3-Adrenoceptor subtypes and the opening of plasmalemmal

Transcription

1 Br. J. Phrmcot. (1993), Br. J. Phrmcol. (1993), 109, '." Mcmilln Press Ltd, Adrenoceptor subtypes nd the opening of plsmlemml K+-chnnels in trchelis muscle: electrophysiologicl nd mechnicl studies in guine-pig tissue S.J. Cook, IR.C. Smll, J.L. Berry, P. Chiu, S.J. Downing & R.W. Foster Smooth Muscle Reserch Group, Deprtment of Physiologicl Sciences, University of Mnchester, Oxford Rod, Mnchester Ml 3 9PT 1 Mechnicl nd electrophysiologicl studies of guine-pig isolted trchelis hve been mde with the objectives of () identifying which of the P-drenoceptor subtypes medites the opening of plsmlemml K+-chnnels, (b) gining further insight into the properties of the novel, long-cting P- drenoceptor gonist, slmeterol nd (c) clrifying the role of K+-chnnel opening in mediting the relxnt ctions of gonists t P-drenoceptors. 2 Nordrenline (10 nm- 100 JM) cused concentrtion-dependent increse in the rte of beting of guine-pig isolted tri. The selective P1-drenoceptor blocking drug, CGP 20712A (100nM-10 JM) cused concentrtion-dependent ntgonism of nordrenline. The selective P2-drenoceptor blocking drug, ICI , lso produced concentrtion-dependent ntgonism of nordrenline, but only when used in concentrtions greter thn 300 nm. 3 Cromklim (100nM-l10 M), isoprenline (1-100nM), procterol (0.1-30nM), slbutmol (1 nm-i JlM), slmeterol (1-100 nm) nd theophylline (1IlM- 1 mm) ech cused concentrtiondependent suppression of the spontneous tone of guine-pig isolted trchelis. 4 ICI (10 nm- 1 JAM) ntgonized isoprenline, procterol nd slmeterol in suppressing the spontneous tone of the isolted trche. The ntgonism ws concentrtion-dependent. In contrst, ICI (1 JAM) ntgonized neither cromklim nor theophylline. CGP 20712A (up to 1 JAM) filed to ntgonize cromklim, isoprenline, procterol, slmeterol or theophylline. In trche treted with indomethcin (2.8 JM) nd crbchol (10 JAM), slmeterol (1 JAM) ntgonized the effects of isoprenline but not minophylline. 5 Intrcellulr electrophysiologicl recording from guine-pig isolted trchelis showed tht the relxnt effects of cromklim (10 JM), isoprenline (100 nm), procterol (10 nm) nd slbutmol (10 nm- 1 JAM) were ccompnied by the suppression of spontneous electricl slow wves nd by cellulr hyperpolriztion. In contrst, the relxnt effects of slmeterol (10 nm- 1 JiM) were not ccompnied by significnt cellulr hyperpolriztion. 6 CGP 20712A (1 AM) inhibited the hyperpolriztion but not the relxtion induced by isoprenline (100 nm). In contrst ICI (100 nm) inhibited both the hyperpolriztion nd the relxtion induced by isoprenline (100 nm). Neither CGP 20712A (1 JAM) nor ICI (100 nm) inhibited the hyperpolriztion induced by cromklim (10 JM). Slmeterol (1I jm) inhibited the hyperpolriztion induced by isoprenline (100 nm) but not tht induced by cromklim (1O JAM). 7 It is concluded tht ctivtion of either l- or P2-drenoceptors cn promote the opening of K+-chnnels in the trchelis plsmlemm. The poor bility of slmeterol to hyperpolrize trchelis muscle reflects neither its selectivity in ctivting P2-drenoceptors s opposed to P1-drenoceptors nor non-specific ction in stbilizing the cell membrne. Insted, it my reflect low intrinsic efficcy of the drug t P2-drenoceptors. The opening of plsmlemml K+-chnnels plys supportive rther thn crucil role in mediting the trchel relxnt ctions of gonists t P-drenoceptors. Keywords: Trchelis muscle; electrophysiology; P-drenoceptor subtypes; K+-chnnels; isoprenline; procterol; slbutmol; slmeterol; CGP 20712A; ICI Introduction In bovine nd cnine trchelis the mechno-inhibitory effects of gonists t P-drenoceptors (drenline, isoprenline nd procterol) re ssocited with hyperpolriztion of the muscle cells (Suzuki et l., 1976; Kirkptrick, 1981; Ito & Tjim, 1982; Cmeron et l., 1983; Fujiwr et l., 1988). In humn nd guine-pig trchelis, gonists t P-drenoceptors, such s isoprenline nd terbutline, suppress spontneous mechnicl tone, suppress electricl slow wves nd evoke cellulr hyperpolriztion (Allen et l., 1985; Hond et l., 1986; Hond & Tomit, 1987). Since the slow wve suppression nd the hyperpolriztion re inhibited by proprnolol (Kirkptrick, 1981; Ito & Tjim, 1982; Allen et l., 1985; Hond et l., 1986) it seems cler 'Author for correspondence. tht these electrophysiologicl chnges re medited by the ctivtion of P-drenoceptors. However, the drenoceptor subtype (Pi- or p2-) responsible for mediting these electrophysiologicl chnges hs yet to be determined. Electrotonic potentils induced by trnsmembrne current pulses re reduced in mplitude during the hyperpolriztion of irwys smooth muscle induced by gonists t P-drenoceptors (Ito & Tjim, 1982; Cmeron et l., 1983; Fujiwr et l., 1988). This my suggest tht the hyperpolriztion is consequence of n increse in membrne conductnce. Furthermore, since isoprenline-induced hyperpolriztion pproches the predicted K+ equilibrium potentil for trchelis muscle (Kirkptrick, 1981; Allen et l., 1985) it is likely tht this conductnce chnge involves the opening of membrne K+-chnnels. Direct support for this suggestion comes from the ptch clmp studies of Kume et l. (1989).

