Ryanodine Inhibits the Release of Calcium from Intracellular Stores in Guinea Pig Aortic Smooth Muscle

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1 730 Ryandine Inhibits the Release f Calcium frm Intracellular Stres in Guinea Pig Artic Smth Muscle Katsuaki It, Sachik Takakura, Khichi Sat, and Jhn L. Sutk Frm the Department f Veterinary Pharmaclgy, Faculty f Agriculture, University f Miyazaki, Miyazaki, Japan, and the Departments f Physilgy and Internal Medicine (Cardilgy Divisin), The University f Texas Health Science Center, Dallas, Texas SUMMARY. We have examined the effects f ryandine, an inhibitr f the release f sarcplasmic reticulum calcium in cardiac muscle, n cntractile tensin and calcium-45 mvement in artic smth muscle f guinea pigs t learn whether this agent als mdifies the release f stred calcium in vascular smth muscle. Ryandine (3-100 /*M) suppressed the phasic cntractins induced by caffeine and nrepinephrine in calcium-free medium and prevented the stimulatin f calcium-45 efflux by these agnists. Ryandine did nt significantly alter either the cntractile respnse r the increased cellular influx f calcium-45 caused by high ptassium in mre than 1 mm calcium, suggesting that this agent des nt affect deplarizatin-induced calcium entry int the cells. In a calcium-free, high ptassium slutin, the additin f calcium at cncentratins f 1 mvi and less resulted in a cntractin which appeared t depend largely n the release f calcium frm intracellular stres. This cntractin was blcked by ryandine. These data are cnsistent with the hypthesis that ryandine causes a diminished release f calcium frm the intracellular stre in vascular smth muscle, as it des in cardiac muscle. Mrever, ur results indicate that a calcium-induced calcium release may exist in smth muscle, and that this release is antagnized by ryandine. (Circ Res 58: , 1986) IT HAS been suggested that mbilizatin f calcium frm intracellular stres, presumably sarcplasmic reticulum (SR), in vascular smth muscles, underlies the cntractile effects f agents such as vasactive amines and peptides 0hanssn and Smly, 1980). Nevertheless, the rle f the SR in regulating intracellular calcium cncentratin in vascular smth muscle is nt well understd, due t its lw density and distributin in the cell, and t difficulties in islating SR membranes frm vascular smth muscles. Pharmaclgical interventins which mdify the release f intracellularly stred calcium have been used t investigate the cntributins made by intracellularly stred calcium t smth muscle cntractins. These interventins include caffeine and lcal anesthetics, such as prcaine (End, 1977; Ith et al., 1981; Saida, 1982). Hwever, a limitatin t the use f these agents is that bth caffeine and prcaine decrease the influx f calcium frm the extracellular space (Jacbs and Keatinge, 1974; Leijten and Van Breemen, 1984; Spedding and Berg, 1985). Therefre, it is difficult t differentiate the effect f these agents n the calcium release frm that n the calcium influx when intact smth muscle is used. Ryandine, a naturally ccurring alkalid, has been demnstrated t alter specifically calcium mvements acrss SR membranes in cardiac and skeletal muscles (Jenden and Fairhurst, 1969; Sutk et al., 1979; Sutk and Willersn, 1980; Fabiat, 1985; Sutk et al., 1985). Mrever, this agent alters the mechanical respnses f certain smth muscle preparatins in a manner that suggests that it might prduce similar effects in this tissue (Steinsland et al., 1973). If ryandine selectively mdifies the functin f the SR, it will be a pwerfull tl fr the study f the rle f the SR in vascular smth muscle cntractin. The present study was undertaken t learn hw ryandine mdifies the release f calcium frm intracellular stres in guinea pig artic smth muscle. The results suggest that this agent