single smooth muscle cells from guinea-pig and rabbit jejunum

Transcription

1 BrRish Journl of Phrmology (1995) B 1995 Stokton Press All rights reserved 71188/95 $12. Effet of UK84149 on voltgetivted lium urrents of single smooth musle ells from guinepig nd rbbit jejunum nd rbbit oronry rtery 'H. Zhng & T.B. Bolton Deprtment of Phrmology & linil Phrmology, St. George's Hospitl Medil Shool, rnmer Terre, London SW17 ORE 1 Smooth musle ells of the longitudinl musle of the guinepig nd the rbbit jejunum nd rbbit oronry rtery were dispersed by enzyme tretment nd reordings of membrne urrent were mde in the stndrd whole ell voltgelmp mode nd by perforted'pth tehnique. The effet of UK84149 on the voltgedependent lium inwrd urrent of single isolted smooth musle ws studied t room temperture. 2 When inwrd nd outwrd urrents were evoked by stepping to mv or + 1 mv from 8 mv every 2 s, UK84149 redued the voltgedependent inwrd urrent in guinepig jejunum smooth musle ells with ID5 of 2.3 JLM, nd lso redued voltgedependent outwrd urrent with ID5 of 7.2;LM. The effet of UK84149 ws diffiult to wsh out. 3 The intivtion of the inwrd urrent ws studied by holding t vrious potentils for 1 s before evoking inwrd urrent t test potentil of mv. The voltgeintivtion urve ws shifted to the left by 3 JLM UK The voltge t whih urrent ws 5% vilble (Vh) ws hnged from 37 ± 2 mv to 5 mv ± 2.2 (n = 6, mens ± s.e.men). 4 When inwrd urrent ws evoked by stepping to mv from 8 mv for 5 ms, every 2s, nirdipine immeditely, nd verpmil nd UK449 slowly, redued the pek mplitude. Inhibition by the ltter two drugs depended on the number of stimultions pplied. So, like verpmil, UK84149 hd usedependent tion. 5 The effet of UK84149 on inwrd urrent from rbbit jejunum nd oronry rtery smooth musle ells ws ompred with the effet of nirdipine nd D6. When inwrd urrent ws evoked by stepping to mv from 8 mv every 2 s, UK84149 inhibited inwrd urrents from both types of smooth ell with n ID5o of 1.7 pm for jejunum nd n ID5o of 3.2 pm for oronry rtery. ID5 vlues of D6 were 8.1 pm for jejunum nd 3.9 ftm for oronry rtery. ID5 vlues of nirdipine were 24 nm for jejunum nd 12 nm for oronry rtery. 6 The results of the present studies indite tht UK84149 hs liumntgonist tion tht n explin its inhibitory tion on bowel motility. Its bowel seletivity my rise due to its usedependent, nd persistent, tion. Keywords: UK84149; nirdipine; D6; lium hnnel; smooth musle Introdution UK84149 (S5,1 ldihydro5[1(4 methoxyphenethyl)2pyrrolidinylmethyl]dibenzo [b.e] [1,4] thizepinemlete]), newly developed lium ntgonist drug, showed signifint tissue seletivity for intestine smooth musle in preliminry experiments (Quinn & Wllis, 1994; Wllis et l., 1994). UK84149 potently inhibited smll nd lrge bowel motility stimulted by holesytokinin otpeptide, with rdiovsulr effets being evident only t doses 1 times higher. This ontrsted with lium hnnel blokers suh s nifedipine nd diltizem whih produed doserelted rdiovsulr effets over the sme dosernge s those required to inhibit motility (Quinn & Wllis 1994). UK84149 ws ble to inhibit liumindued ontrture of depolrized guinepig ileum whih ould be reversed by the ddition of exess lium, inditing tht lium ntgonist effet might be involved in the effets of UK84149 (Wllis et l., 1994). The purpose of the present experiments ws to investigte the tion of UK84149 on lium urrents from the guinepig jejunum, rbbit jejunum, nd oronry rtery smooth musle ells, nd to ompre it with verpmil, nirdipine nd ' Author for orrespondene. D6 by using the wholeell voltge lmp reording tehnique. Methods Single ell