ejp ELSEVIER European Journal of Pharmacology 284 (1995)

Transcription

1 ejp ELSEVIER Eurpean Jurnal f Pharmaclgy 284 (1995) Characterizatin f the psitive and negative intrpic effects f acetylchline in the human mycardium Xia Y. Du a, Regien G. Schemaker a, Egbert Bs b, Pramd R. Saxena a,. a Department f Pharmaclgy, Cardivascular Research Institute 'CEUR" Faculty f Medicine and Health Sciences, Erasmus University Rtterdam, P.. Bx 1738, 3000 DR Rtterdam, Netherlands b Department f Thracic Surgery, Cardivascular Research Institute 'CEUR', Faculty f Medicine and Health Sciences, Erasmus University Rtterdam, P.. Bx 1738, 3000 DR Rtterdam, Netherlands Received 9 February 1995; revised 20 April 1995; accepted 20 June 1995 Abstract In the human islated mycardium, acetylchline (10-9 t 10-3 M) elicited a biphasic intrpic effect (a decrease in the lwer and an increase in the higher cncentratin range) in atrial and a psitive intrpic effect in ventricular trabeculae. Hwever, under cnditins f raised cntractility achieved by expsure t nradrenaline (10-5 M), nly negative intrpic effects were bserved in bth atria and ventricles. Atrpine (10-6 M), but nt prpranll (10-6 M), antagnized bth psitive and negative intrpic effects f acetylchline, thus shwing that the respnses were mediated by muscarinic acetylchline receptrs. The use f subtype selective muscarinic receptr antagnists (10-7 t 10-5 M), pirenzepine (M 1 > M 3 > M2), AF-DX 116 ( -({2-[(diethy amin )-methy ]- -piperidy }acety )-5 -dihydr -6H-pyrid [2 3-b][ 4]benz diazepine-6- ne base; M 2 > M 1 > M 3) and HHSiD (p-flurhexahydr-siladifenidl hydrchlride; M 3 >_ M 1 >> M 2) revealed that the negative intrpic effect f acetylchline in atrial as well as the psitive intrpic effect in ventricular trabeculae were best antagnized by AF-DX 116 and nt by pirenzepine, suggesting the invlvement f the muscarinic M 2 receptr subtype, pssibly linked t different secnd messenger systems. n the ther hand, the psitive intrpic effect f acetylchline (10-6 t 10-3 M) in the atrial tissue, bserved nly in preparatin with depressed cntractility, was nt effectively antagnized by either AF-DX 116 r HHSiD, but was significantly reduced by pirenzepine. Furthermre, the selective muscarinic M 1 receptr agnist McN-A-343 (4-(m-chlrphenylcarbamylxy)-2-butynyltrimethyl ammnium chlride; 10-9 t 10-3 M), which failed t significantly change the baseline cntractility in either atrial r ventricular trabeculae, prduced a psitive intrpic effect in atrial preparatins when cntractility had been depressed by prir treatment with acetylchline (10-9 t 10-7 M). This effect f McN-A-343 was effectively antagnized by pirenzepine (10-5 M). These data shw that, besides the muscarinic M 2 receptr mediating bth negative (atria) and psitive (ventricle) intrpic effects, muscarinic M 1 receptrs, capable f reversing depressed atrial cntractility, are present in the human heart. Keywrds: Acetylchline; Atrium AF-DX 116, human; HHSiD (p-flurhexahydr-siladifenidl hydrchlride); McN-A-343; Muscarinic receptr; Mycardial cntractility; Pirenzepine; Ventricle I. Intrductin It is well knwn that acetylchline causes negative intrpic and chrntrpic effects in the mammalian heart, including humans (Brdde et al., 1992; Caulfield, 1993; B6hm et al., 1994; Landzberg et al., 1994). In additin, studies have shwn that acetylchline can * Crrespnding authr. Tel. (31) (0) /47, fax (31) (0) , SAXENA@FARMA.FGG.EUR.NL. elicit a psitive intrpic respnse in islated cardiac tissue f sme species (Endh and Blinks, 1984; Tajima et al., 1987; Eglen et al., 1988). In ur previus study in the human islated mycardium, we reprted that acetylchline elicited a biphasic respnse (an initial decrease fllwed by an increase in cntractility back t baseline values) in the atrial trabeculae, whereas nly a psitive intrpic effect was nticed in the ventricular trabeculae (Duet al., 1994). This latter effect was nt bserved earlier (Jakb et al., 1989; Deightn et al., 1990; B6hm et al., 1994) /95/$ Elsevier Science B.V. All rights reserved SSDI (95)