blcks the release f intracellularly stred calcium caused by nrepinephrine (NE) and caffeine, but des nt affect deplarizatin-dependent calcium entry frm the extracellular space. Methds Artas were islated frm 250- t 400-g male guinea pigs fllwing a blw n the neck. Fr tensin experiments, the helical strip f arta was suspended in an rgan bath filled with 10 ml Tyrde's slutin cntaining (mm): NaCl, 136.8; KC1, 5.4; MgCl 2, 0.5; NaHCO 3, 11.9; and dextrse, 5.5; gassed with 95% O 2 and 5% CO 2 (ph 7.4; 37 C). High ptassium slutin was prepared either by hypertnically increasing KG t 60 mm r by istnically substituting KC1 fr NaCl. Nrepinephrine (Sigma) and ryandine (lt 704 RWP-1, S.P. Penick Crp.) were added t the Tyrde's slutin frm 1 mm stck slutin, while caffeine (Nakarai Chemicals) was disslved directly in the

2 lt et al./effect f Ryandine n Vascular Smth Muscle 731 buffer slutin. The muscle strip was placed under 1 g f resting tensin, and its ismetric tensin was recrded. The muscle was equilibrated fr 30 minutes and then was treated alternately with 60 mm KC1 and 1 fim NE until stable reprducible cntractins were btained. Fr 45 Ca flux experiments, the slutins were buffered with tris(hydrxymethyl)aminmethane (Tris) and gassed with 100% O 2 t avid precipitatin in the medium due t the presence f lanthanum. We had ensured, in preliminary experiments, that the results f tensin experiments in Tris buffer were identical with thse in a bicarbnate buffer. Uptake f 45 Ca by artic muscles was measured by a cld lanthanum wash prcedure (Karaki and Weiss, 1979). First, the muscles were depleted f calcium by 20 minutes f expsure t 10 /*M NE in calcium-free medium, and then were transferred t calcium-free, ptassiumdeplarizing slutin cntaining KG, 142; MgCU, 0.5; dextrse, 5.5; 5 mm Tris (ph 7.4). Fifteen minutes later, 1 mm calcium cntaining 45 Ca (1 /ici/ml) was added. After incubatin with 45 Ca fr 5 r 30 minutes, the muscles were transferred t lanthunum slutin (LaCl 3, 80.8; dextrse, 11; and Tris, 5 mm; ph 6.8) cled t 0.5 C. The residual 45 Ca cntent after 60 minutes f incubatin in cld lanthanum slutin was determined as the net cellular 45 Ca uptake. Fr the measurement f 45 Ca efflux, the muscles were laded with 45 Ca during a 150-minute incubatin perid in Tyrde's slutin cntaining 45 Ca (2 jici/ml; ttal calcium was 2.5 mm). The muscles then were placed in a calcium-free medium, and the efflux f 45 Ca was initiated. The efflux medium was changed every 10 minutes. After minutes, the muscles were expsed t 10 HM NE r 20 mm caffeine. The radiactivity in the efflux medium was cunted in a liquid scintillatin cunter. Data are expressed as rate cefficient, which means the percent lss f 45 Ca cntent during each washing interval. Data represent the mean ± SEM. Differences were cnsidered t be significant at the level f P < 0.05 when tested by Student's /-test. Results When 2.5 HIM calcium was present in the external medium, pretreatment f artic muscles with 30 r 100 HM ryandine did nt significantly affect the rate f tensin develpment and the peak amplitude f the cntractin elicited by 60 mm ptassium (n = 8 fr each cncentratin). On the ther hand, 30 JUM ryandine slwed the develpment f respnses t 1 fim NE (the time fr half-maximal respnse increased frm 19 ± 3 secnds t 96 ± 17 secnds; P < 0.05) and decreased its peak tensin by 22 ± 8% (P<0.05;H = 8). In calcium-free slutins cntaining 0.5 mm EGTA, bth caffeine (5-10 mm) and NE (1 I*M) induced an initial phasic cntractin which, in the case f NE, was fllwed by a tnic ne. Ryandine selectively suppressed the phasic cntractin due t bth agents in a dse-dependent manner (Fig. 1). The ryandine-insensitive tnic cntractin prduced by NE was decreased by elevating the cncentratin f EGTA in the medium t 2 mm, supprting the view that this