figure 8 ffets of panuronium on steady-state Na+ inativation (h.,) at different voltages. The peak of the Na+ urrent was measured during a 30 ms long depolarizing test pulse to 0 mv, following 70 ms long onditioning prepulses to various potentials (Vp). Current values, averaged over five ells and normalized to the value at -100 mv, were fitted by a Boltzmann equation (see text). Differenes between ontrol (0) and panuronium (0) are not signifiant. s.e.means are shown. VH = -80 mv. en 1.6-1.4-1.2-1.0-10 - 8- b -30-20 -10 0 The steady-state Na' inativation was measured in ontrol onditions and in presene of panuronium. The perentage of non-inativated sodium urrent (ha,,) was measured at 0 mv, after a 70 ms long onditioning prepulse ranging between - 100 mv and -20 mv. The voltage-dependene of ha., both under ontrol onditions and during perfusion with 250 fim panuronium, is shown in Figure 8. xperimental data were fitted by a smooth urve derived from the Boltzmann relation: ha = 1/[1 + exp((vp - Vj)/k)] where VP is the prepulse value, V is the potential of halfinativation and k is the slope of the urve. The best fit gave V =-45 mv, k = mv in ontrol and V =-47 mv, k = 10 mv in panuronium, indiating that panuronium does not affet the steady-state inativation of Na+ urrent. u) 6-4 - 2- -30-20 -10 0 Figure 7 Voltage-dependene of ativation and inativation timeonstants in ontrol solution (V), in panuronium (0) and after reovery (V). (a) a,, is larger in panuronium than in the ontrol at membrane potentials <0 mv; (b) the inativation time-onstant Th is redued by panuronium at membrane potentials <-10 mv. Data were averaged over six ells; s.e.means are shown. Disussion The present results show that, when externally perfused, panuronium bromide seletively affets the amplitude of the tetrodotoxin-sensitive sodium urrent in hik embryo DRG sensory neurones. Na' urrent inhibition is voltage-independent and onentration-dependent. Some interesting differenes between our data and results obtained by Yeh & Narahashi (1977) on squid giant axons should be onsidered. Panuronium strongly affets sodium ondutane in externally perfused DRG neurones, but interats with squid axon Na' hannels only by internal perfusion. A similar effet has been reported for the general anaestheti ketamine: Shrivastav (1977) found that ketamine bloks the Na+ urrent very effiiently when applied intraellularly to squid axons. However, the same drug in frog isolated nodes of Ranvier inhibits Na+ urrents when externally perfused (Benoit et al., 1986). These authors suggest a diret ation of ketamine from the outside of the nodal membrane.
PANCURONUM AFFCTS Na CURRNT N DRG NURONS 287 The ativation time-onstant, Tm, of the Na+ urrent is affeted by panuronium in DRG neurones, and beomes larger during drug perfusion than in ontrol onditions. This effet was not observed in squid axon membranes. On the ontrary, the Na+ urrent inativation seems to be aelerated by panuronium in both preparations, due to an apparent redution in the inativation time-onstant, th. Yeh & Narahashi (1977) inferred that, in squid axons, panuronium binds to a site inside the pore of the open hannels, bloking the Na' flux and reduing the number of onduting hannels. Therefore, the derease in urrent amplitude during membrane depolarizations, is due to both the real Na' hannel inativation and the hannel blok by panuronium. However, in DRG neurones, panuronium affets the sodium urrent ativation mehanism, suggesting the possibility of interation between panuronium and Na+ hannels also at rest. n order to explain these differenes, two hypotheses ould be put forward. The presene of an external binding site for panuronium in DRG Na+ hannels might aount for the effets of the drug during external perfusion. Appreiable strutural differenes were observed by Sato & Matsumoto (1992) among Na' hannels of squid and vertebrates. These authors found a subunit amino-aid sequene of squid Na hannel onsiderably shorter (25%) than those of the three main rat brain a-subunits. Moreover, a similar effet was observed for the 'delayed retifier' K+ hannel of vertebrate neurones: unlike the other lasses of K+ hannels, the delayed retifier is bloked by the quaternary ammonium tetraethylammonium (TA) through an external binding site. This inhibition is voltage-independent but it does not modify the K+ urrent kinetis (Hille, 1967). Diffiulties enountered by the drug in diffusing from the external to the ytoplasmi side of the squid axon membrane ould be another possibility. Panuronium ionization is highly insensitive to hanges in ph, due to pka> 13 (Bertrand & Conina, 1980). This moleule bears positive harges also at physiologial ph values, and annot ross lipid membranes easily in spite of the lipophili androstane moiety. Differenes in ell membrane ompositions (glia sheet thikness, presene of peuliar membrane surfae omponents, surfae harge