dispersion Rbbits were killed by overdose of pentobrbitone nd guinepigs by ervil dislotion nd exsnguintion. Strips of the longitudinl smooth musle lyer of the guinepig or rbbit jejunum were ut into smll piees (34 mm) nd inubted for 1 min in low (1 tim) physiologil slt solution (PSS) nd then trnsferred to low medium ontining.4 mg ml l ollgense (Sigm, type XI) nd.4 mg ml' protese (Sigm, type E) for 2 min t 37'. At the end of digestion, the tissue ws wshed with low PSS without enzyme nd gitted mildly by suking in nd out of widebore pipette. The suspension of ells ws then entrifuged nd the pellet resuspended in PSS ontining bout.4 mm. ells were mintined t 4 nd used within 6 h. All experiments were t room temperture (226'). The left desending oronry rtery of rbbit ws identified under the binoulr mirosope nd ws isolted refully. The onnetive tissue ws removed nd the rtery ws ut into smll piees (23 mm). The proedure for dispersion

2 1658 ws similr to tht for jejunum exept higher levels of enzymes (ollgense.6 mg ml, protese.6 mg ml ) nd longer inubtion time (3 min) were used. Eletril reordings ells were voltgelmped by use of the wholeell onfigurtion of the pthlmp tehnique (Hmill et l., 1981) or perforted pth reording tehnique in whih nysttin (1I pg ml') ws dded to the pipette solution (Horn & Mrty, 1988). The urrentvoltge onverter ws pthlmp mplifier (Biologi). Anlogue signls were reorded on FMtpe (Rl) t tpe speed of 3.75 in s'. Firepolished pipettes were prepred from borosilite glss using pth eletrode puller (Nrishige Si. Inst. Lb.). The resistnes of the pipettes were 24 MQ for jejunum smooth musle ell reordings nd 35 M for rbbit oronry rtery smooth musle ell reordings. ells were pled in the hmber (.3 ml) on the stge of n inverted mirosope, H. Zhng & T.B. Bolton lium hnnels bloked by UK84149 whih ws perfused ontinuously with solution t rte of bout 2 ml min'. ommnd pulses were pplied nd signls were digitized t 2 khz using ED 141 interfe in onjuntion with 386 P or BB miroomputer. Solutions The norml PSS (physiologil slt solution) in the bth hd the following ioni omposition (mm): Nl 13, Kl 5, Mgl2 1.2, l2 1.7 or 2.5, gluose 1, HEPES 1, nd ws titrted to ph 7.4 with NOH. The pipette solutions hd the following ioni omposition (mm): (1) s solution; sl 135, Mgl2 5, EGTA 5, HEPES 5. To mke n ATPgenerting solution ATP (3 mm), GTP (.1 mm), retine phosphte (15 mm), nd retine phosphokinse (5 u ml') were dded or nysttin (1 jig ml') ws dded for perforted pth reording. (2) K solution: Kl 13, Mgl2 2, EGTA 5, HEPES 2. The solutions were titrted to ph 7.4 with soh or KOH. In some ses, 2 mm TEA hloride ws inluded in the pipette nd bth solution b W mini ~~ 3 min 3s K I3 s mv 1.75 d 8OmV 5ms 1.5 o 1.25 :3 A A (U E.75 < 5 (.25 S * L Figure 1 Time ourse of hnges in size of pek inwrd urrent in guinepig jejunum smooth musle ell reorded using three types of sontining pipette solution. () onventionl whole ell reording with ATPfree pipette solution; (b) onventionl whole ell reording with 3 mm ATP, 15 mm retine phosphte nd 5 u mlp retine phosphokinse in the peptide solution; () 'perforted pth' whole ell reording with 1 jig ml nysttin in the pipette solution. The times fter the ell membrne ws ruptured re indited. ells were held t 8 mv nd stepped to mv for 5 ms every 2 s s shown below. (d) Reltive mplitude of pek inwrd urrent reorded with the three types of solution. The inwrd urrent mplitude evoked by the first pulse fter rupture of the ell membrne ws normlized s 1.. ATPgenerting pipette solution (A), nysttin pipette solution (), nd ATPfree pipette solution (@). Mens nd I s.e.men from 45 ells. 4 I. I 5 6