2 P-ADRENOCEPTOR SUBTYPES AND TRACHEALIS K+-CHANNELS 1141 Using cells freshly dispersed from rbbit trchelis smooth muscle, nd recording from cell-ttched ptches, these uthors showed tht the ddition of isoprenline (0.2 JM) to the bthing medium stimulted the opening of plsmlemml K+-chnnels of lrge conductnce. The bility of chrybdotoxin, in guine-pig trchelis nd humn bronchus, to ntgonize gonists t P-drenoceptors such s isoprenline nd slbutmol (Jones et l., 1990; Murry et l., 1991; Miur et l., 1992) suggests tht the opening of the lrge conductnce, C2'-dependent K+chnnel (BKc) is involved in the relxnt ctivity of the gonists t P-drenoceptors. However, whether BKc chnnel opening plys crucil or merely supportive role in mediting the mechno-inhibitory effects of the gonists t B-drenoceptors is currently mtter of ctive debte (Jones et l., 1990; Murry et l., 1991; Cook & Smll, 1991; 1992). In the present study we hve employed gonists (e.g. procterol nd slmeterol) nd ntgonists (e.g. CGP 20712A nd ICI ) showing high selectivity for Pl- or P2- drenoceptors in experiments designed to determine which of the P-drenoceptor subtypes medites plsmlemml K+chnnel opening in trchelis muscle. Other objectives of the present experiments were to shed more light on the role of K+-chnnel opening in mediting the mechno-inhibitory effects of the gonists t B-drenoceptors nd to chrcterize further the phrmcologicl properties of the novel, longcting P2-drenoceptor gonist, slmeterol (Bll et l., 1991; Dougll et l., 1991). The present electrophysiologicl nd mechnicl studies of guine-pig trchelis were crried out in prllel with studies of mechnicl ctivity nd 86Rb+ efflux in bovine trchelis (Chiu et l., 1993). Some of our findings hve been the subject of communictions to the British Phrmcologicl Society (Chiu et l., 1992; Cook et l., 1992; Cook & Smll, 1992). Methods Preprtion of isolted tissues Guine-pigs of either sex (weight g) were killed by stunning nd bleeding. The hert ws quickly excised from ech niml nd immersed in Krebs solution. Following removl of the pericrdium, ventriculr tissue ws dissected from the tri tking cre to preserve the integrity of the trioventriculr septum. Trchee were excised from the nimls, clened of dhering dipose nd connective tissue nd opened by cutting longitudinlly, dimetriclly opposite the trchelis. Tissue bth studies with isolted tri Isolted, spontneously-beting tri were set up in Krebs solution (37.5 C) under n initil resting tension of 1 g. Atril tension chnges were recorded with force displcement trnsducer coupled to Grss 7P1 premplifier mounted in Grss model 79D polygrph. The tension chnge signl ws used to trigger Grss model 7P44 tchogrph so tht recordings of tril tension nd rte of beting were mde simultneously. Once tril rte hd stbilized, test tissues were treted with CGP 20712A (100 nm, 1 JlM or 10 JM) or ICI (100 nm, 300 nm, 1 JAM or 10 JAM) for 20 min prior to constructing cumultive log concentrtion-chronotropic effect curve for nordrenline (10 nm-100 JAM) in the presence of one of the ntgonists t P-drenoceptors. Concentrtion increments for nordrenline were mde t 3 min intervls. Time-mtched control tissues were treted identiclly to test tissues except tht they were not exposed to CGP 20712A or ICI Tissue bth studies with isolted trche Smll segments of trche were set up for the isometric recording of tension chnges essentilly s described by Foster et l. (1983). At the onset of ech experiment, tissues were subjected to n imposed tension of 1.5 g. Approximtely 20 min lter, minophylline (1 mm) ws dded in order to determine the recorder pen position t zero tissue tone. The minophylline ws removed from the tissue (initil wsh followed by two further wshes t 10 min intervls) nd when spontneous tone subsequently becme mximl (40 min fter initil wsh), study of bronchodiltor drugs commenced. A cumultive log concentrtion-effect curve ginst spontneous tone ws constructed for one of the following: cromklim (100 nm- 10 JiM), isoprenline (1-100 nm), procterol ( nm), slbutmol (1 nm- IlJM) or theophylline (1 JAM-I mm) in ech test tissue. The concentrtion of isoprenline ws incresed t 4 min intervls, tht of procterol, slbutmol or theophylline t 5 min intervls nd tht of cromklim t 8 min intervls. Once the response to the highest concentrtion of the relxnt gonist hd equilibrted, minophylline ws dded to determine the recorder pen position t zero tissue tone. All relxnt responses were mesured in terms of the mximl response to minophylline. Following wshout of the relxnt gonist, CGP 20712A (100nM, IiJM or 10,UM) or ICI (10nM, 100 nm or 1 JAM) ws incubted with the tissue for period of 40 min. The log concentrtion-effect curve for the relxnt gonist ws then reconstructed in the presence of the ntgonist. Time-mtched control tissues were treted identiclly to test tissues but were not exposed to CGP 20712A or ICI Responses of the isolted trche to slmeterol required protrcted period (>30 min) to equilibrte nd such responses proved very difficult to reverse by repeted chnges of the bth fluid. Accordingly, slmeterol ws studied by modified protocol. Test tissues were preincubted (40min) with CGP 20712A (l00nm, IiJM or 101M) or ICI (10 nm, 100 nm or 1 JAM) for 40 min prior to the ppliction of single concentrtion of slmeterol (1 nm, 10 nm, 100 nm, 1 JAM, 10 JAM or 100 JAM). The relxnt response to slmeterol ws llowed to develop for period of 45 min before the ddition of minophylline (1 mm) in order to determine the pen position corresponding to zero tissue tone. Timemtched control tissues were treted similrly but were not exposed to CGP 20712A or ICI A further group of experiments ws performed in order to determine whether slmeterol might (s reported by Dougll et l., 1991) ct s prtil gonist t 112-drenoceptors. In these experiments, indomethcin (2.8 JAM) ws dded to the Krebs solution 20 min fter the initil setting up of the trchel segments. Following indomethcin-induced suppression of spontneous tissue tone (60 min), crbchol (10 JAM) ws dded to the bth fluid. Once the spsmogenic response to crbchol hd equilibrted (40 min), cumultive log concentrtion-relxtion curve for isoprenline (1 nm- 10 JAM) ws constructed, essentilly s described bove. Following isoprenline wshout (40 min), test tissues were incubted with slmeterol (1 JiM) for 45 min before reconstructing the log concentrtion-relxtion curve for isoprenline in the presence of the slmeterol. Time-mtched control tissues were treted similrly but were not exposed to slmeterol. Similr experiments were performed using minophylline (1 AM- 30 mm) insted of isoprenline. Intrcellulr electrophysiologicl recording from trchelis Simultneous recording of intrcellulr electricl ctivity nd mechnicl chnges of contiguous segment of trche ws performed with the technique nd tissue holder described by Dixon & Smll (1983). In brief, prt of the trchelis ws immobilized to permit long-term electricl recording while mechnicl ctivity of contiguous muscle bundles ws mesured under n initil, imposed tension of 1.5 g. The recording microelectrodes were filled with 3 M KCI nd were of resistnce greter thn 40MQ1.