2 120 X. Y. Duet al. / Eurpean Jurnal f Pharmaclgy 284 (1995) Five muscarinic acetylchline receptr subtype genes have been clned and expressed, but nly muscarinic M 2 receptrs have been detected in the human heart (Maeda et al., 1988; Frd et al., 1992; Caulfield, 1993). Crrespndingly, the respnses t muscarinic receptr agnists in the cardiac muscle appear t be mediated by the activatin f well-characterized muscarinic M 2 receptrs, which are cupled t different signal transductin pathways (Schimerlik, 1989). Hwever, the cardiac effects f muscarinic receptr stimulatin are nt yet fully understd and evidence is emerging that, besides the muscarinic M 2 receptr, ther muscarinic receptr subtypes are als expressed in the heart. Fr example, the muscarinic M 1 receptr seems t mediate psitive intrpic effects in cells islated frm adult guinea-pig ventricles (Gall et al., 1993) as well as the increase in autmaticity in canine Purkinje fibres (Rsen et al., 1990). Furthermre, it has been reprted that lw dses f pirenzepine ( < 3 rag) cause bradycardia that cincides with the blckade f muscarinic M1 receptrs in humans (Pitschner and Wellstein, 1988). Thus, it is suggested that althugh the predminant ppulatin f muscarinic receptrs at pstsynaptic sites in the heart is f the M 2 subtype, a small ppulatin f M 1 subtype is als present (Watsn et al., 1983; Evans et al., 1985). The purpse f the present study was t characterize the receptrs invlved in the bserved intrpic effects f acetylchline in the human cardiac tissue (Duet al., 1994) by using relatively selective antagnists at the muscarinic M1, M 2 r M 3 receptrs (Dds et al., 1987) as well as a selective agnist at the muscarinic M 1 receptr. A part f this investigatin has been presented at the last winter meeting f the British Pharmaclgical Sciety (Du et al., 1995). 2. Material and methds 2.1. Preparatins Right atrial and left ventricular trabeculae were btained frm 61 heart beating rgan dnrs (43 males, 18 females aged 2-55 years), wh died f nn-cardiac disrders (36 cerebrvascular accident, 20 plytrauma, 5 hypxia) less than 24 h befre the tissue was brught t the labratry. The hearts were kindly prvided by the Rtterdam Heart Valve Bank (Bi Implant Services Fundatin/Eurtransplant Fundatin) after remval f the artic and pulmnary valves fr hmgraft valve implantatin. The hearts were stred at 0-4 C in a sterile rgan prtecting slutin (UW, Eurcllins, r HTK-Brettschneider, see Pleg et al., 1992) immediately fllwing circulatry arrest. After excisin, tissue samples were placed in ice-chilled xygenated Krebs buffer (cmpsitin in Mm: NaC1 118, KCI 4.7, CaC12 2.5, MgS 4 1.2, NaHC 3 25, KHP and glucse 8.3) and atrial and ventricular trabeculae (< 1 mm thickness) were carefully dissected free. The trabeculae were munted in rgan baths cntaining Krebs buffer (37 C, gassed with 95% 0 2 and 5% C 2) and paced at 1 Hz using electrical field stimulatin (3 ms, vltage 20% abve threshld). The develped tensin was recrded using ismetric Harvard transducers and Gerz flatbed recrders (Schemaker et al., 1993; Duet al., 1994). Based n preliminary experiments, the chsen ptimal resting lad, yielding the highest develped tensin, was 7 mg and 19 mg fr atrial and ventricular trabeculae, respectively Experimental prtcl After stabilizatin, the resting tensin and baseline cntractile frce were measured. A cncentratin-respnse curve fr nradrenaline was btained t check the viability f the tissues (Schemaker et al., 1993; Du et al., 1994). Tissues with less than 25 mg respnse t 10-5 M nradrenaline were excluded frm further analysis. After washing (6 times) and stabilizatin, 10-6 M physstigmine was added t the Krebs buffer in experiments invlving acetylchline t prevent its rapid degradatin by chlinesterase (see Chatnnet and Lckridge, 1989). ne cumulative cncentratin-respnse curve fr acetylchline (10-9 t 10-3 M) was cnstructed in each atrial and ventricular preparatin, in the absence (cntrl) r after 30 min incubatin with ne f the fllwing antagnists: prpranll (10-6 M, nn-selective /3-adrenceptr antagnist), atrpine (10-6 M, nn-selective muscarinic receptr antagnist), pirenzepine (10-7, 10-6 r 10-5 M, muscarinic M 1 receptr antagnist, Hammer et al., 1980), AF-DX 116 (10-7, 10-6 r 10-5 M, muscarinic M e receptr antagnist, Giachetti et al., 1986) and HHSiD (10-6 r 10-5 M, muscarinic M 3 receptr antagnist, Lambrecht et al., 1989). In additin, the effects f acetylchline n trabeculae pre-stimulated with nradrenaline (10-5 M) were investigated either in the absence r presence f 10-6 M atrpine. Since nt all effects f acetylchline culd be fully explained after the use f antagnists, we als investigated the effects f a muscarinic M 1 receptr agnist, McN-A-343. ne cumulative cncentratin-respnse curve fr McN-A-343 (10-9 t 10-3 M) was btained in each atrial and ventricular preparatin. In additin, in the atrial tissue, cumulative cncentratin-respnse curves fr McN-A-343 (10-9 t 10-3 M) were als cnstructed fllwing a maximal negative intrpic effect induced by acetylchline (10-9 t >_ 10-6 M), in the absence r presence f pirenzepine (10-5 M). There was n apparent difference in the respnses t nradrenaline r acetylchline bserved in trabecu-

3 X.Y. Du et al. / Eurpean Jurnal f Pharmaclgy 284 (1995) lae btained frm subjects f different age r dying frm different causes. This is als true fr a number f ther subtances, including 5-hydrxytryptamine and histamine (Schemaker et al., 1993; Du et al., 1993,1994) Data presentatin and analysis Data are presented as means + S.E.M. Baseline values fr atrial and ventricular tissue were cmpared using an unpaired t-test. The effects f acetylchline and McN-A-343 in the absence r presence f varius antagnists were analyzed using an analysis f variance fr repeated measurements. Differences were regarded statistically significant, if P < Since the negative intrpic respnse in atrial and the psitive intrpic respnse in ventricular tissues reached their maximum, the curves were fitted t a fur-parameter lgistic functin (De Lean et al., 1978) t calculate, where applicable, apparent pd 2 values (negative lgarithm f the mlar cncentratin eliciting half-maximal effect). Apparent pk B values were derived frm pd 2 values, using the fllwing equatin: pk B =-Lg[B] + Lg{([Az]/[At])-1}, where [B] is the mlar cncentratin f antagnists and [AI] and [A2] represent mlar cncentratins f agnists eliciting half-maximal effect in the absence and presence f antagnists, respectively Chemicals used The chemicals used in the present study were: acetylchline chlride (Ciba, Breda, Netherlands), AF- DX 116 (ll-({2-[(diethylamin)-methyl]-l-piperidyl} acetyl)-5,11-dihydr-6 H-pyridl[ 2,3-b ][1,4]benzdiazepine-6-ne base; Thmae, Biberach/Riss, Germany), atrpine sulphate (Centrafarm, Etten-Leur, Netherlands), HHSiD (p-flurhexahydr-siladifenidl hydrchlride; Research Bichemicals Internatinal, Natick, MA, USA), McN-A-343 (4-(m-chlrphenylcarbamylxy)-2-butynyltrimethylammnium chlride; McNiel, Frt Washingtn, PA, USA), nradrenaline bitartrate (Sigma, St. Luis, M, USA), physstigmine salicylate (Sandz, Basel, Switzerland), pirenzepine base (Thmae, Biberach/Riss, Germany), prpranll hydrchlride (Imperial Chemical Industries, Macclesfield, UK). 3. Results 3.1. Viability f the tissues The baseline cntractile frce was significantly lwer in the atrial ( mg, n = 57) than in ventricular ( mg, n = 54) tissue. In bth tissues, nr- -~ m JQ E z. q.. - ~ C U. : -75 (J Right atrium -Lg [Aeh] M Left ventricle -Lg [Aeh] M Fig. 1. Cumulative cncentratin-respnse curves f acetylchline (Ach) n the baseline frce f cntractin, btained in the atrial (left panel) and ventricular (right panel) trabeculae in the absence (, n = 5 and 6, respectively) r presence f 10-6 M atrpine (A, n = 6 each) r prpranll (11, n = 7 and 6, respectively). adrenaline (10-8 M t 10-5 M) increased cntractile frce in a cncentratin-dependent manner. After expsure t 10-5 M nradrenaline, the frce f cntractin went up t mg (n = 57) and mg (n = 54) in the atrial and ventricular trabeculae, respectively Intrpic respnses t acetylchline in atrial and uentricular trabeculae Effect n the baseline cntractility The effect f acetylchline n the baseline atrial and ventricular cntractility as well as the influence f atrpine and prpranll are shwn in Fig. 1. In right atrial trabeculae, acetylchline exhibited a biphasic cntractile respnse cnsisting f an initial decrease fllwed by an increase. At lw cncentratins (10-9 t 10-7 M), acetylchline clearly decreased the baseline cntractile frce (maximum decrease frm baseline values: % at 10-7 M), but with higher cncentratins the cntractile frce started t increase twards baseline values (0 _+ 23% change frm baseline values at ]0-3 U). If after expsure