cmpnent was due t entry f superficially bund calcium r t the release Caffeine? Ryandine Caffeine 7 T CCa-1, 2.5mM 0 mm 2.5mM OmM Ryandine (um) ]0.2g FIGURE 1. Suppressin f the phasic cntractins induced by caffeine and nrepinephrine (NE) in calcium-free medium by ryandine. After a 15-minute incubatin in calcium-free medium cntaining 0.5 mm EGTA, artic strips were cntracted with either 10 nim caffeine r 1 HM NE. After the agnists frm the rgan bath were washed, the muscles were equilibrated in 2.5 nim calcium fr 30 minutes. The external medium was then changed t the calcium-free medium cntaining ryandine. Panel A: ablitin by 30 HM ryandine f caffeine-induced cntractin. Panel B: suppressin by 10 HM ryandine f NE-induced phasic cntractin. Panel C: cncentratin-dependence f the effects f ryandine n the phasic (clsed circle) and tnic (pen circle) cntractins induced by 1 HM NE in calcium-free medium cntaining 0.5 mm EGTA. The rdinate represents the relative amplitude f the cntractin as cmpared t the maximum cntractin induced by 60 mm KCI in 2.5 nim calcium-tyrde's slutin. Each pint represents a mean f seven preparatins with SEM. f calcium frm intracellular site which can be readily depleted by extracellular EGTA (Wheeler and Weiss, 1979; Karaki and Weiss, 1980a, 1980b). NE (10 IIM) and caffeine (20 mm) increased the 45 Ca efflux frm artic muscles int calcium-free medium, which effect is thught t result frm an acceleratin f release f stred calcium by these agents (Gdfraind, 1976; Deth and Casteels, 1977; Casteels and Drgmans, 1981). As shwn in Figure 2, the presence f 30 fim ryandine prevented this effect caused by NE. Similarly, ryandine prevented the increase f 45 Ca efflux caused by 20 mm caffeine (data nt shwn). Thus, the results shwn in Figures

3 732 Circulatin Research/Vl. 58, N. 5, May 1986 ^ 5 E "; 4 I 3 O 0 L Time (min) FIGURE 2. Inhibitin by ryandine f the stimulatin by 10 IXM NE f the i5 Ca efflux frm the artic muscles. After the muscles were laded with 4S Ca fr 150 minutes, the muscles ivere expsed t the efflux medium which was changed every 10 minutes. After minutes muscles were expsed t 10 \IM NE. When present, 30 fim ryandine was added 20 minutes prir t NE, as indicated by arriv (filled circle). Open circle: cntrl. Each curve represents the average f fur preparatins. 1 and 2 clearly indicate that ryandine inhibits the release f calcium frm the intracellular stres sensitive t NE r caffeine. In the next series f experiments, we examined c Q L (mm) N.S Ry T 5.A K2 N.S. T Ry 5 min 30 min FIGURE 3. Cellular uptake f 45 Ca int artic muscles in the presence f 5.4 HIM r 142 mm ptassium. After the intracellular calcium stre was depleted by 10 HMNE in calcium-free medium, the muscles were expsed t calcium-free medium cntaining 5.4 HIM r 142 mm ptassium. Twenty minutes later, 1 HIM calcium with >5 Ca was added. The muscles were allwed t take up <5 Ca fr 5 r 30 minutes, and were then transferred t the lanthanum slutin at 0.5 C t measure cellular * 5 Ca uptake. In the ryandine-treated grup (Ry), 30 HM ryandine was present in the calcium-free, 142 mm ptassium slutin. Each bar represents a mean f eight preparatins with SEM. N.S. = nt significant. the effects f ryandine n the cntractin f artic smth muscle resulting frm the reintrductin f calcium t muscles previusly incubated in calciumfree, high ptassium (140 mm) slutin. Ryandine (30 HM) significantly decreased the calcium-induced cntractin f the muscles by 73 ± 6%, 57 ± 6%, and 42 ± 4% (n = 7) when the cncentratin f calcium added was 0.1, 0.3, and 1.0 mm, respectively. Hwever, ryandine did nt significantly inhibit the cntractin when the added calcium exceeded 2 mm. The suppressin f this cntractin culd be due t inhibitin by ryandine f either calcium influx int the cell r f calcium-induced calcium release