Effects of organophosphorus agents on sarcoplasmic reticulum in skinned skeletal muscle fibers

These experiments were designed to determine whether skinned skeletal muscle fibers could be useful in screening new antidotes to organophosphorus poisons. Isometric force and fiber diameter were measured in mechanically skinned fibers from mice and frogs. Fibers were depleted of calcium and placed in a calcium loading solution that contained 0.5 mM EGTA with pCa 6.25. The elapsed time (zero time) before a contracture began and the maximum rate of force development (slope) were measured and divided by the square of the diameter (normalized zero time, normalized slope). The zero time was assumed to be the time required for the sarcoplasmic reticulum to attain a threshold concentration for calcium-induced calcium release, and the slope was assumed to indicate primarily the rapidity of the release of calcium from the sarcoplasmic reticulum. Organophosphorus agents, sarin, soman, tabun, and VX were also placed in the loading solutions. Only sarin failed to shorten the normalized zero times of mouse fibers compared to controls, and all agents decreased the normalized slopes. The normalized zero times of frog fibers were not altered by the agents, but the normalized slopes were altered by some agents. Pralidoxime chloride (PAM) and 3-Cl-2,5,6-trimethylbenzoic acid (TBA) were also added to the loading solution for mouse fibers; PAM was marginally effective in moderating some actions of the organophosphates. Because the effects of the agents on the fibers were so definite, we concluded that the skinned muscle fiber might indeed be useful as a screening tool for developing and testing new antidotes to organophosphorus poisons.     

Inhibition of calcium release from the sarcoplasmic reticulum of rabbit aorta by hydralazine.

1. The mechanism of hydralazine-induced vasorelaxation was investigated in rabbit isolated aorta, by determining its ability to interfere with force development under a variety of conditions. 2. Hydralazine relaxed phenylephrine-contracted aorta with half maximal relaxation at 17 microM and maximal relaxation above 100 microM. At 200 microM, hydralazine had little effect on contractions induced by 25 mM or 50 mM K+. 3. Hydralazine was equally effective at inhibiting contractile responses to phenylephrine in the absence or presence of extracellular Ca2+. Responses to phenylephrine in Ca(2+)-free solution were blocked to the same degree whether hydralazine was applied during filling of the sarcoplasmic reticulum (SR) Ca2+ stores or after filling was complete. Caffeine-induced contractions were less sensitive to block by hydralazine. 4. Thapsigargin, cyclopiazonic acid, ryanodine, nifedipine and diltiazem all failed to block the inhibitory effect of hydralazine on tonic contractions to phenylephrine in the presence of extracellular Ca2+. However, when cyclopiazonic acid was applied either with diltiazem or ryanodine, substantial inhibition of the hydralazine response was observed. 5. We propose that tonic contractions to phenylephrine are largely maintained by Ca2+ cycling through the SR, with Ca2+ entering the smooth muscle cell being sequestered by the SR eventually to leak out through IP3-activated channels close to the contractile proteins. Sequestration of Ca2+ would employ two pathways, one sensitive to inhibitors of the SR Ca(2+)-ATPase and the other to Ca antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of calcium release from sarcoplasmic reticulum of skeletal muscle by xanthone and norathyriol.

1. Effects of xanthone and its derivative, 1,3,6,7-tetrahydroxyxanthone (norathyriol), on Ca2+ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle. 2. Both xanthone and norathyriol dose-dependently induced Ca2+ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca2+ release inhibitor, and Mg2+. 3. Xanthone and norathyriol also dose-dependently increased apparent [3H]-ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine. 4. In the presence of Mg2+, which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg2+. 5. Xanthone activated the Ca(2+)-ATPase activity of isolated SR vesicles dose-dependently reaching 70% activation at 300 microM. 6. When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca(2+) -free bathing solution or one containing 2.5 mM Ca2+. These norathyriol-induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine. 7. These data suggest that xanthone and norathyriol can induce Ca2+ release from the SR of skeletal muscle through a direct interaction with the Ca2+ release channel, also known as the ryanodine receptor.     