3 1142 S.J. COOK et l. After implement of trchelis cell, severl minutes were llowed to elpse to check the stbility of the record of electricl ctivity. Trchel relxnt drugs (cromklim, 10ItM; isoprenline, 100 nm; procterol, 10 nm; slbutmol, 10 nm, 100 nm or 1 glm; slmeterol, 10 nm, 100 nm or 1 tlm) were then studied by their ddition to the Krebs solution superfusing the tissue. Whenever possible, the effects of relxnt drugs on the electricl ctivity of the impled cell were monitored for periods of 4 (isoprenline), 5 (slbutmol), 6 (procterol), 8 (cromklim) or 14 min (slmeterol). When the effects of the trchel relxnt drugs were exmined in the presence of modifying gent (CGP 20712A, ICI or slmeterol), the modifying gent ws llowed to preequilibrte with the tissue for t lest 20min. Drugs nd solutions/sttisticl nlysis of results Drug concentrtions re expressed in terms of the molr concentrtion of the ctive species. The following substnces were used: minophylline (BDH), CGP 20712A (2-hydroxy- 5-(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoro-methyl)-1H-imidzole-2-yl)-phenoxy)propyl)mino)ethoxy)-benzmide monomethnesulphonte; Cib-Geigy Phrmceuticls), cromklim (SmithKline Beechm Phrmceuticls), ICI (erythro - DL (7 - methylindn yloxyl) (isopropylmino - butn-2-ol hydrochloride); ICI Phrmceuticls), (-)- isoprenline hydrochloride (Sigm), (-)-nordrenline bitrtrte (Sigm), procterol (Sigm), slbutmol (Glxo Group Reserch), slmeterol (Glxo Group Reserch), theophylline (Sigm). Stock solutions of most drugs were prepred in twice-distilled wter. Tht of cromklim ws prepred in 70% ethnol. Isoprenline, nordrenline, slbutmol nd slmeterol stock solutions were prepred in 0.1 M HCI. Dilutions of isoprenline nd nordrenline were mde with distilled wter contining 0.57 mm scorbic cid. The Krebs solution used in the tissue bth experiments nd for the microelectrode recording of membrne potentil chnges hd the following composition (mm): N , K+ 5.9, C2+ 2.6, Mg2+ 1.2, Cl , HC03-25, S42-1.2, H2P nd glucose 1.1 This solution (ph 7.4) ws mintined t 37 C nd ws gssed with mixture of 95% 02:5% CO2. The significnce of differences between mens ws ssessed by use of two-tiled, unpired t test. The null hypothesis ws rejected when P <0.05. Results Studies with isolted tri Nordrenline (10 nm- 100 gm) cused concentrtiondependent increse in the rte of tril beting. CGP 20712A (100 nm- 10 JM) did not significntly lter the resting tril rte but ntgonized nordrenline in concentrtiondependent fshion. At concentrtion of 1 JAM, CGP 20712A cused rightwrd shift of the nordrenline log concentrtion-effect curve which ws greter thn 100 fold (Figure l). ICI (100 nm- 10 JAM) cused no chnge in the resting rte of tril beting. At concentrtions in the rnge nm, ICI filed to modify the chronotropic ction of nordrenline. However, t concentrtions in the rnge 1-10IM, ICI ntgonized nordrenline in concentrtion-dependent mnner (Figure lb). Studies of the mechnicl ctivity of isolted trchelis Isoprenline (1-100nM; Figure 2), procterol (0.1-30nM; Figure 2), slbutmol (1 nm- 1 JM) nd slmeterol (1-100nM; Figure 3) ech cused concentrtion-dependent suppression of the spontneous tone of guine-pig isolted trchelis. Ech of these gents ws ble to cuse full suppression of the spontneous tone of the trche. The rnk._ D Cu -o cn 4) L- CU E n -W 4-o CU._ CU CU log [Nordrenline] I I I I * * log [Nordrenline] Figure 1 The effects of () CGP 20712A nd (b) ICI ginst the chronotropic ction of nordrenline cting on guine-pig isolted tri. In ech pnel the bsciss scle indictes the molr concentrtion of nordrenline on loglo scle while the ordinte scle indictes tril rte in bets min '. In both pnels (@) indictes the time-mtched control log concentrtion-effect curve for nordrenline. () Shows log concentrtion-effect curves for nordrenline obtined in the presence of 100 nm (0), I gm (U) nd 10 jm (0) CGP 20712A; (b) shows log concentrtion-effect curves for nordrenline obtined in the presence of 100 nm (0), 300 nm (M), I JiM (0) nd 10 JM (A) ICI All dt points indicte the men ( ± s.e.men) of vlues obtined from t lest 6 tissues. The dt points shown on the extreme left in ech pnel indicte the resting tril rte prior to the dministrtion of nordrenline. order of relxnt potency for the gonists t P-drenoceptors ws procterol > isoprenline = slmeterol > slbutmol (Tble 1). Cromklim (100 nm- 10 gm) nd theophylline (1 JAM-I mm) lso produced concentrtion-dependent suppression of trchel tone. The mximl relxnt effect of theophylline ws equivlent to tht of minophylline, wheres the mximl effect of cromklim ws 80-90% of tht of minophylline (dt not shown). CGP 20712A (in concentrtion up to 1 JAM) did not lter the spontneous tone of the trche, nor did it ntgonize isoprenline, procterol, slbutmol or slmeterol. However, t concentrtion of 10JM, CGP 20712A cused minor (<four fold) ntgonism of procterol nd slbutmol (Figures 2 nd 3; Tble 1). CGP 20712A (1 JuM) did not modify the relxnt ctions of either cromklim or theophylline (Tble 1). Like CGP 20712A, ICI (10 nm- 1 FAM) did not lter the spontneous tone of the trche, nor did it (t concentrtion of 100 nm) ntgonize either cromklim or theophylline (Tble 1). However, ICI