t ]0-7 M acetylchline higher cncentratins were nt used, the cntractile frce remained depressed withut cming back twards baseline values. Hwever, when 10-4 M acetylchline was administrated withut the preceding part f the cncentratin-respnse curve, n psitive intrpic effect was bserved (data nt shwn). In cntrast t atria, acetylchline caused nly a psitive intrpic effect in left ventricular trabeculae. The maximum increase in cntractile frce (53 _+ 17% frm baseline values) was bserved at 10-3 M. Atrpine (]0-6 M) effectively antagnized bth the negative and psitive intrpic effects f acetylchline in atrial as well as the psitive intrpic effect in q

4 122 X. Y. Duet al. / Eurpean Jurnal f Pharmaclgy 284 (1995) z T- 1/I t- D. 1/I Right atrium \4\ \, 0 10 Left ventricle ~~_ ~ -Lg [Aehi M -Lg laehl M Fig. 2. Cumulative cncentratin-respnse curves f acetylchline (Ach) n frce f cntractin after pre-stimulatin with nradrenaline (NA; 10-5 M), btained in the atrial (left panel) and ventricular (right panel) trabeculae in the absence (, n = 7 and 6, respectively) r presence f atrpine (10-6 M; A, n = 7 and 5, respectively). ventricular trabeculae. Since in the presence f atrpine a cmplete cncentratin-respnse curve t acetylchline culd nt be cnstructed, pd 2 values f acetylchline were nt calculated. Hwever, frm Fig. 1 it can be seen that atrpine shifted the cncentratin-respnse curve t the right by mre than 3 and 4 lg units in atrial and ventricular trabeculae, respectively. Prpranll (10-6 M) did nt mdify the respnses t acetylchline. Effect n nradrenaline-stimulated cntractility In the presence f nradrenaline (10-5 M), acetylchline prduced nly a negative intrpic effect in bth atrial and ventricular trabeculae (Fig. 2). The reductin f the cntractile frce by 10-3 M acetylchline was frm t mg (86 _ 4%) in atrial trabeculae (n = 7) and frm t mg (39 + 5%) in ventricular trabeculae (n = 6). The pd 2 value fr acetylchline was (n = 5) and (n = 6) in the atrial and ventricular tissues, respectively. Atrpine shifted the cncentratin-respnse curve fr acetylchline t the right in bth atrial and ventricular tissue fllwing pre-stimulatin with 10-5 M nradrenaline (Fig. 2). The pd 2 values fr acetylchline were decreased in bth atrial ( ; n = 7) and ventricular ( ; n = 5) trabeculae Effect f the muscarinic receptr antagnists n acetylchline-induced intrpic respnses Right atrium Fig. 3 presents cncentratin-respnse curves t acetylchline in atrial trabeculae in the absence (cntrl) r presence f the three relatively selective muscarinic receptr antagnists, whereas the relevant parameters f these curves are summarized in Table 1. The muscarinic M t receptr antagnist pirenzepine (10-7, 10-6 r 10-5 M) did nt significantly change the negative intrpic effect f acetylchline (10-9 t 10-6 M), whereas the psitive intrpic effect f acetylchline (10-6 t 10-3 M) was significantly reduced by pirenzepine at the highest cncentratin (10-5 M; pk B culd nt be calculated). The muscarinic M e receptr antagnist AF-DX 116 did nt significantly affect the negative intrpic effect f acetylchline at 10-7 M, but 10-6 M AF-DX 116 prduced a parallel shift t the right f the acetylchline curve (apparent PKB: ; n = 8) and 10-5 M AF-DX 116 cmpletely ablished the negative intrpic effect. AF-DX 116 either did nt affect (10-7 and 10-6 M) r even seemed t increase (10-5 M) the psitive intrpic Table 1 Maximum negative (with apparent pd 2) and psitive intrpic effects in atrial and maximum psitive intrpic effect (with apparent pd 2) in ventricular trabeculae induced by acetylchline Antagnist Iz M Atrium Ventricle n Negative a pd 2 Psitive b n Psitive c pd 2 Cntrl : : : Pirenzepine ± : :15 ~ : :0.2 Pirenzepine :11 d : _+ 0.6 Pirenzepine : : :3 d 7 5: Cntrl ± : AF-DX : _ _ AF-DX :0.2 d :0.6 AF-DX d,e d,~ : :0.1 d Cntrl ± HHSiD : HHSiD _+ 28 d a Percent change frm baseline values by acetylchline (10-6 M r 10-5 M in the presence f the tw highest cncentratins f pirenzepine); b percent change by 10-3 M acetylchline, calculated as the difference frm the maximal negative effect (pd 2 nt calculable); c percent change frm baseline values by acetylchline (10-4 M r 10-3 M); d significantly different frm values in cntrl experiments run in parallel; e nly psitive intrpic respnse.