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

A Ca(2+)-sensitive electrode was used to measure the Ca2+ concentration of the medium containing the heavy fraction of the fragmented sarcoplasmic reticulum (SR) prepared from guinea-pig psoas muscle. Among K(+)-channel blockers tested, 4-aminopyridine (4-AP), tetraethylammonium (TEA) and charybdotoxin elicited Ca2+ release from the SR, but apamin and glibenclamide did not. These results suggest that a reduction of SR K+ conductance leads to Ca2+ release from the SR.     

The effects of drugs on calcium uptake and calcium release by mitochondria and sarcoplasmic reticulum of frog skeletal muscle

The effects of quinidine, chlorpromazine and caffeine on Ca uptake and Ca release by mitochondria and fragmented sarcoplasmic reticulum (FSR) of frog muscle were studied. Quinidine (1–2 mM) released considerable Ca from preloaded mitochondria but had little effect on preloaded FSR. The uptake of Ca both by mitochondria and FSR was inhibited by higher concentrations (2 or 1 mM) of quinidine but the inhibition of mitochondrial Ca uptake was much greater. With lower concentration (0.4 mM), there was no significant effect on Ca uptake by FSR, but a 48 per cent inhibition of mitochondrial Ca uptake was observed.Chlorpromazine (0.01–0.1 mM) inhibited Ca uptake by both mitochondria and FSR, but the inhibition in the case of FSR was weaker than mitochondria. Only the highest concentration (0.1 mM) of chlorpromazine caused a release of Ca from mitochondria or FSR. Caffeine (2–10 mM) inhibited Ca uptake both by mitochondria and FSR and again the inhibition of Ca uptake by mitochondria was greater than of FSR. Caffeine (10 mM) in contrast to quinidine released Ca from FSR and not from mitochondria. Ca releasing effect of both caffeine and quinidine increased when the ratio of the drug and FSR protein increased.The Ca releasing concentrations of these drugs were comparable to those reported to elicit contractures of living muscle. The lower concentrations which inhibited Ca uptake were comparable to those which potentiate twitch.     

MCI-154对大鼠皂苷蜕膜心肌收缩系统Ca~(2 )敏感性和肌浆网Ca~(2 )释放的作用(英文)

目的:探讨MCI-154的正变力机制.方法:用皂苷500或50 nag·L~(-1)破坏或保留肌浆网(SR)的蜕膜心肌标本.皂苷500 mg·L~(-1)蜕膜标本的张力-pCa关系曲线描述了心肌收缩蛋白Ca~(2 )敏感性,pCa_(50)是Ca~(2 )敏感性的指标;皂苷50 mg·L~(-1)蜕膜标本的咖啡因挛缩幅值是SR Ca~(2 )释放的指标.结果:1)在相同Ca~(2 )浓度下,MCI-154(0.1mmol·L~(-1))增强心肌Ca~(2 )激活张力,对收缩蛋白的Ca~(2 )敏感性具有明显的增敏效应,pCa_(50)由对照的5.54(5.30-5.79)升为5.84(5.54-6.14) (P<0.01,n=8);Hill系数n降低了0.29(P<0.01,n=8);2)在保留了SR的标本上,MCI-154不能引起SR内Ca~(2 )释放,并对咖啡因引起的挛缩幅值无显著影响(P>0.05).结论:MCI-154直接增强心肌收缩蛋白的Ca~(2 )敏感性,但对SR的Ca~(2 )释放无明显作用.     

Effects of MCI-154 on calcium sensitivity of contractile system and calcium release from sarcoplasmic reticulum in saponin-skinned rat myocardium.

AIM: To explore the possible mechanisms underlying the positive inotropic effect of MCI-154. METHODS: Skinned fibers with disrupted or preserved sarcoplasmic reticulum (SR) were prepared by saponin 500 or 50 mg.L-1. The tension-pCa relationship and pCa50 of saponin (500 mg.L-1)-skinned fibers were taken as the indices of Ca2+ sensitivity of contractile proteins. The amplitude of caffeine-induced contracture was an index of Ca2+ release from SR in saponin (50 mg.L-1)-skinned fibers. RESULTS: 1) MCI-154 (0.1 mmol.L-1) showed a Ca2+ sensitizing effect on contractile proteins. The pCa50 was increased to 5.84 (5.54-6.14) compared with control value 5.54 (5.30-5.79) (P < 0.01, n = 8). Hill coefficient n was decreased by 0.29 (P < 0.01, n = 8); 2) No contracture was produced by MCI-154 in preparations with preserved SR. Caffeine-induced contracture before and after MCI-154 treatment were not changed (P > 0.05, n = 4). CONCLUSION: MCI-154 directly enhances the Ca2+ sensitivity of contractile protein but has little effect on Ca2+ release from SR in rat skinned cardiac fibers.     

