Enhancement by benzodiazepines of the inhibitory effect of adenosine on skeletal neuromuscular transmission

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1 Bridsh Joumal of Phamaology (1995) 116, B 1995 Stokton Press All rights reserved /95 $12. x Enhanement by benzodiazepines of the inhibitory effet of adenosine on skeletal neuromusular transmission L.. hiou, J.Y. Ling & 1.. hang Department of Pharmaology, ollege of Mediine, National Taiwan University, Jen-Ai Rd., No. 1, Se. 1, Taipei, Taiwan Keywords: 1 Interations of benzodiazepines with adenosine on the neuromusular transmission were studied in mouse diaphragm preparations. 2 In tubourarine (.6-.8 gm)-partially paralyzed preparations, diazepam (35 gm) and Ro (3 3 jm), a peripheral type benzodiazepine reeptor agonist, potentiated the inhibitory effet of adenosine on indiret twith responses. 3 The entral type reeptor agonist, lonazepam did not affet the inhibitory effet of adenosine. 4 The peripheral benzodiazepine reeptor antagonist, PK1 195 (1-1 gm) attenuated the adenosine inhibition and antagonized the potentiation by Ro Ro failed to enhane further the inhibitory effet of adenosine in the presene of dipyridamole, an adenosine uptake inhibitor that also potentiated adenosine inhibition. 6 Neither Ro nor PK affeted the inhibition produed by a stable adenosine analogue, 2- hloroadenosine, whih is not a substrate for the adenosine uptake system. 7 Ro did not affet endplate potentials (e.p.ps) in the absene of adenosine, but redued the amplitude of e.p.ps in the presene of adenosine without affeting miniature e.p.ps. 8 It is suggested that benzodiazepines potentiate the adenosine-effeted presynapti inhibition of neuromusular transmission by an inhibition of adenosine uptake through ativation of peripheral type benzodiazepine reeptors. Benzodiazepines; adenosine; neuromusular transmission; adenosine uptake; benzodiazepine reeptors Introdution In addition to anxiolyti, antionvulsant and sedative therapeuti effets, benzodiazepines are also used as musle relaxants. Most of these effets are believed to be mediated by the entral type benzodiazepine reeptors that onstitute the modulatory site of y-aminobutyri aid (GABA)A reeptorhloride hannel omplex (Olsen, 1982; Haefely et al., 1985). Nevertheless, several lines of evidene suggest that benzodiazepines exert some of their effets, if not all, through interation with adenosine in the entral nervous system (Dragunow et al., 1985; Stone, 1986; Phillis & O'Regan, 1988a; ontreras & Germany, 1991; O'onnor et al., 1991; Sierralta & Miranda, 1992). There are also reports (Roahe & Griffiths, 1987; Roa et al., 1988; Ruiz et al., 1988) referring to interations between benzodiazepines and methylxanthine adenosine reeptor antagonists (Snyder et al., 1981). Benzodiazepines have been shown to interfere with adenosine uptake by inhibiting the purine transporter system (Phillis et al., 198; Hammond et al., 1983; Moritoki et al., 1985; Bender & Hertz, 1986; Morgan & Stone, 1986) and hene to potentiate the pharmaologial responses of adenosine in the entral nervous system (Stone 1986; Phillis & O'Regan, 1988b) as well as in peripheral tissues, suh as ardia musle (lanahan & Marshall, 198; Kenakin, 1982; Moritoki et al., 1985), trahea (Devillier et al., 1992), vas deferens (lanahan & Marshall, 198; Esubedo et al., 1991), taenia oli (Moritoki et al., 1985) and skeletal neuromusular transmission (Mendona & Ribeiro, 1989). In addition, benzodiazepines have also been reported to affet the binding of