3 Drugs UK84149 s its mlete slt UK (S5,1 1dihydro 5[l(4 methoxyphenethyl)2pyrrolidinylmethyl] dibenzo [b.e] [1,4]thizepinemlete]) ws obtined from Pflizer entrl Reserh, Sndwih, Kent nd ws dissolved first in dimethylsulphoxide (DMSO) nd diluted with PSS to the finl onentrtion given in the text. The finl onentrtion of DMSO ws less thn.3%. Nirdipine, verpmil nd methoxyverpmil (D6) were ll from Sigm, nd were dissolved diretly in wter to mke stok solutions. ollgense (type XI), protese (type E), EGTA, N2ATP, retine phosphte, retine phosphokinse, nd GTP were from Sigm. All results re expressed s men ± s.e.men. Sttistil signifine ws evluted by nlysis of vrine with repeted mesurements or Student's bilterl unpired t tests, when pproprite. Differenes with vlues of P<.5 were onsidered to be signifint. H. Zhng & T.B. Bolton lium hnnels bloked by UK84149 Results urrent rundown 1659 Figure 1 shows inwrd urrent from rbbit jejunum smooth musle ell evoked by stepping from holding potentil of 8 mv to mv. With esium hloride in the pipette, outwrd K urrent ws pprently bolished beuse no differene ws observed when 2 mm TEA ws inluded in the pipette nd bth solution (dt not shown). In stndrd whole ell reording mode when no ATP ws inluded in the pipette solution, the pek mplitude of the voltgetivted urrent grdully deresed with time fter trnsient inrese; the pek inwrd urrent deresed to 19 ± 14% (n = 5) of its initil vlue 2 min fter rupture of the membrne pth. ATP (5 mm) mrkedly delyed the rundown of inwrd urrent (dt not shown). When the ATPgenerting pipette solution (see Methods) ws used, the (i) ontrol 3 gm UK84149 (ii) pa 2, 8 mv 3 ms o mv 3 Ms 4 mvmy l 4~~~~~~~~~ 2 mv mv mv O pa 2 mv t 45 5ms 3 ms 1 b (ii) L.) u ~ ~~ 5 * 3) 2 5. vi. *, \K Am 6.1 Time (s) Figure 2 Effet of UK84149 on inwrd urrents in guinepig jejunl single smooth musle ells. The ells were bthed in PSS ontining 1.7 mm 21 nd perfused ontinuously t the rte of 2 ml min1. ()(i) ells were held t 8 mv nd stepped to vrious potentils for 3 ms, every 2 s. Open () nd losed (@) irles indited the pek inwrd urrents in the ontrol nd with UK84149 (3 gsm), respetively. ()(ii) urrentvoltge urves in the bsene () nd presene () of UK81149 (3 sm). Open nd losed irles indited pek inwrd urrent. (b)(i) Pek inwrd urrents in the bsene nd presene of UK84149 (I 1 gm) plotted ginst time. ells were held t 8 mv nd stepped to mv for 3 ms, every 2 s. (b)(ii) The reltive mplitude of inwrd urrent ginst vrious onentrtions of UK The pek inwrd urrent mplitude in the bsene of the drug ws normlised s 1%. The ID5o ws bout 2.3 rm. Men ± s.e.men (n = 6). * UK (gm) 1

4 166 rundown ws delyed further nd the initil inrese of the pek inwrd urrent persisted even s long s 2 min; therefter the urrent eventully delined to 58 ± 17% (n = 3) of its initil pek mplitude t 6 min. With I jg ml' nysttin in the pipette solution ('perforted pth' reording mode), run down of inwrd urrent ws usully negligible for 3 min or longer but by 6 min run down ws pprent. Thus, run down ws very slow ompred with stndrd whole ell reordings. The pek inwrd urrent 5 min fter gining eletril ess to ell (series resistne H. Zhng & T.B. Bolton lium hnnels bloked by UK Mfl) ws still 96 ± 16% of its initil vlue (n = 3). Rundown of inwrd urrent is problem when onstruting onentrtionresponse reltionships for lium ntgonists, beuse the blok often took long time to reh stedystte. Thus, nysttin pipette solution ws used for onstruting onentrtionresponse reltionship for the effets of UK 84149, D6 nd nirdipine on inwrd lium urrent from