frm the intracellular stres. T test these pssibilities, we first examined the effect f ryandine n the uptake f calcium by the muscles (Fig. 3). After the muscles had been incubated in calcium-free, istnic ptassium slutin fr 20 minutes, 1 mm calcium with 45 Ca was added. High ptassium significantly increased 45 Ca uptake int the muscle cells. Ryandine (30 /J.M) did nt significantly affect the 45 Ca uptake increased by high ptassium. The results suggest that ryandine des nt affect deplarizatin-dependent calcium entry frm the external medium. This hypthesis is cnsistent with the fact that ryandine did nt affect the cntractin induced by 60 mm ptassium in the presence f 2.5 mm calcium. T assess the secnd pssibility, we examined the dependency f the calcium-induced cntractin n the extent f calcium lading f the intracellular stre (Fig. 4). In ne grup f muscles, the intracellular stre was filled with calcium by expsing the muscles t a medium cntaining 60 mm KG and 2.5 mm calcium just befre the intrductin f calciumfree, high ptassium slutin. The ability f this prcedure t lad intracellular calcium stres was cnfirmed in separate experiments in which NEinduced phasic cntractins btained in calciumfree medium were shwn t be augmented by this prcedure. In a secnd grup, the intracellular calcium stre was depleted f calcium by three successive expsures t 1 HM NE in calcium-free buffer. Cntractins that develped during the initial 5 minutes after the additin f 0.1 mm calcium were cmpared, since the calcium stre might be significantly replenished after lnger times. Figure 4 shws that bth the rate f develpment and the amplitude f the cntractins induced by 0.1 mm calcium were greater in the calcium-laded than in the calciumdepleted muscles. Ryandine suppressed the calcium-induced cntractin develped fr 5 minutes after expsure t calcium in the calcium-laded muscles, but nt in thse that had been depleted f calcium. These results suggest that intracellularly stred calcium makes a significant cntributin t the calcium-induced cntractin elicited frm deplarized muscles with intact calcium stres, and that a calcium-induced calcium release mechanism may be perable in this preparatin.

4 lt et fl/./effect f Ryandine n Vascular Smth Muscle g 20 O c r CQ - depleted 2 Time 3 (min) FIGURE 4. Effects f ryandine n calcium-induced cntractins in ptassium-deplarized artic smth muscles. Upper panel: calciumdepleted muscles. The muscles were depleted f calcium by three successive applicatins f NE in the calcium-free medium cntaining 0.5 HIM ECTA. Subsequently, the external medium was changed t the calcium-free, ptassium-deplarizing medium (142 nim KC1, n ECTA). Lwer panel: calcium-laded muscles. Artic muscles were laded with calcium by applicatin f 60 IUM ptassium and 2.5 nim calcium just befre changing t a calcium-free, high-ptassium slutin. In bth panels, when present (pen circle), 30 /JM ryandine was added when the external medium ivas changed t the calciumfree, high ptassium slutin. Clsed circle: cntrl. Data represent mean f seven preparatins with SEM. 'Significantly different frm cntrl (P < 0.05). Discussin The results btained in this study suggest that ryandine can diminish the release f calcium frm intracellular stres in vascular smth muscle withut affecting the calcium influx frm the extracellular space. The selective inhibitin by ryandine f nly thse cntractins which invlve the release f calcium frm intracellular sites means that this agent has an advantage ver caffeine r lcal anesthetics as a tl fr the study f smth muscle cntractin. This selectivity als argues against an effect n subsequent steps in the smth muscle cntractile mechanism, such as mysin light chain kinase, which culd be cmmn t all f the agents tested. Althugh experimental verificatin is required, we are