adenosine (Hawkins et al., 1989; Kaplan et al., 1992). Two types of benzodiazepine reeptor have been identified; the entral type displaying nanomolar affinity for diazepam and the peripheral type reeptors with affinity for diazepam in the 1 nanomolar range. The latter were first demonstrated in Author for orrespondene. nonneuronal tissues (Braestrup & Squires, 1977) and later also in the entral nervous system (Shoemaker et al., 1981). Benzodiazepine binding sites appear to exist in the motor nerveskeletal musle system (Roeske & Yamamura, 1982; Wilkinson et al., 1982) and diazepam interated synergistially with adenosine-indued inhibition of neuromusular transmission (Mendona & Ribeiro, 1989). We have previously shown that peripheral type benzodiazepine reeptor agonists but not entral type agonists inreased musle ontratility and antagonized the regenerative toni endplate depolarization indued by train pulses in neostigmine-treated preparations (hiou & hang, 1993; 1994). In the present study, we further show that a peripheral benzodiazepine reeptor agonist but not a entral one potentiates adenosine-indued inhibition of mouse neuromusular transmission. The mehanism of synergism was also examined. Methods Musle ontrations Phreni nerve-hemidiaphragm preparations isolated from IR mie (2-25 g) of either sex were used. The preparation was inubated in an organ bath ontaining 15 ml Tyrode solution (omposition in mm: Nal 137, Kl 2.8, al2 1.8, Mgl2 1.2, NaH2PO4.33, NaHO and dextrose 11.2) kept at and oxygenated with 95% 2 plus 5% O2- Indiret musle ontations were eliited by stimulation of the phreni nerve with supramaximal retangular pulses of.5 ms width at.1 Hz and reorded isometrially with a transduer (BG25, Gould) oupled to a physiologial reorder (Gould 3). The safety fator for neuromusular transmission in phreni nervediaphragm preparations is high, being 3-5 (Paton & Waud, 1967; hang et al., 1975). In order to visualize the small magnitude of the inhibitory effet of drugs, the safety fator was deliberately dereased by adding.6-.8 gm tubourarine

2 L.. hiou et al that inhibited indiret twith responses to 73-78% of ontrol. This proedure redued the safety margin lose to one so that any inhibitory effet on neuromusular transmission would result in a further derease of the indiret ontration. Inhibitory effets were alulated in terms of perentage inhibition taking the response before treatment with adenosine as ontrol. In the study of dose-inhibition relationships, adenosine or 2-hloroadenosine was applied umulatively. The I5 of adenosine or 2-hloroadenosine was determined by the interpolation from dose-response urves. Eletrophysiologial studies Transmembrane potentials and endplate potentials (e.p.ps) were measured by lassial intraellular reording tehnique with 3 M Kl-filled miroeletrodes (1-4 Mfl). Preparations were mounted horizontally in a perfusion hamber ontaining 4 ml oxygenated Tyrode solution and were perfused at a rate of 6-8 ml min- at Signal inputs to the miroeletrode were registered through a high impedane amplifier (Axolamp-2A) and a omputer-aided digitizer (D61, Analogi). Endplate potentials were evoked by stimulation of the nerve at 1 Hz and monitored in ut musle preparations (Barstad & Lilleheil, 1968) or in unut musle preparations treated with gm tubourarine. The amplitude of e.p.ps of ut musle preparations was orreted for non-linear summation to -4 mv, assuming a reversal potential of mv (hang et al., 1986). hemials Diazepam, lonazepam and flumazenil were generous gifts