rbbit jejunum nd oronry rtery ells nd ATPgenerting pipette solution ws used for the rest of the experiments whih involved observtions on inwrd lium urrent. (i) ontrol UK gm I 83 ms L 3 8 mv 8 mv 4 mv o nl V 15 1' (ii) V pa 5 mv 6 N.3 3 mv 15 pa 2 mv I V b (ii) 4 = 8 4 < ' 6 ORI 4 s2 o 2 4 tv ns~~~~~~ w X~~~~~~~~~I. Z ** * So p%7 =~~~~4. o~~~ 6~~~~~~~ E g 2 o3 6 soo ss 12 ~~~~4 Time (s) o n~~~~~~b UK~~~~~~~rM~ ~b O.1 1 UK (gm) Figure 3 Effet of UK84149 on inwrd nd outwrd urrents in guinepig jejunum smooth musle ells. ()(i) ells were held t 8 mv nd stepped to vrious potentils for 3 ms, every 2 s. Pek inwrd urrent (irles), pek (squres) nd end of pulse (tringles) outwrd urrent in the bsene (open symbols) nd presene (losed symbols) of 3 plm UK84149 were mesured. ()(ii) urrentvoltge urve obtined in the bsene nd presene of 3 gm UK Symbols re s in (Xi). (b)(i) Inwrd nd outwrd urrent in one ell, plotted ginst time. ells were held t 8 mv nd stepped to + 1 mv for 3 ms, every 2 s. (b)(ii) The reltive mplitude of inwrd nd outwrd urrent (evoked by step to + 1 mv) ginst vrious onentrtions of UK84149 (mens ± s.e.men, n 4). The mplitude of inwrd nd outwrd urrent in the bsene of the drug were normlized s 1%. the = ID5s were 2.3 pm for pek inwrd (), 7.2 jum nd 3.3 Sim for pek () nd end (V) of pulse outwrd urrent, respetively. 1

5 Effets of UK84149 on inwrd nd outwrd urrent from guinepig jejunum smooth musle ells Figure 2()(i) nd ()(ii) show the effet of UK84149 on the urrentvoltge reltionships of the inwrd urrent. ells were held t 8 mv nd stepped to vrious potentils. The inwrd urrent ws evoked by stepping more positive thn 3 mv from 8 mv. Outwrd urrent ws inhibited by inluding s insted of K in the pipette solution. Figure 2 () (i) shows inwrd urrent before nd 1 min fter pplition of UK84149 (3 pm). UK84149 onsistently inhibited the pek inwrd urrent mplitude observed t ny given potentil (from 3 to + 4 mv) (Figure 2()(i) nd (ii)). Figure 2(b)(i) nd (ii) show the reltionship between the mplitude of inwrd urrent nd onentrtions of UK In these experiments, the inwrd urrent ws evoked by stepping to mv for 3 ms from holding potentil of 8 mv, every. 6, L 45 L m 2 = 1 E < 1.. W D.8, ( %I. Q 4 'o.2 E o~o, b 8 mv I I mv I I I mv 1s H. Zhng & T.B. Bolton kium hnnels bloked by UK ms 2s. Figure 2(b) (i) shows the effet in ell of different onentrtions of UK84149 on pek inwrd urrent. About 5 min or longer ws needed for the drug to reh its stedystte effet t the lower onentrtions. Figure 2(b)(ii) shows dt from 6 ells. The pek inwrd urrent before pplition of UK84149 ws normlized t 1%. The ID5 vlue for the effet of UK84149 on pek inwrd urrent ws 2.3 gm. Figure 3 shows the effets of UK84149 on inwrd nd outwrd urrents from guinepig jejunum smooth musle ells. In these experiments, high K solution ws used in the pipette solution. When the ell ws stepped more positive thn 2mV from 8 mv, both inwrd nd outwrd urrents were observed (Figure 3()(i) nd (ii)). Figure 3()(i) nd ()(ii) shows the effet of UK84148 on the urrentvoltge reltionship of inwrd nd outwrd urrents whih were inhibited t ll potentils ( 2 to + 3 mv) fter pplition for 1 min. Figure 3(b)(i) nd (ii) shows the reltionship between the mplitude of inwrd nd outwrd urrents nd onentrtions of UK Inwrd nd outwrd urrents were evoked by stepping to + 1 mv for 3 ms from 8 mv, every 2 s. Figure 3(b)(i) shows the effet of UK84149 on pek inwrd, pek nd end of pulse outwrd urrent from one ell. It seems tht UK84149 ws muh slower to tke effet on inwrd urrent thn on outwrd urrents, with delyed strt nd delyed stedystte (Figure 