presently assuming frm analgies with striated muscles that the SR is the intracellular calcium stre affected by agents such as ryandine and caffeine. This cnclusin has been reached by ther authrs (Casteels and Drgmans, 1981; Ith et al., 1982; Bnd et al., 1984a, 1984b). Relatively high cncentratins f ryandine are required t affect vascular smth muscle. This apparent insensitivity may be explained by the use- r deplarizatin-dependence exhibited by the actins f this agent n bth skeletal and cardiac muscles (Sutk et al., 1985), as well as by the actual characteristics f the ryandine-binding site. Calcium-induced cntractin f deplarized muscles seemed t depend n calcium lading f the cells. Hwever, we must cnsider an alternative pssibility that this cntractin might depend n superficially bund calcium. Since NE treatment, used t deplete calcium, can remve superficially bund calcium (Wheeler and Weiss, 1979; Karaki and Weiss, 1980a, 1980b), the different amunts f superficially bund calcium in tw grups may explain the difference between the respnses t 0.1 mm calcium if calcium at this site cntributes t the calcium-induced cntractin. As fr this fractin f calcium, ryandine des nt seem t affect the mbilizatin f superficially bund calcium, since this agent did nt inhibit the tnic cntractin induced by NE in calcium-free medium. On the cntrary, ryandine greatly inhibited the cntractin induced by calcium in calcium-laded muscles. Therefre, we can cnclude that calcium-induced cntractin f deplarized muscle did nt depend n superficially bund calcium, but n intracellularly stred calcium. It has been suggested that calcium-induced calcium release underlies the caffeine-induced cntractin in certain vascular smth muscles (Ith et al., 1981,1982; Saida and Van Breemen, 1984). Caffeine is thught t increase the affinity f SR fr calcium (Yamamt and Kasai, 1982; Kirin et al., 1983) and t induce calcium release at physilgical levels f free calcium in the cytplasm even in the absence f calcium influx (Saida, 1982). Hwever, the release f calcium frm intracellular stres by transmembrane calcium influx has nt yet been demnstrated in vascular smth muscle. Calcium-induced cntractin in deplarized muscles has lng been believed t be caused directly by transmembrane calcium influx. The present data indicate that this cntractin is dependent n stred calcium rather than n calcium entry, especially when the external calcium is lw. Since ryandine des nt inhibit calcium influx, inhibitin f this cntractin by ryandine suggests that transmembrane calcium influx can trigger calcium release frm intracellular stres in vascular smth muscle as it des in cardiac muscles (Fabiat, 1983). The present findings suggest that ryandine is a useful tl t assess a cntributin f calcium release frm intracellular stres t mechanical activity f vascular smth muscle. Our results suggest that ryandine inhibits SR calcium release in guinea pig artic smth muscle. Ryandine can bth increase

5 734 Circulatin Research/Vl. 58, N. 5, May 1986 and decrese the calcium permeability f cardiac and skeletal SR membranes, depending n the ryandine cncentratin and experimental cnditins used (Lattanzi and Sutk, unpublished bservatins; G. Meissner, persnal cmmunicatin). Cnsequently, the actual effects f ryandine will have t be established fr different experimental prtcls and types f smth muscles. ILS is an Established Investigatr f the American Heart Assciatin. Address fr reprints: Dr. Katsuaki H, Department f Veterinary Pharmaclgy, Faculty f Agriculture, University f Miyazaki, Miyazaki , ]apan. Received Nvember 11, 1985; accepted fr publicatin February 14, References Bnd M, Kitazawa T, Smly AP, Smly AV (1984a) Release and recycling f calcium by the sarcplasmic reticulum in guinea-pig prtal vein smth muscle. ] Physil (Lnd) 355: Bnd