4 13-ADRENOCEPTOR SUBTYPES AND TRACHEALIS K+-CHANNELS ,1 IC ii 0. log [lsoprenlinel 80-0 e E 60' so - C-W 40- x 20 - I A , I n~qw log [Slmeteroll b 100,1 x 0E I-R c 0 x log [Procterol] 80- x E * 60._2 *X i o I log [Isoprenlinel log [Procteroll I. I Figure 2 The effects of CGP 20712A nd ICI ginst the ctions of isoprenline nd procterol in suppressing the spontneous tone of guine-pig isolted trchelis muscle. In ech cse the bsciss scle indictes the logl0 molr concentrtion of isoprenline or procterol while the ordinte scle indictes relxtion expressed s percentge of the mximl relxtion induced by minophylline. In ll pnels (0) indictes the log concentrtioneffect curve of the gonist t P-drenoceptors s observed in the bsence of ny ntgonist. () nd (b) show log concentrtion-effect curves for the gonists t P-drenoceptors s observed in the presence of 100 nm (0), 1 JlM (U) or IO gm (0) CGP 20712A; (c) nd (d) show log concentrtion-effect curves for the gonist t frdrenoceptors s observed in the presence of 10nm (0), 100nM (U) or 1 gam (0) ICI In ech pnel ll dt points indicte the mens of vlues from t lest 6 issues ± s.e.men. o0 -ic log [Slmeteroll Figure 3 The effects of () CGP 20712A nd (b) ICI ginst the ction of slmeterol in suppressing the spontneous tone of guine-pig isolted trchelis muscle. In ech cse the bsciss scle indictes the logl0 molr concentrtion of slmeterol nd the ordinte scle indictes relxtion expressed s percentge of the mximl relxtion induced by minophylline. In both pnels the solid lines indicte the liner regression of response versus the log molr concentrtion of slmeterol while the broken lines indicte the upper nd lower 95% confidence limits of the regression lines. In () responses to slmeterol were obtined in the bsence (v) nd presence of 100 nm (0), 1 JLM (U) nd 10 JAM (0) CGP 20712A. In (b) responses to slmeterol were obtined in the bsence (0) nd presence of lonm (0), loonm (U) nd IAM (L) ICI ntgonized isoprenline, procterol, slbutmol nd slmeterol in concentrtion-dependent mnner (Figures 2 nd 3, Tble 1). In trchel segments treted with indomethcin (2.8pJM) nd crbchol (10 JAM), isoprenline (1 nm-10 JM) nd minophylline (1 gmm-30 mm) ech evoked concentrtiondependent relxtion. The incubtion of test tissues with slmeterol (1 gam) cused some reduction (pproximtely 20%) in the crbchol-induced tone. This reduction in tone did not render the tissues more sensitive to isoprenline. On the contrry, the log concentrtion-effect curve for isoprenline seen in the slmeterol-treted tissues ws shifted to the right (- log EC50 vlues for control nd test tissues were 6.62 ± 0.06 nd 5.59 ± 0.11 respectively; men ± s.e.men, n = 6) compred with tht seen in the time-mtched control tissues. Furthermore, the mximl response to isoprenline ws slightly reduced in the test tissues (dt not shown). Slmeterol (1 JAM) did not, however, ntgonize minophylline (- log EC,0 vlues for control nd test tissues nd 3.64 ± 0.10 respectively) in suppressing crbcholinduced contrction of the trche.

5 1144 S.J. COOK et l. Tble 1 The effects of CGP 20712A nd ICI ginst the ctions of cromklim, theophylline nd some gonists t P-drenoceptors in suppressing the spontneous tone of guine-pig isolted trchelis Agonist Cromklim Theophylline Isoprenline Procterol Slbutmol Slmeterol Agonist Cromklim Theophylline Isoprenline Procterol Slbutmol Slmeterol Time-mtched control (no ntgonist) 6.50 ± ± ( ) Time-mtched control (no ntgonist) ± ( ) 100 nm t 8.27 ± ± ± ( ) 10 nm tt 7.27 ± 0.07* 8.07 ± 0.07* 6.54 ± 0.22* 7.18* ( ) CGP 20712A 1 ILM 6.53 ± ± ± ± ± ( ) ICI nm 5.79 ± ± ± 0.12* 7.47 ± 0.25* 5.50 ± 0.32* 6.29* ( ) 10 JAM t 8.23 ± ± 0.08* 7.26 ± 0.09* 8.20 ( ) For most gents the dt indicte men (± s.e.men) vlues of - log10 ECio. For slmeterol the men - log10 EC50 nd its lower nd upper 95% confidence limits re shown. For ll gents, dt from t lest 6 tissues contribute to the men. tindictes prmeter not mesured. *indictes vlue significntly (P <0.05) different from tht observed in the bsence of the relevnt ntgonist. 1 JAM t 6.18 ± 0.12* 5.84 ± 0.05* 4.36 ± 0.15* 5.09* ( ) Intrcellulr electrophysiologicl recording from trchelis The ddition of either cromklim (10 pm) or isoprenline (100 nm; Figures 4 nd 6) to the Krebs solution superfusing the trchelis muscle reduced the mechnicl tone of the tissue, suppressed spontneous electricl slow wves nd cused hyperpolriztion of the impled cell. In this respect we hve been ble to confirm the results of our erlier studies (Allen et l., 1986). Procterol (10 nm) nd slbutmol (10 nm- 1 tlm) lso produced relxtion ccompnied by slow wve suppression nd by cellulr hyperpolriztion (Figure 4 nd Tble 2). As nticipted from the results of the tissue bth studies, slmeterol (10 nm- 1 jlm) produced relxnt effects, but these were very slow to develop compred with those of the other gonists t P-drenoceptors. Slmeterol (10 nm- 1 JLM) filed to evoke significnt hyperpolriztion of the trchelis cells (Figure 5 nd Tble 2). At concentrtions of 