Pimobendan increases calcium sensitivity of skinned human papillary muscle fibers

The calcium sensitizing effects of the cardiotonic agent pimobendan on force production were investigated on chemically skinned human papillary muscle fibres. The papillary muscles were obtained from patients undergoing mitral valve replacement operations. Tension was produced by different free calcium concentrations and 100 mumol/l of pimobendan shifted the pCa-tension relationship significantly to the left. The effects of pimobendan were concentration dependent (10-300 mumol/l) but additionally also correlated with the state of left ventricular performance of the patients. Skinned papillary muscle fibres obtained from patients with reduced ventricular performance were more sensitive to calcium as well as pimobendan compared with fibres from patients with normal left ventricular performance. No differences in response to pimobendan were observed between fibres from NYHA II and NYHA III heart failure patients.     

Effect of diltiazem on the release of calcium from the canine fragmented cardiac sarcoplasmic reticulum

Fragmented sarcoplasmic reticulum fraction (SR) was prepared from the ventricle of canine heart, and the effect of diltiazem on its Ca2+ binding and Ca2+ release was examined by centrifugation and filtration methods using 45Ca. Cardiac SR bound 45-55 nmoles/mg of Ca ions in the presence of Mg-ATP. Diltiazem in concentrations up to 10(-4) M had little effect on the Ca2+ binding of SR. The membrane of cardiac SR was "depolarized" by either changing propionate to chloride (anionic) or potassium to Tris (hydroxymethyl) aminomethane (Tris) (cationic). About 12% of the maximum Ca2+ bound to the SR was released by cationic "depolarization", but no Ca2+ was released by anionic "depolarization". The Ca2+ release induced by the cationic "depolarization" was inhibited by diltiazem, and the inhibitory effect of diltiazem on the Ca2+ release was dependent on the incubation time. Incubation of the SR with 3 X 10(-6) M diltiazem for 30 sec almost completely inhibited the release of Ca2+, while incubation with 3 X 10(-7) M diltiazem incompletely inhibited the Ca2+ release. About 20% of the maximum Ca2+ bound to the SR was released by the addition of 5.1 mM caffeine. The Ca2+ release induced by caffeine was inhibited by increasing the concentration of MgCl2 from 5 to 10 mM, but was not inhibited by 10 mM procaine. An increase of ATP concentration accelerated the time course of the caffeine-induced release of Ca2+ from the SR and subsequent rebinding of Ca2+. Diltiazem up to 10(-5) M had no effect on the caffeine-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-Aminopyridine can induce release of calcium from the sarcoplasmic reticulum of frog heart

4-Aminopyridine (4-AP), a K(+)-channel blocker, has been associated with cellular Ca2+ movements in various excitable tissues but its interaction with intracellular Ca2+ pools has not been described. Spontaneously beating ventricular strips of frog hearts responded to 4-AP (1.6 x 10(-2) M) by contracture that was susceptible to tolerance development when 4-AP was applied in a repetitive manner after washout periods. Under full mechanical inhibition by Ca(2+)-free Ringer or high-K+ Ringer or a combination of both, 4-AP could still produce contracture, a finding indicating that the source of Ca2+ for such an effect should be intracellular Ca2+ pools, namely the sarcoplasmic reticulum. It was concluded that 4-AP could induce release of Ca2+ from internal stores that might be of importance in its cardiac actions.     

Effects of bipyridylium compounds on calcium release from triadic vesicles isolated from rabbit skeletal muscle.