from Hoffmann-La Rohe (Basle, Switzerland) and PK (also oded as RP 5228) (1-(2-hlorophenyl)-N-methyl-N-(1- methylpropyl)-3-isoquinoline arboxamide) was from Rhone- Poulen Rorer (edex, Frane). Adenosine, 2-hloroadenosine, dipyridamole and tubourarine hloride were purhased from Sigma (U.S.A and Ro (7-hloro-1,3- dihydro-1-methyl-5-(p-hlorophenyl)-2h-1,benzodiazepine- 2-one) from Fluka (Switzerland). All benzodiazepines and PK11195 were dissolved in ethanol as stok solutions. Final vehile onentrations added to the organ bath were less than.2% and had little effet on neuromusular transmission. Dipyridamole was dissolved in a minimum volume of 1 N HO and then diluted to 1 mm as a stok solution. Statistis All data are expessed as mean + s.e.mean. In eletrophysiologial experiments, data from 6-7 endplates for eah preparation treated with tubourarine were pooled for omparison. In ut musle preparations, responses of the same endplate before and after drug treatment were ompared. n refers to the number of preparations tested. Differenes between means were analyzed by Student's paired t test. ontratile responses among different treatments were analyzed by non-paired t test. Results Effets on the inhibitory ation of adenosine on twith responses In mouse diaphragm preparations partially paralyzed with.6-.8 gm tubourarine, adenosine inhibited the indiret twith response within 1 min. The I5 obtained from the umulative onentration-inhibition urve was 74+7 JM (n=22). Diazepam, at 35 JM, inreased the indiret twithes by 35 ± 2% (n = 16) when applied alone, but potentiated the inhibition aused by adenosine. The onentration-inhibition urve of adenosine was shifted to the left in the presene of 35 gm diazepam (Figure 1) and the I5 for adenosine was Benzodiazepines and adenosine synergism dereased to gm (n = 16). Interestingly, diazepam at a lower onentration, 1 gm, shifted the inhibition urve rightward (Figure 1), indiating a biphasi nature of ation. A higher onentration of diazepam (1 gm) was not used beause axon ondution blok ourred (hiou & hang, 1993). Like 35 gm diazepam, Ro , a seletive peripheral benzodiazepine reeptor agonist (Braestrup & Squires, 1977), enhaned the inhibitory effet of adenosine on neuromusular transmission although it inreased musle ontatility. At 3, 1 and 3 gm, Ro inreased the twith responses by , and 51+3%, respetively. Unlike diazepam, Ro potentiated the adenosine-indued inhibition dose-dependently at all onentrations tested (3-3 gm) (Figure 2). The I5 for adenosine in the presene of 3 and 3 gm Ro was dereased from 74+7 to 55+1 (n=7) and 38+3 gim (n= 1), respetively. Ro also aused a ondution blok at onentrations higher than 5 gim. In ontrast to diazepam and Ro , lonazepam, a 'a.r4 :5 1 r 8 F- 6F- 4 H Figure 1 Effets of diazepam on adenosine-indued inhibition of indiret twith responses. Indiret twith responses were evoked at.1 Hz in mouse diaphragm preparations pretreated with tubourarine (.6-.8 gm). Inhibitions (%) of twith amplitude after umulative addition of adenosine were obtained in the presene of vehile (alohol.1%, v/v) (), 1O gm (V) and 35 gm diazepam (A). P<.5 vs. ontrol. n = ar._4 %._ r 1 r 8 [- 6 I- 4 F- 2 V -/I I Adenosine (gim) Figure 2 Effets of Ro and lonazepam on adenosineindued inhibition of indiret twith responses. Same experimental onditions as desribed in Figure 1 legend. Vehile ontrol (); 3 (V) or 3 (A)pM Ro ; 1 IM lonazepam (V). P<.5 vs. ontrol. n =