3(b)(i)). Figure 3(b)(ii) shows the dt from 4 ells. UK84149 ws more potent inhibitor of the end of pulse outwrd urrent thn the pek outwrd urrent nd elerted the dey of the outwrd urrent. The IDms for the effets of UK84149 on pek inwrd, pek outwrd nd end of pulse outwrd urrent were 2.3, 7.2 nd 3.3 EM, respetively. The effet of UK84149 on the vilbility of the inwrd urrent The vilbility of the inwrd urrent ws tested by mens of 2 2 double pulse protool. The pek inwrd urrent ws evoked by test potentil of mv fter holding for 1 s t vrious onditioning potentils. The holding potentil ws 8 mv. Double pulses were pplied t 2 s intervls. In the ontrol, the inwrd urrent mplitude evoked by test pulse ws redued when onditioning potentils were more positive thn 6 mv. Figure 4 shows tht the urrent ws hlf vilble t Vh = 3 mv, nd the slope, Vs, ws 5.5, when Boltzmnn eqution of the form I/II = (1 + exp((vvh)/ V,))1 ws fitted to the intivting omponent of the urrent. V is the onditioning potentil nd IIImx the frtion of the urrent vilble reltive to tht vilble from 8 mv. UK84149 (3 JsM) redued the inwrd urrent mplitude from ll potentils nd shifted the stedy stte intivtion urve to the left (Figure 4b). Vh ws shifted to 58 mv, nd the slope vlue to 7 in this ell. Similr hnges were observed in nother 5 ells, with men vlue of Vh shifted from 37± 2 mv (ontrol, n = 5) to 5± 2.2 mv (n = 5), nd 2 o 2 of V, from 6.2+±.4 (ontrol, n = 5) to 7.3 ±.1 (n = 5) Figure 4 Effet of UK84149 on voltgedependent intivtion of the inwrd urrent in guinepig jejunl smooth musle ell. The durtion of the onditioning pulse (prepulse) ws 1 s. () Absolute mplitude of inwrd urrent evoked by the test pulse (5ms in durtion, stepped to mv from the pproprite membrne potentil set by the onditioning pulse). The intervl between onditioning pulses ws 2 s. (b) Reltive mplitude of inwrd urrent. The inwrd urrent mplitude evoked by the test pulse, without onditioning (the holding potentil ws 8 mv) ws normlized s 1.. (U) ontrol; (@) 3 tm UK. Solid lines were drwn from Boltzmnn's eqution (see text). The vlue of Vh in this ell ws 39 mv for ontrol nd 52 mv in 3 #LM UK The slope ws 5.5 for ontrol, nd 7 in 3 gm UK Averge Vh vlues from nother 5 ells were 37 ± 2 mv (men ± s.e.men) for ontrol nd 5 ± 2.2 mv in 3 pm UK84148, nd for the slope, 6.2 ±.4 for ontrol nd 7.3 ±.1 in 3 jim UK Usedependent tions of UK84149 Inwrd urrent blok by verpmil nd D6 is known to be usedependent; UK84149 seemed to hve similr property. As shown in Figures 2 nd 3, it took long time for UK84149 to reh its stedystte effet. Figure 5 shows the results of n investigtion of the usedependene of UK 84149, verpmil nd nirdipine on inwrd urrent in guinepig jejunum smooth musle ells. Inwrd urrent ws evoked by stepping from 8 mv to mv for 5 ms, every 2 s. The mplitude of the pek inwrd urrent ws plotted ginst time. After rupture of the ell membrne the drugs were not pplied until the inwrd urrent beme stble. Figure 5 shows the effet of ontrol PSS nd verpmil (3 gm) on inwrd urrent. The ell hd n inwrd urrent size

6 A2w~ H. Zhng Zhn & T.B. Bolton of bout 6 pa. When the ommnd pulse ws stopped for 3 min in ontrol PSS, whih is indited by the first interruption between the open irles, the pek inwrd urrent mplitude ws temporrily very slightly inresed. When the ommnd pulse ws stopped for nother 3 min in verpmil (3 pm), the inwrd urrent mplitude evoked by the first ommnd pulse fter rest ws not hnged, ompred with tht before rest. Therefter the urrent grdully deresed in the ontinuous presene of 3 pm verpmil nd rehed stedy stte fter bout 3 min. When pulses were