5 X.Y. Du et al. / Eurpean Jurnal f Pharmaclgy 284 (1995) Right atrium Left ventricle q) r" U) (U.Q E im q.. ~ C m p Pirenzepine 100 [HHSiD! 100 AF-DX 116 /i 5 ~ - -T////i -Lg lach] M s Cntrl -5 ~1.~ ~ --Ia-- 10 "sm -- ~' "s M e",m m. E k. q,. e- ra t = Pirenzepine i//~..s,0 II 25 AF-DX 116 I Cntrl 10.7 M --,-- 10"e M "s M Fig. 3. Cumulative cncentratin-respnse curves f acetylchline (Ach) n baseline frce f cntractin in the atrial trabeculae in the absence r presence f antagnists. Upper left panel: acetylchline alne (n = 16) r in the presence f pirenzepine (10-7, 10-6 and 10-5 M; n = 6, 8 and 6, respectively). Upper right panel: acetylchline alne (n = 16) r in the presence f AF-DX , 10-6 and 10-5 M; n = 6, 8 and 6, respectively). Lwer left panel: acetylchline alne (n = 10) r in the presence f HHSiD (10-6 and 10-5 M; n = 6 each). cmpnent f the effects f acetylchline. The preferential muscarinic M 3 receptr antagnist HHSiD (10-6 M) failed t mdify the effects f acetylchline, but its higher cncentratin (10-5 M) reduced the negative intrpic cmpnent (apparent pkb: 6.0 -t- 0.6; n = 6), withut affecting the psitive intrpic cmpnent. Left ventricle The effects f muscarinic receptr antagnists n acetylchline-induced psitive intrpic effects in ventricular trabeculae are shwn in Fig. 4 and Table 1. Pirenzepine did nt efficiently antagnize the respnses t acetylchline; nly at 10-5 M pirenzepine, the curve fr the acetylchline seemed t be slightly shifted t the right (apparent pkb: ; n = 6). n the ther hand, AF-DX 116 (10-6 and 10-5 M) caused a cncentratin-dependent antagnism f the respnses t acetylchline (apparent pkb: and 6.2 _+ 0.3, respectively; n = 6 each). HHSiD failed t mdify the respnses t acetylchline Effects f MeN-A-343 Baseline cntractility The effects f MeN-A-343 (10-9 t 10-3 M), a relatively selective muscarinic M 1 receptr agnist, n the baseline cntractility in the atrial and ventricular trabeculae are presented in Fig. 5. MeN-A-343 failed t significantly alter the frce f cntractin in either Fig. 4. Cumulative cncentratin-respnse curves f acetylchline (Ach) n baseline frce f cntractin in the ventricular trabeculae in the absence r presence f antagnists. Upper left panel: acetylchline alne (n = 14) r in the presence f pirenzepine (10-7, 10-6 and 10-5 M; n= 5, 4 and 7, respectively). Upper right panel: acetylchline alne (n = 16) r in the presence f AF-DX , 10-6 and 10-5 M; n = 6, 6 and 7, respectively). Lwer left panel: acetylchline alne (n = 10) r in the presence f HHSiD (10-6 and 10-5 M; n = 6 and 5, respectively). tissue; the values befre and after MeN-A-343 (10-3 M) were and mg in atrial trabeculae and and mg in ventricular trabeculae. Acetylchline-induced depressed cntractility in atrial trabeculae Fig. 6 presents examples f riginal tracings shwing the effects f acetylchline and MeN-A-343 in atrial Q~ c 25,n 4) (U.Q E 2 I C m -e- Atrium -'- Ventricle N -25 i i i i -Lg [McN-A-343] M Fig. 5. Cumulative cncentratin-respnse curves f McN-A-343 n baseline frce f cntractin, btained in atrial (e) and ventricular ( ) trabeculae (n = 6 each).