100nM nd 1 ylm this gent did, however cuse some reduction in slow wve mplitude or frequency (Figure 5b,c). Pretretment of the trchelis with slmeterol (1 lam) bolished the mechnicl tone of the tissue, filed to modify the hyperpolriztion induced by cromklim (10I1M) but reduced the bility of isoprenline (100 nm) to cuse hyperpolriztion of the impled cell (Tble 2). The ddition of CGP 20712A (1 AIM) to the Krebs solution superfusing the trchelis muscle modified neither the mechnicl tone of the tissue nor the electricl behviour of the impled cell. CGP 20712A (1 t4m) did not modify the relxnt ction of cromklim (10 M) nor the bility of cromklim to hyperpolrize the trchelis cells (Tble 2). CGP 20712A (1 lim) did not reduce the relxnt ction of isoprenline (100 nm) but significntly reduced the hyperpolriztion induced by isoprenline (Figure 6 nd Tble 2). ICI (100 nm) did not itself modify either the mechnicl tone of the tissue or the ongoing electricl behviour of the impled cell. ICI (100 nm) lso filed to modify electricl nd mechnicl responses of the trche to cromklim (Tble 2). In contrst ICI (100 nm) significntly reduced both the relxtion induced by isoprenline nd the bility of isoprenline to hyperpolrize the trchelis cells (Figure 6 nd Tble 2). -40 mrvj I_ I 30s -I _45 b lt mvi mi X _ 0.3 Figure 4 The effects of () isoprenline (100 nm) (b) slbutmol (I jlm) nd (c) procterol (10 nm) on the electricl nd mechnicl ctivity of guine-pig isolted trchelis. In ech row of recordings the upper trce represents membrne potentil chnges recorded from single cell while the lower trce represents tension chnges recorded from contiguous segment of trche. In ech row the left hnd pnel represents control ctivity recorded immeditely prior to the dministrtion of the gonist t,-drenoceptors. The centre pnels show ctivity recorded 45 s fter the dministrtion of the P-drenoceptor gonist. The right hnd pnels indicte ctivity recorded 4 (isoprenline), 5 (slbutmol) or 6 min (procterol) fter the dministrtion of the P-drenoceptor gonist.

6 P-ADRENOCEPTOR SUBTYPES AND TRACHEALIS K+-CHANNELS 1145 Tble 2 Some drug effects on the electricl nd mechnicl ctivity of guine-pig isolted trchelis muscle Agent (s) Isoprenline (100 nm) Isoprenline (100 nm) + CGP 20712A (I JiM) Isoprenline (100 nm) + ICI (100 nm) Isoprenline (100 nm) + slmeterol (I gam) Procterol (10 nm) Slbutmol (10 nm) Slbutmol (100 nm) Slbutmol (1 gam) Slmeterol (10 nm) Slmeterol (100 nm) Slmeterol (1 JiM) Cromklim (10 gm) Cromklim (10 JAM) + CGP 20712A (1 JM) Cromklim (10 JM) + ICI (100 nm) Cromklim (10 JM) + slmeterol (1 JM) Hyperpolriztion 13.6 ± 1.3* 5.8 ± 1.4*t ± 0.8t -4.1 ± 1.4*t 6.8 ± 2.4* -0.3 ± ± 1.5* 9.7 ± 1.6* 0.3 ± ± ± ± 3.7* 21.0 ± 2.6* 22.3 ± 0.9* 23.0 ± 2.7* Dt indicte the mens (± s.e.men) of vlues from the indicted (n) number of tissues. Mesurements of membrne potentil nd tension chnges were mde 4, 5, 8, 6 nd 14 min respectively fter the dministrtion of isoprenline, slbutmol, cromklim, procterol nd slmeterol. *indictes men vlue of membrne potentil chnge significntly (P <0.05) different from zero. tindictes significnt (P < 0.05) difference from the corresponding vlue for the gonist mesured in the bsence of ny ntgonist. (mv) Relxtion (g) 1.20 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.18 (n) mv r 0L. 130s 9 O- m\/-6 b -40 "AmMlW 2.0 I s I -45 m\rni 1.5T 0-6 Figure 5 The effects of slmeterol 10 nm (), 100 nm (b) nd I JAM (c) on the electricl nd mechnicl ctivity of guine-pig isolted trchelis. In ech row of recqrdings the upper trce represents membrne potentil chnges recorded from single cell while the lower trce represents tension chnges recorded from contiguous segment of trche. In ech row the left hnd pnel represents control ctivity recorded immeditely prior to the dministrtion of slmeterol. The subsequent pnels show ctivity recorded 1, 4 nd 14 min respectively fter the dministrtion of slmeterol. Note tht, t the outset, the cell illustrted in () lcked spontneous electricl slow wve ctivity. Note lso the filure of slmeterol (10 nm- 1 JAM) to cuse cellulr hyperpolriztion. -50 ##HAWAA AitIIIIjIJIwp mv ~~~~.,f,. O-_ Figure 6 The effects of CGP 20712A nd ICI on the electricl nd mechnicl responses of guine-pig isolted trchelis to isoprenline (100 nm). In ech row of recordings the upper trce represents membrne potentil chnges recorded from single cell while the lower trce represents tension chnges recorded from contiguous segment of trche. In ech row the left hnd pnel represents control ctivity recorded immeditely prior to the ddition of isoprenline (100 nm). The subsequent pnels show ctivity recorded 45 s nd 4 min, respectively, fter the ddition of isoprenline. () Shows the response to isoprenline recorded in the bsence of ny modifying gent; (b) nd (c) show responses to isoprenline (100 nm) recorded from tissues equilibrted with CGP 20712A (1 JM) nd ICI (100 nm) respectively. Note tht CGP 20712A inhibited the hyperpolriztion but not the relxtion induced by isoprenline. Note lso tht ICI ttenuted both the hyperpolriztion nd the relxtion induced by isoprenline.