gin stopped in the presene of verpmil (3 pm), inwrd urrent evoked by the first pulse fter stopping hd reovered to bout 8% of the ontrol size. Figure Sb shows tht high onentrtion of verpmil (1 JLM) lso hd usedependent tion. Both ells showed good reovery of inwrd urrent liumhnnelsblokedbyuk84 hnnels bloked by UK84149 fter wshout of verpmil. Figure 5 shows the effet of nirdipine on inwrd urrent. In ontrst to verpmil, nirdipine hd little usedependent tion on the inwrd urrent. Upon resting in the presene of nirdipine (.1 JiM) for 3 min, the inwrd urrent 'evoked by the first pulse ws 33% of urrent size before rest, whih ws lmost the sme vlue s tht fter 5 min stimultion. UK84149, more like verpmil, showed similr usedependent tion on inwrd urrent (Figure 5d). After resting in the presene of 3 ptm UK84149 for 3 min, the mplitude of the inwrd urrent evoked by the first pulse did not hnge, ompred with tht before rest. Therefter it deresed grdully nd rehed stedy stte bout 1min lter. However unlike verpmil, resting in the ontinuous presene of UK84148 (3pM) did not improve inwrd urrent, nd lso it took long time to I I I I I I b I * * *I. I *I. *_. X 2 2. U ) E E' 6 _ D Go o % Verpmil 3 gm A' N Verpmil 1 gm I. I. I d I. Im wi W WLI L m._ 1 o._ ' E d9"=q2 Nirdipine.1 gm v~g 34 1 E 6 9 D d9 UK 3 1kM 8 8 Figure 5 Time ourses of inhibition by verpmil (,b), nirdipine () nd UK84149 (d) of inwrd urrent in guinepig jejunum smooth musle ells. Inwrd urrent ws evoked by stepping from 8 mv to mv for 5 ms every 2 s. Pek inwrd urrent before nd fter pplition of 3 1M () nd 1 1M (b) verpmil,.i1 LM nirdipine () nd 31M UK84149 (d) were plotted ginst time. The interruption between the irles indites tht ommnd pulses were stopped for 3 min. Some reovery of inwrd urrent ourred fter rest periods in the ses of verpmil (3 nd 1 gm) but not nirdipine or UK84149.

7 wsh out the effet of UK84149 on inwrd urrent. The poor reovery of the inwrd urrent fter stopping stimultion in the ontinuous presene of UK84148 (31M) ws probbly due to the strong binding of UK84148 to the reeptor. To lrify the usedependent effet of UK84148, ells from guinepig jejunum were exposed to UK84148 (3LM) for 1min before stimulting, nd the blok of the inwrd urrent ws ompred with tht hieved with ontinuous repetitive simultion for 1min in the presene of UK84148 (3 AIM). Figure 6 shows the results of these experiments. When ells were exposed to UK84148 (3 1M) for 1min before stimulting, the inwrd urrent ws only slightly inhibited, lthough the inwrd urrent grdully deresed fter the stimultion ws repplied to the ell in the ontinuous presene of UK84148 (Figure 6). However, when the ell ws exposed to UK84148 (3tM) for 1min nd ws repetitively simulted without ny rest period the inwrd urrent ws gretly inhibited (Figure 6b). Figure 6 shows the summrized dt. In 4 ells, the inhibition of the inwrd urrent by UK84148 (3 1M) without stimultion ws 5.3 ± 3.6% (from 486 ± 6 pa to 47 ± 51 pa) whih ws signifintly (P <.1) less thn tht with repetitive stimultion (52± 8%; from 455 ± 59pA to 219± 5pA, n=4). Effets of UK84149, D6 nd nirdipine on inwrd urrents from rbbit jejunum nd oronry rtery smooth musle ell Figure 7 shows the effet of UK84149 on inwrd urrent from rbbit jejunum nd oronry rtery smooth musle ells, ompred with the effets of D6 nd nirdipine. The nysttin reording method ws used in these experiments. The ell ws held t 8 mv nd stepped to mv for 5 ms, every 2 s. A 1 ms 1 mv depolrizing pulse ws used to monitor the series resistne. The drugs were bth pplied nd perfusion ws ontinuous t rte of 2 ml min'. dmium (1 1M) ws used to bolish inwrd urrent ompletely t the end of the experiments nd rete bse line for mesurement. Figure 7()(i) shows the effet of UK84149 (3 1M) on inwrd urrent in jejunum (left) nd oronry rtery (right) smooth musle ells. UK84149 (3 1M) inhibited inwrd urrent from both types of smooth musle ells nd with similr poteny. At this onentrtion, the effet ould be reversed by wshing, lthough it took long time (bout 15 min). Figure 7()(ii) shows the 1 <2 mi Jr.