1. The effects of 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP), a viologen for electrochromic memory display agent, on calcium release and ryanodine binding were studied with triad-rich sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscle. 2. DHBP inhibited the calcium release induced by 2 mM caffeine and 2 micrograms ml-1 polylysine with an IC50 value of 5 micrograms ml-1 and 4 micrograms ml-1 respectively. 3. DHBP inhibited [3H]-ryanodine binding in a dose-dependent manner with an IC50 of 2.5 micrograms ml-1 and 90-100% inhibition at 20-30 micrograms ml-1. 4. Calcium uptake by SR was inhibited in the presence of caffeine and this inhibition was antagonized by concomitant addition of DHBP. 5. The effect of DHBP on muscle twitches was studied on the mouse diaphragm. Muscle twitches elicited by direct electrical muscle stimulation and contractions induced by either 10 mM caffeine or 1 microM ryanodine were blocked by pretreatment with DHBP. 6. Data from this study provided evidence that DHBP blocked the calcium release from SR by direct interaction with the calcium release channel, also known as the ryanodine receptor. A possible use of this agent as a specific inhibitor for calcium release and as a muscle relaxant was suggested.     

Effect of bupivacaine enantiomers on Ca2+ release from sarcoplasmic reticulum in skeletal muscle

Local anesthetics affect intracellular Ca2+ movement in the myocyte. The use of isomers may help to reveal specific mechanisms of action, such as receptor mediation. In the present study, we used skinned fibers from mammalian skeletal muscle to test whether bupivacaine enantiomers had different effects on Ca2+ release and uptake by the sarcoplasmic reticulum, and on the Ca2+ sensitivity of the contractile system. Ca2+-induced Ca2+ release was enhanced by S-bupivacaine 1 approximately 3 mM, but inhibited by R-bupivacaine 3 mM, remaining unaffected at lower doses. These enantiomers inhibited Ca2+ uptake to different degrees, with R-bupivacaine having a stronger effect. Ca2+ sensitivity of the contractile system was equally enhanced by R- and S-bupivacaine. These findings might help to explain the myoplasmic Ca2+ elevation induced by bupivacaine. The observed stereoselectivity suggests effects on specific proteins, the ryanodine Ry1 receptor and the Ca2+-ATPase pump, rather than non-specific increase in Ca2+ permeability.     

Amitriptyline activates cardiac ryanodine channels and causes spontaneous sarcoplasmic reticulum calcium release

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proarrhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2), causing premature Ca(2+) release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured the effect of AMT on RyR2 channels from mice and sheep and on intact mouse cardiomyocytes loaded with the Ca(2+) fluorescent indicator Fura-2 acetoxymethyl ester. AMT induced trains of long channel openings (bursts) with 60 to 90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT], voltage, and open probability (P(o)) dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT also activated RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P(o) by increasing cytoplasmic [Mg(2+)] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca(2+) releases and decreased the SR Ca(2+) content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 muM) compared with its effect in lipid bilayers (5-10 muM). Increasing extracellular [Mg(2+)] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca(2+) leak may contribute to AMT's proarrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.     

Effects of anti-oestrogens and beta-estradiol on calcium uptake by cardiac sarcoplasmic reticulum

1. Tamoxifen and a group of structurally similar non-steroidal, triphenolic compounds inhibit the oestrogen receptor. In addition to this action, these anti-oestrogens are known to inhibit some types of plasma membrane ion channels and other proteins through mechanisms that do not appear to involve their interactions with the estrogen receptor but could be the result of their effect on membrane lipid structure or fluidity. 2. We studied the effects of beta-estradiol and three anti-oestrogens (tamoxifen, 4-hydroxytamoxifen and clomiphene) on Ca(2+) uptake into sarcoplasmic reticulum (SR) vesicles isolated from canine cardiac ventricular tissue. 3. The antiestrogens all inhibit SR Ca(2+) uptake in a concentration-dependent manner (order of potency: tamoxifen > 4-hydroxytamoxifen > or = clomiphene). Although these compounds rapidly inhibit net Ca(2+) uptake they do not have a similar rapid effect on the ATPase activity of the SR Ca pump. beta-estradiol has no effect on Ca(2+) uptake nor does it alter the inhibitory action of tamoxifen on the SR. 4. The differences in the effects of beta-estradiol and the anti-oestrogens on cardiac SR Ca(2+) uptake do not correlate with differences in the ways in which these compounds have been reported to interact with membrane lipids. Our results are consistent, however, with direct effects on a membrane protein (possibly an SR Cl(-) or K(+) channel).     