7 1146 S.J. COOK et l. Discussion Properties of CGP 20712A nd ICI In the present study, CGP 20712A (100 nm- 10 glm) filed to modify the spontneous tone or spontneous electricl ctivity of guine-pig trchelis muscle. CGP 20712A (1 AtM) filed to inhibit the trchel relxnt ction of theophylline (Tble 1). Furthermore, the sme concentrtion of CGP 20712A filed to inhibit the trchel relxtion or the cellulr hyperpolriztion induced by the potssium chnnel opener, cromklim (Tbles 1 nd 2). Collectively, these findings suggest tht CGP 20712A (100 nm- 10 LM) does not directly inhibit the plsmlemml K+-chnnels controlling the excitbility of the trchelis cells nor the K+-chnnel (KKco) involved in the ction of cromklim. The fct tht CGP 20712A (100 nm- 1 lm) ntgonized nordrenline in incresing tril rte (Figure 1) without ntgonizing isoprenline, procterol, slbutmol or slmeterol in suppressing trchel tone (Figures 2 nd 3; Tble 1) is entirely consistent with reports (Dooley & Bittinger, 1984; Lemoine et l., 1985) tht CGP 20712A is n ntgonist hving very much higher (t lest 10,000 times) ffinity for l- thn for P2-drenoceptors. ICI (10nM-100nM) filed to modify the spontneous tone or the spontneous electricl ctivity of guinepig trchelis muscle. ICI (100 nm) filed to inhibit the trchel relxnt ction of theophylline (Tble 1). Furthermore, the sme concentrtion of ICI filed to inhibit the trchel relxtion or the cellulr hyperpolriztion induced by cromklim (Tbles 1 nd 2). Collectively, these observtions suggest tht, like CGP 20712A, ICI does not directly inhibit the plsmlemml K+-chnnels controlling the excitbility of the trchelis cells nor does it directly inhibit KKCO. Our observtion tht ICI ( nm) ntgonized isoprenline, procterol, slbutmol nd slmeterol in suppressing trchel tone without ntgonizing nordrenline in incresing tril rte (Figures 1, 2 nd 3; Tble 1) is entirely consistent with reports (O'Donnell & Wnstll, 1980; Bilski et l., 1983; Rimele et l., 1988) tht ICI is n ntgonist hving much higher ( times) ffinity for P2-drenoceptors thn for,i-drenoceptors. The results of our mechnicl studies in the tri nd trche suggest tht CGP 20712A (1 pm) produces mrked ntgonism t PI-drenoceptors with negligible concurrent ntgonism t P2-drenoceptors. Similrly, it my be suggested tht ICI (100 nm) produces mrked ntgonism t P2-drenoceptors with negligible concurrent ntgonism t P1-drenoceptors. Effects of slmeterol on the electricl nd mechnicl ctivity of trchelis muscle Erlier reports (Bll et l., 1991; Dougll et l., 1991) tht slmeterol exerts relxnt effects in trchelis muscle, tht such effects require protrcted (>20 min) period to rech equilibrium nd re poorly reversible by removl of the drug from the bthing medium hve been confirmed in the present study. The clim (Bll et l., 1991) tht slmeterol is n gonist tht is highly selective for P2- s opposed to l- drenoceptors is supported by the present findings tht the trchel relxnt ction of slmeterol ws ntgonized by ICI but not by CGP 20712A (Figure 3 nd Tble 1). Other gonists (i.e. slbutmol nd procterol) with reported selectivity for P2- s opposed to P1-drenoceptors hd relxnt effects in guine-pig trchelis tht were ssocited with hyperpolriztion of the trchelis cells (Figure 4 nd Tble 2). In view of this, we were surprised to observe (Figure 5 nd Tble 2) tht relxnt concentrtions of slmeterol, while cusing some inhibition of slow wve ctivity, filed significntly to modify the membrne potentil of the trchelis cells. We considered the possibility tht slmeterol might, in ddition to cting s n gonist t P2-drenoceptors, stbilize the trchelis cell membrne in non-specific mnner. A secondry ction of this kind might prevent expression of ny membrne hyperpolriztion induced by the ctivtion of P2-drenoceptors. However, slmeterol filed to reduce cromklim-induced hyperpolriztion (Tble 2) suggesting tht, t concentrtions cusing suppression of the spontneous tone of guine-pig trche, slmeterol does not stbilize the plsmlemm by non-specific mechnism. Severl groups of investigtors (e.g. Bll et l., 1991; Dougll et l., 1991; Wldeck & Kllstrom, 1991) hve reported tht slmeterol is prtil rther thn full gonist t P2-drenoceptors. Our finding tht slmeterol ntgonized the relxnt ction of isoprenline (but not tht of minophylline) in crbchol-treted trche supports this ide. It my lso explin our observtion tht slmeterol could inhibit the hyperpolriztion of trchelis cells induced by isoprenline (Tble 2). We therefore propose tht the filure of slmeterol to induce significnt hyperpolriztion of the trchelis cells is reflection of its low intrinsic efficcy (Dougll et l., 1991) t P2-drenoceptors. Subtypes of P-drenoceptor nd the ctivtion of plsmlemml K+ -chnnels Electrophysiologicl studies of guine-pig trchelis hve shown tht the relxnt ction of isoprenline is ccompnied by cellulr hyperpolriztion (Allen et l., 1985; Hond et l., 1986) wheres no such membrne potentil chnge ccompnies the relxnt ction of slmeterol (Cook & Smll, 1992; Figure 5). In view of the high selectivity of slmeterol s n gonist t P2- rther thn,i-drenoceptors (Bll et l., 1991) nd the filure of slmeterol to evoke