<_ UK3NsM No stimultion b H. Zhng & T.B. Bolton lium hnnels bloked by UK84149 min Stimultion UK3 gm Figure 6 Usedependeny of inhibition of UK84149 of inwrd urrent in guinepig jejunum smooth musle ells. Inwrd urrent ws evoked by stepping from 8 mv to mv for 5 ms every 2 s. () Stimultion ws stopped (s indited by interruption between the irle symbols) for O min in the presene of UK84149 (3 gm). (b) UK84149 (3 jtm) ws pplied to repetitively stimulted ell for 1 min. () The pek inwrd urrent mplitude is shown (men ± s.e.men) in the bsene (solid olumns) nd presene (stippled olumns) of 3 gim UK84149 with nd without stimultion. onentrtioneffet reltionship for the effet of UK84149 on inwrd urrent from the two types of smooth musle ells. The mplitude of inwrd urrent before pplition ws normlised s 1%. The IDM vlues were not signifintly different (nlysis of vrine, P>.5) (1.7 1M for jejunum (squres) nd 3.2 1M for oronry rtery (irles) smooth musle ells). Figure 7(b)(i) shows the effet of D6, nd Figure 7()(i) shows the effet of nirdipine on the two types of smooth musle ell. Agin, no signifint differenes were found. The ID5 vlues for the effet of D6 on inwrd urrent were 8.1 1AM for jejunum nd 3.9 1AM for oronry rtery smooth musle ells (Figure 7(b)(ii)). The ID5 vlues for the effet of nirdipine on inwrd urrent were 24 nm for jejunum nd 12 nm for oronry rtery smooth musle ells. Among the three ntgonists, nirdipine ws most potent in both jejunum nd oronry rtery ells. UK84149 ws 25 times more potent thn D6 but 23 times less potent thn nirdipine. Disussion 1663 The min finding of the present experiments ws tht UK inhibited the inwrd urrent evoked in smooth musle ells from the guinepig nd rbbit jejunum nd rbbit oronry rtery ell with poteny similr to tht of D6 lthough it ws less potent thn nirdipine. The effet of UK84149 ws longlsting nd voltgedependent. Like verpmil, UK84149 showed usedependent blok. UK inhibited voltgedependent outwrd urrent s well s inwrd urrent. This ws not surprising beuse mny ntgonists, inluding nirdipine, verpmil nd diltizem hve been shown to be blokers of voltgedependent outwrd urrents in smooth musle ells (Terd et l., 1987). Sine 5 mm EGTA ws inluded in the pipette in the present experiments nd the outwrd urrent ws evoked by stepping only to + 1 mv, there should be little tivtion of lrgeondutne, tivted potssium hnnels. Therefore the effet of UK84149 on outwrd urrent is unlikely to be due to the inhibition of 2+ influx vi voltgedependent hnnels. The rtio of ID5 vlues for the inhibition of UK of pek outwrd nd inwrd urrent ws bout 3 (7.2 1AM/2.3 1M) whih ws muh lower thn tht of nirdipine on rbbit jejunum ell whih nd hd similr rtio to D6 (rtio of ID5 vlues ws 11, Terd et l., 1987). Like the effet of verpmil nd nirdipine on rbbit ileum smooth musle ells (Terd et l., 1987), UK84149 elerted the outwrd urrent dey. In generl, the seletivity of the ntgonists for the inwrd urrent in smooth musle ells ws low in omprison with tht seen in rdi ells (Hume, 1985). However, the ID5 vlues for the inhibition of outwrd urrent by different ntgonists in different tissues (smooth musle, rdi