3 r- L.. hiou et al Benzodiazepines and adenosine synergism 1 r 2- )._ :2r._ Figure V 4-2k Effet of PK11195 on the adenosine-indued inhibition of indiret twith responses in the presene or absene of Ro Same experimental onditions as desribed in Figure 1 legend. Vehile ontrol (), 1 M PK1195 (Q), 3gM Ro (A); lojim PK11195 plus 3J±M Ro (). P<.5 vs. ontrol. #P <.5 vs. Ro alone. n = entral benzodiazepine reeptor agonist (Braestrup & Squires, 1977) affeted neither the indiret twith responses by itself, nor the inhibitory effet of adenosine (Figure 2). To exlude the possibility that the interations of benzodiazepines with adenosine desribed above were due to a nonspeifi pharmaologial ation of these agents, unrelated to their binding to peripheral benzodiazepine reeptors, we further studied the effet of PKl 1195, a peripheral reeptor antagonist with a nonbenzodiazepine struture (Le Fur et al., 1983a,b). PKl 1195 slightly attenuated the inhibition by adenosine and shifted the adenosine-inhibition urve to the right (Figure 3). PKl 1195, at 1 and 1 gm, aused the same magnitude of attenuation. The I5 inreased from (n = 22) to 98 ± 9 (n = 12) and to gm (n = 24), respetively, after treatment with 1 and 1 gm PKII195. PK11195 also diminished the potentiation by Ro of adenosine inhibition (Figure 3). The I5 for adenosine in the presene of 3 g.m Ro was inreased from (n = 1) to (n = 1) and 55 ± 8 JM (n = 1 1), respetively, by 1 and 1 gm PKI Effets on the inhibitory ation of 2-hloroadenosine 2-hloroadenosine, a nonhydrolyzable adenosine analogue whih is not taken up by the nuleotide transporter system (Daly, 1983), inhibited indiret twith responses dose-dependently, like adenosine, in preparations partially paralyzed with tubourarine. The I5 was gm, being about 4 fold more potent than adenosine. In ontrast to adenosine-indued inhibition, Ro (3 gm) did not affet the inhibitory effet of 2-hloroadenosine on the indiret twithes (Figure 4). PK 11195, at 1 gm whih attenuated the adenosine inhibition no matter whether Ro was present or not, had no signifiant effet on 2-hloroadenosine-indued inhibition (Figure 4). U R ). 8 H 'r- :_ 61-4 V 2 - i, hloroadenosine (gm) 1. Figure 4 Effets of Ro and PK 1195 on the inhibition by 2- hloroadenosine of indiret twith responses. Similar experiments to those in Figure 3 using 2-hloroadenosine in plae of adenosine. ontrol (); 3 gm Ro (A); IOgM PK11195 (El). n= o ) 'ad. Bo a to 1 r 8 1-6k 4 H 2 H o I111 IUI Figure 5 Effets of dipyridamole and Ro on adenosineindued inhibition of indiret twith responses. Same experimental onditions as desribed in Figure 1. Vehile alone (); 1 IgM dipyridamole (A); 1IRM dipyridamole plus 3gM Ro (A). P <.5 vs. ontrol. n = Effet on adenosine inhibition in the presene of dipyridamole In the presene of 1 JIM dipyridamole, an adenosine uptake inhibitor (Huang & Daly, 1974; Jarvis, 1986), the inhibitory effet of adenosine on the indiret twithes was markedly enhaned and the I5 was redued from to gm. In the presene of dipyridamole, Ro failed to enhane further the inhibitory effet of adenosine (Figure 5). Table 1 Effets of adenosine on the endplate responses in the absene or presene of Ro Treatment ontrol Adenosine, 3gM Ro , 3 gm Ro adenosine ut musle ± ± 2.3 e.p.p. (mv) urare-treated m.e.p.p. (my) Intat musle ±.3 Endplate potentials (e.p.ps) were evoked at 1 Hz in um tubourarine-treated preparations with membrane potentials within 75-8 mv, or in ut musle preparations with membrane potentials at mv. Miniature e.p.ps (m.e.p.ps) were reorded in normal musle preparations with membrane potential at mv. P<.5 vs. ontrol; P<.5 vs. adenosine alone. n=7-12.