6 124 X.Y. Du et al. / Eurpean Jurnal f Pharmaclgy 284 (1995) trabeculae. Acetylchline (]0-7 M) prduced a sustained negative intrpic effect (panel A), which was reversed by higher cncentratins f acetylchline (> 10-6 M; panel B) as well as by McN-A-343 (> 10 8 M; panel C). Pre-treatment f the atrial trabeculae with pirenzepine (10-5 M) antagnized the psitive intrpic effect f McN-A-343 (panel D). The mean data btained in atrial trabeculae with McN-A-343 in the absence r presence f 10-5 M pirenzepine (after maximum negative intrpic effect was reached by lw cncentratins f acetylchline) are presented in Fig. 7. Acetylchline decreased the baseline cntractile frce frm 868 -t- 169 mg t 140 _+ 63 mg (-84_+ 5%) at 10 7 M (n = 5). McN-A-343 (10-9 t 10-3 M) increased the cntractile frce back t baseline values (902 _+ 170 mg at 10-3 M; n = 5) in a cncentratin-dependent way (PD2:6.75 _+ 0.42). Althugh pirenzepine (10-5 M) slightly shifted the acetylchline curve t the right, it strngly antagnized the McN-A-343-induced psitive intrpic effect. In the presence f pirenzepine, the frce f cntractin was nt changed by McN-A-343 (10-9 t 10-4 M). nly at 10-3 M, McN-A-343 increased the cntractility frm t mg, clse t baseline values 4) e- 4) (n t~ r, E k. v,- 4) ~ r. t- ~ Atrium,=%, i/ - -,9 /I \l\ U / -100, ~,,,,,, I -Lg [McN-A-3431 M Fig. 7. Cumulative cncentratin-respnse curves f McN-A-343 n the frce f cntractin in atria] trabeculae after depressin f cntractility by acety]chline (Ach; ]0-9 t 10-7 r 10 6 M), btained in the absence (; n = 5) r presence (e; n = 4) f pirenzepine (]0-5 M). (380 _+ 110 mg) (n = 4); the pd 2 value f McN-A-343 based n the last respnse was The apparent pk B value f pirenzepine against McN-A-343 was fund t be 8.57 _ (n = 4). A D Lg [McN-A-343] M Lg [McN-A-343] M 400 mg Fig. 6. Recrdings f frce f cntractin in fur right atrial trabeculae shwing the effects f acetylchline (Ach) in different cncentratins. Nte that the negative intrpic effect f acetylchline (10-7 M), which was maintained fr several min (panel A), was reversed by higher cncentratins f acetylchline (panel B) and McN-A-343 (panel C). The reversal f the negative intrpic effect f acetylchline by McN-A-343 was nt bserved in atrial trabeculae pretreated with pirenzepine (10-5 M) (panel D). 4. Discussin 4.1. Invlvement f the muscarinic receptrs in acetylchline-induced respnses The results frm the present and previus (Duet al., 1994) studies shw that in the human islated mycardium acetylchline elicits cmplex intrpic effects, cnsisting f a biphasic respnse (decrease fllwed by increase at high cncentratins) in the atrial baseline cntractility and nly an increase in the ventricular baseline cntractility. Hwever, in cnfrmity with previus bservatins (Jakb et al., 1989; Deightn et al., 1990; B6hm et al., 1994) in the isprenaline-augmented human islated mycardium, acetylchline decreased cntractility in bth atrial and ventricular preparatins after prir expsure t nradrenaline. The negative intrpic effect f muscarinic chlinergic agents (antagnized by atrpine) is well knwn, but their psitive intrpic actin, thugh reprted in sme animal species (rat atria, Imai and hta, 1982; guineapig papillary muscles, Krth and Kiihlkamp, 1987; guinea-pig atria, Eglen et al., 1988; chick ventricles, Tsuji et al., 1987), has nt been reprted in humans (see Jakb et al., 1989; Deightn et al., 1990; B6hm et al., 1994). The reasns fr this discrepancy are nt entirely clear. Hwever, in cntrast t ur investigatins perfrmed in nn-diseased hearts and with acetylchline in ventricular trabeculae, previus au-

7 X.Y. Duet al / Eurpean Jurnal f Pharmaclgy 284 (1995) thrs (Jakb et al., 1989; Deightn et al., 1990; B6hm et al., 1994) studied the effects f carbachl n papillary muscles btained frm hearts remved frm patients underging heart transplantatin surgery. The precise receptr mechanisms invlved in the psitive intrpic effects f muscarinic chlinergic agents are nt well understd. In the rat atria (Imai and hta, 1982), guinea-pig papillary muscle (Krth and Kiihlkamp, 1987) and chick ventricles (Tsuji et al., 1987), the psitive intrpic effect was antagnized by atrpine and, in the last tw tissues, it was als shwn that the effect, being unaffected by prir reserpinizatin (Krth and Kiihlkamp, 1987) r prpranll (Tsuji et al., 1987), was independent f endgenus catechlamines. In