nd neurone) were quite similr, rnging from AM (Nishi et l., 1983; Hume, 1985; Terd et l., 1987; Figure 3), but the IDM vlues for the inhibition of inwrd urrent by different ntgonists in different tissues were drmtilly different, rnging from 16 nm to 9 1AM (Nishi et l., 1983; Hume, 1985; Terd et l., 1987b,; Figure 6). Hene, inhibition of voltgedependent outwrd urrents by ntgonists seems to be due to nonspeifi bloking tion of higher onentrtions. UK84149 shifted the voltgedependent intivtion urve to the left. Nirdipine nd other dihydropyridines hve been shown to shift the voltgedependent intivtion urve to the left when the onditioning pulses were 1 s (the sme ondition s used in the present experiment) (Terd et l., 1987b). Therefore, shifting the voltgedependent intivtion urve to the left seems to be one of the ommon fetures of lium hnnel blokers. In most smooth musles the urrent n be disseted into omponents on the bsis of its voltge nd timedependent properties (Aronson et l., 1986; ffrey et l.,

8 is"4 %%pr (i) mv 8 MV 7MT mf5 pa 25 H. Zhng & T.B. Bolton lium hnnels bloked by UK Zhnl 8 P4 4 mv 7 mvi 5 ms l B d *W e I 4 w ~~ 1 (ii) 5 1 I {E 1l b (ii) UK (M) b (i) W 75 5 D D pa pa 3: 25 25' 4 5 8J 1u.fo ~~~~~ I I I I D6 (gm) (i) pa 4 ;S1;~~~1. Y~~~ I ~~: N.~~ l wi(u I I I I Nirdipine (gm) 8 Figure 7 Effet of UK84149, D6 nd nirdipine on inwrd urrent of rbbit oronry rtery nd jejunum smooth musle ells. ell ws held t 8 mv nd stepped to mv for 5 ms, every 2 s. Nysttin (1 jtg ml 1) ws inluded in s12 pipette solution to mke perforted pth whole ell reordings. ()(i), (b)(i), ()(i): Effet of 3 gm UK84149 (UK), 3 1M D6 (D) nd.3 IM nirdipine (N) on inwrd urrent from jejunum (left pnel) nd oronry rtery (right pnel) smooth musle ells. dmium (d) 1 IAM ws pplied t the end of the experiments. ontrol, W fter wshout. ()(ii), (b)(ii), ()(ii): onentrtioneffet reltionship for the effet of UK84149, D6 nd nirdipine on inwrd urrent from jejunum () nd oronry rtery () smooth musle ell. No signifint differenes were found for ll three drugs mong jejunum nd oronry rtery smooth musle ells. The ID5s: UK84149, 1.7 LM for jejunum ells, nd 3.2 jm for oronry rtery ells (nlysis of vrine, P>.5, 38 ells); D6, 8.1 gm for jejunum ells, nd 3.9 gm for oronry rtery ells (P>.5, 36 ells); nirdipine,.24 gm for jejunl ell, nd.12i1am for oronry rtery ells (P>.5, 27 ells). 1986). The diversity of hnnels might ontribute to the diversity of responses of urrent to ntgonists. The effet of lium ntgonists on urrent my be tissuedependent, voltgedependent nd use or frequenydependent. Nirdipine nd other dihydropyridines re the most potent lium ntgonists nd their effets re voltgedependent. Verpmil nd D6 hve usedependent tion (Terd et l., 1987b; present dt). In the present experiments, UK84149 showed usedependent blok whih my explin, t lest prtilly, why UK84149 ws more potent on the bowel thn on blood vessel (Quinn & Wllis, 1994; Wllis et l., 1994), sine bowel hs high rte of spontneous tivity nd rtery is quiesent under norml onditions. Any blokde rising during spontneous tivity would be only slowly dissipted during rest period; during hypermotility its bloking tion would be expeted to develop prtiulrly strongly. In onlusion, our experiments indite tht UK84149 is lium ntgonist nd is tive in bloking voltgetivted lium urrent. UK84149 might be expeted to be useful in onditions suh s irritble bowel syndrome whih re hrterized by hypermotility of the smooth musle. This work ws supported by Pfizer Ltd. We re grteful to Dr R.M. Wllis for dvie.

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