hyperpolriztion of trchelis muscle, Cook & Smll (1992) proposed tht ctivtion of Pi- but not P2-drenoceptors might promote the opening of plsmlemml K+-chnnels. However, severl observtions now suggest tht this hypothesis is untenble. Procterol, slbutmol nd terbutline re ll gonists selective for P2- s opposed to PI-drenoceptors. Procterol cuses hyperpolriztion of trchelis muscle from the guine-pig (Figure 4) nd the dog (Fujiwr et l., 1988). Terbutline (Hond et l., 1986) nd slbutmol (Figure 4) both induce hyperpolriztion of guine-pig trchelis muscle. Furthermore, s discussed bove, the filure of slmeterol significntly to hyperpolrize guine-pig trchelis my be ttributed not to its gret selectivity in ctivting P2- s opposed to,i-drenoceptors, but insted to its low intrinsic efficcy t P2-drenoceptors. Therefore, lthough slmeterol fils to hyperpolrize trchelis cells, the bility of procterol, slbutmol nd terbutline to induce cellulr hyperpolriztion suggests tht the ctivtion of P2- drenoceptors cn promote plsmlemml K+-chnnel opening. This suggestion receives strong support from our findings (Figure 6, Tble 2) tht ICI (100 nm) blted the trchel hyperpolriztion induced by isoprenline. Isoprenline-induced hyperpolriztion of guine-pig trchelis ws lso significntly ttenuted by CGP 20712A (1 ELM; Figure 6; Tble 2) suggesting tht the ctivtion of P1-drenoceptors cn lso promote K+-chnnel opening in the plsmlemm. We thus conclude tht the ctivtion of either,- or P2-drenoceptors cn promote K+-chnnel opening in trchelis muscle. This conclusion receives strong support from the results of our studies of 86Rb+ efflux from bovine trchelis (Chiu et l., 1993). Role ofplsmlemml K+-chnnel opening in mediting the trchel relxtion induced by gonists t P-drenoceptors In guine-pig trchelis (Jones et l., 1990; Murry et l., 1991) nd humn bronchus (Miur et l., 1992) chrybdotoxin inhibits the relxnt effects of gonists t P-dreno-

8 P-ADRENOCEPTOR SUBTYPES AND TRACHEALIS K+-CHANNELS 1147 ceptors such s isoprenline nd slbutmol. Since chrybdotoxin hs been reported to inhibit BKc. chnnels in irwys smooth muscle (Green et l., 1991; Murry et l., 1991), these findings suggest tht the opening of BKc chnnels contributes to the relxnt ctivity of the gonists t P- drenoceptors. It is commonly ssumed tht BKcI chnnel opening cuses plsmlemml hyperpolriztion nd therefore cuses relxtion by inhibiting C2" influx through voltge-operted chnnels. However, number of findings now suggest tht such mechnism cnnot ply crucil role in mediting the mechno-inhibition induced by the gonists t P-drenoceptors. In guine-pig trchelis, for exmple, low concentrtions of isoprenline evoke relxtion without cusing hyperpolriztion (Allen et l., 1985). In the sme tissue, slmeterol is cpble of fully suppressing spontneous tone without cusing significnt chnge in membrne potentil (Cook & Smll, 1992; Figure 5 nd Tble 2). The K+chnnel inhibitor, procine, cn bolish the trchelis muscle hyperpolriztion induced by isoprenline without mrkedly ffecting its relxnt ctivity (Allen et l., 1985). Furthermore, CGP 20712A, n ntgonist selective for P1-drenoceptors, cn mrkedly ttenute the hyperpolriztion but not the relxtion of guine-pig trchelis induced by isoprenline (Cook et l., 1992; Figures 2 nd 6 nd Tble 2). The relxnt ctivity of isoprenline nd slbutmol in trchelis is lso lrgely retined when the tissue is bthed by K+-rich ( mm) medium of osmollity similr to tht of Krebs solution (Kumr, 1978; Allen et l., 1985; Smll et l., 1989; Cook & Smll, 1991). In this sitution the K+ equilibrium potentil nd the resting membrne potentil re so closely pposed tht K+-chnnel opening cn cuse very little hyperpolriztion. Accordingly, gonists t P- drenoceptors cnnot be expected to cuse hyperpolriztion sufficient to ensure the closure of voltge-operted C2+chnnels nd hence the inhibition of C2+ influx. Unless K+-chnnel opening cn evoke relxtion by mechnisms not depending on hyperpolriztion of the plsmlemm, we cn conclude tht K+-chnnel opening induced by the gonists t P-drenoceptors plys merely supportive nd not crucil role in mediting their mechno-inhibitory effects. S.J.C. ws supported by Glxo Group Reserch nd P.C. by the Medicl Reserch Council, R.C.S., R.W.F. nd S.J.D. were supported by the Ntionl Asthm Cmpign. CGP 20712A, cromklim, ICI , slbutmol nd slmeterol were gifts from their respective mnufcturers. References ALLEN, S.L., BEECH, D.J., FOSTER, R.W., MORGAN, G.P. & SMALL, R.C. (1985). Electrophysiologicl nd other spects of the relxnt ction of isoprenline in guine-pig isolted trchelis. Br. J. Phrmcol., 86, ALLEN, S.L., BOYLE, J.P., CORTIJO, J., FOSTER, R.W., MORGAN, G.P. & SMALL, R.C. (1986). Electricl nd mechnicl effects of BRL in guine-pig isolted trchelis. Br. J. Phrmcol., 89, BALL, D.I., BRITTAIN, R.T., COLEMAN, R.A., DENYER, L.H., JACK, D., JOHNSON, M., LUNTS, L.H.C., NIALS, A.T., SHELDRICK, K.E. & SKIDMORE, I.F. (1991). Slmeterol, novel, long-cting P2- drenoceptor gonist: chrcteriztion of phrmcologicl ctivity in vitro nd in vivo. Br. J. Phrmcol., 104, BILSKI, A.J., HALLIDAY, S.E., FITZGERALD, J.D. & WALE, J.L. (1983). The phrmcology of p2-selective