M, Shuman H, Smly AP, Smly AV (1984b) Ttal cytplasmic calcium in relaxed and maximally cntracted rabbit prtal vein smth muscle. J Physil (Lnd) 357: Casteels R, Drgmans G (1981) Exchange characteristics f the nradrenaline-sensitive calcium stre in vascular smth muscle cells f rabbit ear artery. J Physil (Lnd) 317: Deth R, Casteels R (1977) A study f releasable Ca fractins in smth muscle cells f the rabbit arta. ] Gen Physil 69: End M (1977) Calcium release frm the sarcmplasmic reticulum. Physil Rev 57: Fabiat A (1983) Calcium-induced release f calcium frm the cardiac sarcmplasmic reticulum. Am J Physil 245: C1-C14 Fabiat A (1985) Effects f ryandine in skinned cardiac cells. Fed Prc 44: Gdfraind T (1976) Calcium exchange in vascular smth muscle, actin f nradrenaline and lanthanum. J Physil (Lnd) 260: Ith T, Kuriyama H, Suzuki H (1981) Excitatin-cntractin cupling in smth muscle cells f the guinea-pig mesenteric artery. J Physil (Lnd) 321: Ith T, Kajiwara M, Kitamura K, Kuriyama H (1982) Rles f stred calcium n the mechanical respnse evked in smth muscle cells f the prcine crnary artery. J Physil (Lnd) 322: Jacbs A, Keatinge WR (1974) Effects f prcaine and ligncaine n electrical and mechanical activity f smth muscle f sheep cartid arteries. Br ] Pharmacl 51: Jenden DJ, Fairhurst AS (1969) The pharmaclgy f ryandine. Pharmacl Rev 21: 1-25 Jhanssn B, Smly AP (1980) Electrphysilgy and excitatincntractin cupling. In Handbk f Physilgy, The Cardivascular System, vl II, Vascular Smth Muscle, edited by DF Bhr, AP Smly, HV Sparks. Washingtn, D.C., American Physilgical Sciety, pp Karaki H, Weiss GB (1979) Alteratins in high and lw affinity binding f 45 Ca in rabbit artic smth muscle by nrepinephrine and ptassium after expsure t lanthanum and lw temperature. J Pharmacl Exp Ther 211: Karaki H, Weiss GB (1980a) Effects f stimulatry agents n mbilizatin f high and lw affinity site 45 Ca in rabbit artic smth muscle. J Pharmacl Exp Ther 213: Karaki H, Weiss GB (1980b) Dissciatin f varied actins f nrepinephrine n 45 Ca uptake and release at different sites in rabbit artic smth muscle. J Pharmacl Exp Ther 215: Kirin Y, Osakabe M, Shimizu H (1983) Ca 2+ -induced Ca 2+ release frm fragmented sacrplasmic reticulum: Ca 2+ -dependent passive Ca 2+ efflux. J Bichem 94: Leijten PAA, Van Breemen C (1984) The effects f caffeine n the nradrenaline-sensitive calcium stre in rabbit arta. J Physil (Lnd) 357: Saida K (1982) Intracellular Ca release in skinned smth muscle. JGen Physil 80: Saida K, Van Breemen C (1984) Cyclic AMP mdulatin f adrenceptr-mediated arterial smth muscle cntractin. J Gen Physil 84: Spedding M, Berg C (1985) Antagnism f Ca 2+ -induced cntractins f K + -deplarized smth muscle by lcal anaesthetics. Eur J Pharmacl 108: Steinsland OS, Furchgtt RF, Kirpekar SM (1973) Biphasic vascnstrictin f the rabbit ear artery. Circ Res 32: Sutk JL, Willersn JT (1980) Ryandine alteratins f the cntractile state f rat ventricular mycardium. Cmparisn with dg, cat, and rabbit ventricular tissues. Circ Res 46: Sutk JL, Willersn JT, Templetn GH, Jnes LR, Besch HR Jr (1979) Ryandine: Its alteratins f cat papillary muscle cntractile state and respnsiveness t intrpic interventins and a suggested mechanism f actin. J Pharmacl Exp Ther 209: Sutk JL, It K, Kenyn JL (1985) Ryandine: A mdifier f sarcplasmic reticulum calcium release in striated muscle. Fed Prc 44: Wheeler ES, Weiss GB (1979) Crrelatin between respnses t nrepinephrine and remval f 45 Ca frm high-affinity binding sites by extracellular EDTA in rabbit artic smth muscle. J Pharmacl Exp Ther 211: Yamamt N, Kasai M (1982) Kinetics f the actins f caffeine and prcaine n the Ca 2+ gated catin channel in sarcplasmic reticulum vesicles. J Bichem 92: INDEX TERMS: Ryandine Vascular smth muscle. Calcium release Cntractin Calcium fluxes