accrdance with these bservatins, we als fund that atrpine nt nly blcked the negative intrpic effect in the atria, but als the psitive intrpic respnses in bth atria and ventricles; these respnses remained unaltered after expsure t prpranll. Thus, the results shw that bth the negative and psitive intrpic effects f acetylchline in the human islated mycardium are mediated by muscarinic receptrs Subtypes f muscarinic receptrs invlved in acetylchline respnses Althugh, at present, highly selective antagnists at muscarinic receptrs are nt available, a cmbinatin f relatively selective antagnists can be emplyed t characterize the muscarinic receptr subtypes. In the present investigatin, we used three such antagnists having different selectivity prfiles, namely pirenzepine (M l>m 3>M2), AF-DX 116 (M 2>M I>M 3) and HHSiD (M 3 > M 1 >> M 2) (Dds et al., 1994). Hwever, it shuld be pinted ut that the affinity values f these relatively selective antagnists btained in the present experiments in the human mycardium are further prejudiced by the cmplex nature f acetylchline respnses. Invlvement f the muscarinic M 2 receptr subtype The present study shwed that AF-DX 116 mst effectively antagnized the atrial negative intrpic (Fig. 3) and ventricular psitive intrpic (Fig. 4) effects f acetylchline; the cncentratin-respnse curves were clearly shifted t the right. The calculated apparent pk B values f AF-DX 116 in the atria (6.7 +_ 0.4) and ventricles (6.7 _+ 0.6) against the negative and psitive intrpic respnses, respectively, t acetylchline reasnably match with its pk i values (7.14 _ and 7.18 _+ 0.06, respectively) in the tw tissues fr the displacement f [3H]N-methyl scplamine in guinea-pig membranes (Michel and Whiting, 1987). Thus, it appears that the muscarinic M 2 receptr subtype mediates bth the negative (in atria) and psitive (in ventricles) intrpic respnses. This cnclusin is in agreement with the demnstratin f the muscarinic M 2 receptr mrna and prtein in the human heart (Maeda et al., 1988; Caulfield, 1993). Mrever, it is nw well established that muscarinic M 2 receptrs are cupled t three signal transductin pathways (Schimerlik, 1989): (i) inhibitin f adenylyl cyclase t reduce camp (Fleming et al., 1987), (ii) pening f ptassium channels (Ray and MaCled, 1993), and (iii) hydrlysis f phsphatidylinsitl. The first tw may cntribute t the negative intrpic respnse assciated with muscarinic receptr stimulatin (Hanf et al., 1993), while the third may induce psitive intrpic respnses (Mizushima et al., 1987; Khl et al., 1990). Althugh the exact mlecular prcesses are unclear, it is believed that the tw ppsite functinal respnses are mediated via muscarinic M 2 receptrs cupled t different G-prteins and ccurring in different affinity states: a high affinity state assciated with inhibitin f adenylyl cyclase and a lw affinity state assciated with the phsphatidylinsitl breakdwn (Brwn and Brwn, 1984). A recent study cmparing muscarinic K channels in the human atrium and ventricle (Kumi and Wasserstrm, 1994) shwed that the acetylchline-induced K current in whle cell preparatins frm the tw tissues exhibited essentially similar characteristics, except with regard t the channel density. Acetylchline-induced K current in atria was apprximately 2-3 times higher than that in ventricles. Anther study n the cmparisn f cupling f muscarinic receptrs in guinea-pig atrial and ventricular mycardium shwed that atrial and ventricular receptrs were similar, but receptr cupling t cyclase inhibitin r phsphatidylinsitl hydrlysis was distinguishable (Wdcck et al., 1987). Thus, it is pssible that all three pathways perate t mediate the bserved tissue respnses, but their imprtance may vary in different tissues and/r cnditins. Based n the effect f acetylchline n the baseline cntractility (withut prir stimulatin with, fr example, nradrenaline), it wuld appear that the cupling f the rnuscarinic M 2 receptrs t adenylyl cyclase (negatively) and K channels may be mre imprtant in the atrial tissue, whereas in the ventricular tissue the muscarinic M 2 receptrs prbably cuple preferentially t phsphatidylinsitl hydrlysis. Hwever, it is pssible that the cupling can alter in different cnditins. Indeed, it is interesting t recall that in the human ventricular mycardium a cncentratin-dependent negative intrpic effect was seen after pre-stimulatin with sympathmimetic agents (Jakb et al., 1989; Landzberg et al., 1994; present results). Invlvement f the muscarinic M 1 receptr subtype Experiments with AF-DX 116 and HHSiD in atrial tissue shwed that the psitive intrpic effect f