4 Effet of Ro on the e.p.p. L.. hiou et al Benzodiazepines and adenosine synergism 1873 inhibition by adenosine To eluidate the site of interation between adenosine and benzodiazepines, their effets on endplate responses were examined. Both adenosine and Ro , alone or in ombination, had no effet on the amplitude of miniature e.p.ps (m.e.p.ps) in intat musle endplates (Table 1). Adenosine, 3 gm, dereased the e.p.p. amplitude by % (n = 7) in ut musle preparations and by % (n = 12) in preparations paralyzed with gm tubourarine. In parallel with its effet on indiret twithes, 3 gm Ro inreased the depressant effets of adenosine on e.p.ps in the ut and tubourarine-treated preparations from % to % and from % to %, respetively (Table 1). Ro did not affet the e.p.p. amplitude when adenosine was absent. Disussion We have found previously that benzodiazepine reeptor ligands ating on the peripheral type reeptor (suh as diazepam, Ro and PK1 1195), but not those ating speifially on the entral type reeptor (suh as lonazepam and flumazenil), inhibited the regenerative prolonged endplate depolarization aused by repetitive stimulation in the presene of antiholinesterase agents through a presynapti mehanism (hiou & hang, 1993; 1994). In the present experiments we further showed that diazepam and Ro , but not lonazepam, potentiated adenosine-indued inhibition of neuromusular transmission. The potentiation by diazepam or Ro of adenosine-indued inhibition of indiret twithes was not due to an inrease of musle ontratility (hiou & hang, 1994) sine the latter should result in an attenuation rather than potentiation of adenosine inhibition. Adenosine inhibition of neuromusular transmission is produed through an inhibition of aetylholine release from motor nerve terminals (Ginsborg & Hirst, 1972; Silinsky, 1984). Benzodiazepines at onentrations effetive in potentiating adenosine inhibition have no diret effet on neuromusular transmission at either pre- or postsynapti sites (hiou & hang, 1993; 1994). In the presene of adenosine, the quantal ontent was further dereased by Ro sine it further redued the e.p.p. amplitude while not affeting the postsynapti reeptor sensitivity. The potentiation by benzodiazepines ould reasonably be attributed to an enhanement Referenes of adenosine-indued inhibition of aetylholine release, possibly through an ation at the peripheral-type benzodiazepine reeptors. Similar synergisti interations between adenosine and peripheral benzodiazepine reeptor ligands have been reported in other peripheral tissues (Davies & Huston, 1981; Esubedo et al., 1991; Devillier et al., 1992). It is interesting that PK1 1195, a peripheral benzodiazepine reeptor antagonist (Le Fur et al., 1983a, b), dereased the adenosine inhibition either in the absene or presene of Ro , but not the 2-hloroadenosine-indued inhibition. Therefore, it seems unlikely that PK diretly affets adenosine reeptors. Whether the attenuation by PK of adenosine-indued inhibition in the absene of agonist is due to an antagonism against endogenous benzodiazepine (Verma & Snyder, 1989) remains to be eluidated. The reason why diazepam at 35gM potentiated the effet of adenosine while inhibiting it at lower onentrations is not lear. The ineffetiveness of Ro in potentiating the inhibition indued by 2-hloroadenosine suggests that Ro potentiates adenosine inhibition by affeting the adenosine uptake system. Mendona & Ribeiro (1989) proposed that diazepam potentiated adenosine-indued neuromusular blok in frog musles through an inhibition of adenosine uptake. Diazepam and other benzodiazepines have been shown to inhibit adenosine uptake in neuronal and non-neuronal tissues (Phillis et al., 198; Hammond et al., 1983; Moritoki et al., 1985; Morgan & Stone, 1986). It has been suggested that peripheral type benzodiazepine reeptors, present on human erythroyte membranes, may be involved in the inhibition of nuleoside transport (Hammond et al., 1983; Olson et al., 1988). Our experiments with dipyridamole, whih enhaned the inhibitory effet of adenosine on the neuromusular transmission by itself (hiou et al., 1987; Ribeiro & Sebastiao, 1987), but negated the effet of Ro , are in support of the above inferene. It is inferred that peripheral type benzodiazepine reeptors may modulate in some way the adenosine uptake mehanism at the neuromusular juntion. We greatly appreiate the generous gift of diazepam and lonazepam from Hoffmann-La Rohe (Basle, Switzerland) and PK from Rhone-Poulen Rorer (edex, Frane). 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