drenoceptor ntgonist (ICI 118,551). J. Crdiovsc. Phrmcol., 5, CAMERON, A.R., JOHNSTON, C.F., KIRKPATRICK, C.T. & KIRKPAT- RICK, M.C.A. (1983). The quest for the inhibitory neurotrnsmitter in bovine trchel smooth muscle. J. Exp. Physiol., 68, CHIU, P., COOK, S.J., BERRY, J.L., CARPENTER, J.R., DOWNING, S.J., SMALL, A.M. & SMALL, R.C. (1992). Activtion of Pl- or P2- drenoceptors opens plsmlemml K+-chnnels in irwys smooth muscle: evidence from 86Rb+ efflux studies in bovine trchelis. Br. J. Phrmcol., 107, 250P. CHIU, P., COOK, S.J., SMALL, R.C., BERRY, J.L., CARPENTER, J.R., DOWNING, J.S., FOSTER, R.W., MILLER, A.J. & SMALL, A.M. (1993). P-Adrenoceptor subtypes nd the opening of plsmlemml K+-chnnels in bovine trchelis muscle: studies of mechnicl ctivity nd ion fluxes. Br. J. Phrmcol., 109, COOK, S.J. & SMALL, R.C. (1991). K+-rich medi nd the relxnt ctions of bronchodiltor drugs in guine-pig trchelis. Br. J. Phrmcol., 104, 401P. COOK, S.J. & SMALL, R.C. (1992). Role of K+-chnnel opening in slmeterol-induced relxtion of trchelis muscle. Br. J. Phrmcol., 106, 12P. COOK, S.J., CHIU, P., BERRY, J.L., CARPENTER, J.R., DOWNING, S.J. & SMALL, R.C. (1992). Activtion of P,- or P2-drenoceptors opens plsmlemml K+-chnnels in irwys smooth muscle: evidence from mechnicl nd electrophysiologicl studies in guine-pig trchelis. Br. J. Phrmcol., 107, 379P. DIXON, J.S. & SMALL, R.C. (1983). Evidence of poor conduction of muscle excittion in the longitudinl xis of guine-pig isolted trche. Br. J. Phrmcol., 79, DOOLEY, D.J. & BITTINGER, H. (1984). CGP 20712A: selective P,-drenoceptor ntgonist nd useful tool for quntitting Plnd P2-drenoceptors. In Proceedings of the 19th Interntionl Congress of Phrmcology. p London: McMilln. DOUGALL, I.G., HARPER, D., JACKSON, D.M. & LEFF, P. (1991). Estimtion of the efficcy nd ffinity of the P2-drenoceptor gonist slmeterol in guine-pig trche. Br. J. Phrmcol., 104, FOSTER, R.W., SMALL, R.C. & WESTON, A.H. (1983). Evidence tht the spsmogenic ction of tetrethylmmonium in guine-pig trchelis is both direct nd dependent on the cellulr influx of C2+ ion. Br. J. Phrmcol., 79, FUJIWARA, T., SUMIMOTO, K., ITOH, T. & KURIYAMA, H. (1988). Relxing ctions of procterol, bet2-drenoceptor stimulnt, on smooth muscle cells of the dog trche. Br. J. Phrmcol., 93, GREEN, K.A., FOSTER, R.W. & SMALL, R.C. (1991). A ptch-clmp study of K+-chnnel ctivity in bovine isolted trchel smooth muscle cells. Br. J. Phrmcol., 102, HONDA, K., SATAKE, T., TAKAGI, K. & TOMITA, T. (1986). Effects of relxnts on electricl nd mechnicl ctivities in the guinepig trchel muscle. Br. J. Phrmcol., 87, HONDA, K. & TOMITA, T. (1987). Electricl ctivity in isolted humn trchel smooth muscle. Jpn. J. Physiol., 37, ITO, Y. & TAJIMA, K. (1982). Dul effects of ctecholmines on prend postjunctionl membrnes in the dog trche. Br. J. Phrmcol., 75, JONES, T.R., CHARETTE, L., GARCIA, M.L. & KACZOROWSKI, G.J. (1990). Selective inhibition of relxtion of guine-pig trche by chrybdotoxin, potent C2"-ctivted K+-chnnel inhibitor. J. Phrmcol. Exp. Ther., 255, KIRKPATRICK, C.T. (1981). Trcheobronchil smooth muscle. In Smooth Muscle: An Assessment of Current Knowledge. ed. Bulbring, E., Brding, A.F., Jones, A.W. & Tomit, T. pp London: Edwrd Arnold. KUMAR, M.A. (1978). The bsis of bet drenergic bronchodiltion. J. Phrmcol. Exp. Ther., 206, KUME, H., TAKAI, A., TOKUNO, H. & TOMITA, T. (1989). Regultion of C2"-dependent K'-chnnel ctivity in trchel myocytes by phosphoryltion. Nture, 341, LEMOINE, H., EHLE, B. & KAUMANN, A.J. (1985). On the prticiption of Pl- nd P2-drenoceptors in the relxtion of clf trche cused by (-)-nordrenline, (-)-drenline nd (±)-fenoterol. Nunyn-Schmied. Arch. Phrmcol., 329, R81. MIURA, M., BELVISI, M.G., STRETTON, C.D., YACOUB, M. & BARNES, P.J. (1992). Role of potssium chnnels in bronchodiltor responses in humn irwys. Am. Rev. Dis., 146, MURRAY, M.A., BERRY, J.L., COOK, S.J., FOSTER, R.W., GREEN, K.A. & SMALL, R.C. (1991). Guine-pig isolted trchelis: the effects of chrybdotoxin on mechnicl ctivity, membrne potentil chnges nd the ctivity of plsmlemml K+-chnnels. Br. J. Phrmcol., 103,

9 1148 S.J. COOK et l. O'DONNELL, S.R. & WANSTALL, J.C. (1980). Evidence tht ICI is potent, highly bet2-seleictive drenoceptor ntgonist nd cn be used to chrcterize bet-drenoceptor popultions in tissues. Life Sci., 27, RIMELE, T.J., HENRY, D.E., GIESA, F.R., BUCKLEY, S.K., GEIGER, G., HEASLIP, R.J., LEE, D.K.H. & GRIMES, D. (1988). Comprison of the P-drenoceptor ffinity nd selectivity of cetmolol, tenolol, betxolol nd ICI J. Crdiovsc. Phrmcol., 12, SMALL, R.C., BOYLE, J.P., DUTY, S., ELLIOTT, K.R.F., FOSTER, R.W. & WATT, A.J. (1989). Anlysis of the relxnt effects of AH in guine-pig isolted trchelis. Br. J. Phrmcol., 97, SUZUKI, H., MORITA, K. & KURIYAMA, H. (1976). Innervtion nd properties of the smooth muscle of the dog trche. Jpn. J. Physiol., 26, WALDECK, B. & KALLSTROM, B.L. (1991). Slmeterol inhibits the relxing effect of formoterol on guine-pig trchel smooth muscle: evidence for prtil gonism. Br. J. Phrmcol., 104, 402P. (Received December 7, 1992 Revised Mrch 4, 1993 Accepted Mrch 30, 1993)