8 126 X. Y. Duet al. / Eurpean Jurnal f Pharmaclgy 284 (1995) acetylchline, bserved with high cncentratins and nly when the atrial cntractility had been depressed, was nt attenuated and seemed t be ptentiated by these cmpunds, apparently due t the eliminatin f the negative intrpic effect. n the ther hand, pirenzepine seemed t attenuate this respnse, thereby accentuating the preceding negative intrpic effect (Fig. 3). It therefre appears that the acetylchline-induced increase in depressed cntractility is mediated by the muscarinic M 1 receptr subtype rather than the M 2 r M 3 subtype. Althugh HHSiD has been reprted t be a mre ptent antagnist against M 1 than M 2 receptrs (see Dds et al., 1994), this apparently des nt seem t hld true fr the human heart, where this cmpund blcked M 2 receptr mediated respnses (negative intrpism in atria and psitive intrpism in ventricles) mre effectively. The cnclusin that the muscarinic M 1 receptr subtype mediates acetylchline-induced increases in atrial cntractility is further substantiated by the resuits btained with McN-A-343, which in the radiligand binding assays has similar affinities fr the muscarinic M 1 and M 2 receptr subtypes, but in functinal assays, prbably due t differences in intrinsic efficacy and/r tissue receptr reserve, shws selectivity fr the muscarinic M 1 receptr subtype (Eglen et al., 1987). Indeed, McN-A-343 failed t decrease atrial r increase ventricular cntractility (Fig. 5), thus ruling ut the activatin f muscarinic M 2 receptr subtype in the human heart. This cmpund, hwever, mimicked acetylchline (higher cncentratins) in increasing atrial cntractility, nce this had been depressed by prir administratin f lw cncentratins f acetylchline (Fig. 6). The reversal f the negative intrpic effect f acetylchline by McN-A-343 cannt be due t a blckade f muscarinic M 2 receptrs, since the effect f McN-A-343 was antagnized by pirenzepine. The apparent pk B value f pirenzepine against McN-A-343 ( ) was similar t the binding affinity f the drug at the human clned muscarinic M t receptr (PKi: ; Drje et al., 1991), thus cnfirming the invlvement f muscarinic M 1 receptr subtype. Additinal evidence fr functinal atrial muscarinic M 1 receptrs cmes frm studies f Pitschner and Wellstein (1988), wh bserved that lw dses (< 3 mg) f pirenzepine decrease heart rate in human subjects. Similarly, the bradycardia caused by lw dses f atrpine in humans may als be related t blckade f muscarinic M 1 receptrs (Wellstein and Pitschner, 1988; present results), rather than central vagal stimulatin (Weiner, 1990). Kellar et al. (1985) examined the binding f [3H]acetylchline and suggested that mst f the muscarinic M E sites had a high affinity fr acetylchline, whereas the majrity f muscarinic M l sites had a lw affinity fr acetylchline. This is in agreement with ur bservatins that the psitive intrpic respnse in the atrial trabeculae was btained with high cncentratins f acetylchline (10-4 r 10-3 M). It is pssible that the muscarinic M 1 receptrs mediating increases in atrial cntractility are cupled t phsphatidylinsitl hydrlysis (Gall et al., 1993; Wess, 1993). In cnclusin, the present investigatin in the human islated mycardium shwed that: (i) in additin t the generally accepted negative intrpic effect, acetylchline als causes psitive intrpic effects at similar cncentratins in human ventricular trabeculae and these negative and psitive intrpic respnses are mediated by the muscarinic M 2 receptr subtype, pssibly having preferential cupling t adenylyl cyclase (negatively) and K channels in atria, and t phsphinsitl breakdwn in ventricles, (ii) in trabeculae prestimulated with nradrenaline, acetylchline elicited negative intrpic respnses in bth atrial and ventricular trabeculae, suggesting that the preferential cupling in the ventricles can be altered, and (iii) when atrial cntractility has already been depressed, high cncentratins f acetylchline as well as McN-A-343 can increase the atrial cntractility back twards baseline values via the muscarinic M 1 receptr subtype. 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