density and distribution) might therefore failitate or hinder panuronium permeation. Hille (1992) reported the possibility of quaternary ammonium ompounds reahing their binding site in a pore through a hydrophobi pathway. This allows the drug binding and unbinding even to losed Na' hannels. Aording to this view, panuronium, permeating the DRG ell membrane, may in part reah its binding site through the hydrophobi pathway before the Na' hannel opening. The hannel ativation, therefore, might be apparently delayed by the rate-limiting proess of drug dissoiation, as observed for lipophifi steroids like deoxyholate (Wu et al., 1980). nterestingly, the observed residual outward urrent is also affeted by panuronium. The nature of this urrent is not lear: it might be arried by Cs2" ions rossing potassium hannels insensitive to TA. The possibility of a panuronium interation with potassium hannels was tested in DRG neurones perfused with TA-free solutions. nhibition of the K+ urrent was observed in the same range of drug onentrations affeting the Na' urrent (data not shown). These results, too, differ from squid axon data (Yeh & Narahashi, 1977), aording to whih some inhibitory effet on the K+ urrent was observed only at very high onentrations. We think that it should be interesting to investigate also the effets of panuronium on neuronal potassium ondutane. n onlusion, panuronium redues sodium ondutane when externally perfused in hik embryo DRG sensory neurones. Na' urrent inhibition might enhane the neuromusular bloking ation of panuronium, but it might also be a ritial side-effet in linial appliations. Both these possibilities are of great interest to ntensive Care Unit patients, to whom long-term musle relaxation is provided by large doses of panuronium. Referenes AGOSTON, S. (1990). The use and misuse of neuromusular bloking agents in the intensive are unit. n Neuromusular Bloking Agents: Past, Present and Future, ed. Bowman, W.C., Denissen, P.A.F. & Feldman, S.A. pp. 109-116. Amsterdam: xerpta Media, lsevier. BARD, W.L.M. & RD, A.M. (1967). The neuromusular bloking properties of a new steroid ompound, panuronium bromide. Br. J. Anaesth., 39, 775-780. BARD, Y.A., DGAR, D. & THONN, H. (1980). Sensory neurons in ulture: hanging requirements for survival fators during embryoni development. Pro. Natl. Aad. Si. U.S.A., 77, 1199-1203. BNOT,., CARRATU', M.R., DUBOS, J.M. & MTOLO-CHPPA, D. (1986). Mehanism of ation of ketamine in the urrent and voltage lamped myelinated nerve fibre of the frog. Br. J. Pharmaol., 87, 291-297. BRTRAND, J.C. & CONCNA, D. (1980). Fateurs physiologiques influeneant 'ation des urares. n Curares et Curarisation. ed. Conseiller, C., Desmonts, J.M., Glaser, P., Montagne, J., Nahas, G.G., Salamagne, J.C., Viars, P. & Vour'h, G. pp. 33-54. Amsterdam: xerpta Media, lsevier. BOWMAN, W.C. (1990). Pharmaology of Neuromusular Funtion. ed. Bowman, W.C. London: Wright. BUCKTT, W.R., HWTT, C.L. & SAVAG, D.S. (1967). Potent steroidal neuromusular bloking agents. Chim. Thir., 2, 186-191. CARBON,. & LUX, H.D. (1984). A low-voltage ativated, fully inativating Ca hannel in vertebrate sensory neurons. Nature, 310, 501-502. CARBON,. & LUX, H.D. (1986). Sodium hannels in ultured hik dorsal root ganglion neurons. ur. Biophys. J., 13, 259-271. HAMLL, O.P., MARTY, A., NHR,., SAKMANN, B. & SGWORTH, F.J. (1981). mproved path-lamp tehniques for high-resolution urrent reording from ells and ell-free membrane pathes. Pflfigers Arh., 413, 85-100. HLL, B. (1967). The seletive inhibition of delayed potassium urrents in nerve by tetraethylammonium ion. J. Gen. Physiol., 50, 1287-1302. HLL, B. (1992). oni Channels of xitable Membranes. Sunderland, Massahusetts: Sinauer Assoiates n. NOBL, M., CARBON,., LUX, H.D. & ZUCKR, H. (1990). Temperature sensitivity of Ca urrents in hik sensory neurones. Pfluigers Arh., 415, 658-663. RUBN, P.C., STARKUS, J.G. & RAYNR, M.D. (1992). Steady-state availability of sodium hannels. Biophys. J., 61, 941-955. SATO, C. & MATSUMOTO, G. (1992). Primary struture of squid sodium hannel dedued from the omplementary DNA sequene. Biohem. Biophys. Res. Commun., 186, 61-68. SAVAG, D.S., CAMRON, A.F., FRGUSON, G., HANNAWAY, C. & MACKAY,.R. (1971). Moleular struture of panuronium bromide (3a,17 -diaetoxy-2p,16 -dipiperidino-5a-androstane dimethobrobide), a neuromusular bloking agent. Crystal and moleular struture of the water: methylene hloride solvate. J. Chem. So., B, 410-415. SHRVASTAV, B.B. (1977). Mehanism of ketamine blok of nerve ondution. J. Pharmaol. xp. Ther., 201, 162-170. WU, C.H., SDS, P.J. & NARAHASH, T. (1980). nteration of deoxyholate with the sodium hannel of squid axon membranes. J. Gen. Physiol., 76, 355-379. YH, J.Z. & NARAHASH, T. (1977). Kineti analysis of panuronium interation with sodium hannels in squid axon membranes. J. Gen. Physiol., 69, 293-323. (Reeived August 4, 1993 Aepted September 20, 1993)