Effects of dantrolene and its derivatives on Ca(2+) release from the sarcoplasmic reticulum of mouse skeletal muscle fibres

1. We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca(2+) release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle. 2. In intact muscles, GIF-0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF-0082, an azido-functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF-0185. 3. In skinned fibres, Dan inhibited Ca(2+)-induced Ca(2+) release (CICR) under Mg(2+)-free conditions at room temperature. In contrast, GIF-0082 and GIF-0185 showed no inhibitory effect on CICR under the same conditions. 4. Dan-induced inhibition of CICR was not affected by the presence of GIF-0082, whereas it was diminished in the presence of GIF-0185. 5. GIF-0082 and GIF-0185 significantly inhibited clofibric acid (Clof)-induced Ca(2+) release, as did Dan. 6. Several Dan derivatives other than GIF-0082 and GIF-0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof-induced Ca(2+) release. 7. The magnitudes of inhibition of Clof-induced Ca(2+) release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof-induced opening of the RyR-Ca(2+) release channel may be similar to that of physiological Ca(2+) release (PCR). 8. These results indicate that the difference in opening modes of the RyR-Ca(2+) release channel is recognized by certain Dan derivatives.     

Etiology of sarcoplasmic reticulum calcium release channel lesions in doxorubicin-induced cardiomyopathy.

Alterations in the native function of the ryanodine-sensitive Ca2+ release channel complex of sarcoplasmic reticulum (SR) isolated from rat cardiac ventricles during acute and chronic exposure to doxorubicin are examined. Compared to control SR, actively loaded SR from animals exposed to a single intravenous dose of doxorubicin exhibit faster rates of doxorubicin-induced Ca2+ release and the occupancy of [3H]ryanodine is significantly enhanced with subsequent exposure of SR membranes to doxorubicin in vitro. One week after acute exposure to doxorubicin in vitro, the EC50 for activation of the binding of [3H]ryanodine by Ca2+ is not significantly different from control SR. However, the persistence of doxorubicin-sensitized SR channels appears to be latent since repeated exposure to doxorubicin in vitro significantly enhances receptor occupancy in SR obtained from the treated rats compared to control SR. Ryanodine receptors from rats chronically exposed to doxorubicin consistently exhibit a higher sensitivity to activation Ca2+ which persists at least 4 weeks following the last injection of drug. Chronic exposure produces a concomitant reduction in the capacity of [3H]ryanodine binding sites. The marked decrease in receptor density observed with SR from doxorubicin-treated rats coincides with significant reduction in body weight, suggesting a possible influence of nutrition. However, sodium dodecyl sulfate polyacrylamide electrophoresis indicates no significant loss of the high molecular weight subunit of the ryanodine receptor, suggesting that loss of [3H]ryanodine-binding capacity may be the result of progressive and permanent channel desensitization. Consistent with desensitized receptors, membrane vesicles prepared from rats chronically exposed to doxorubicin take up significantly more Ca2+ and exhibit significantly reduced rates of doxorubicin or Ca2+/ryanodine induced Ca2+ release. The data demonstrates (i) doxorubicin inflicts cumulative SR channel lesions in vivo, (ii) a persistent sensitization of the SR channel to activation by Ca2+ and (iii) a significant and apparently irreversible reduction in the number of functional channel complexes.     

Effects of 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on skinned myocardial fibres of the rat: reversible inhibition of calcium release from the sarcoplasmic reticulum

The effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is reported to inhibit the release of intracellularly stored Ca²⁺ in skeletal and smooth muscles, were examined in ventricular myocardia of the adult rat. In skinned papillary muscle fibres with functional sarcoplasmic reticulum (SR) preserved, application of 100 or 300 M TMB-8 during the Ca²⁺ loading period had no significant effect on the peak tension of subsequent caffeine-induced contraction, but when applied during exposure to caffeine, concentration-dependent reduction of the peak tension was observed. At 1000 M, TMB-8 reduced the peak tension of caffeine-induced contraction when applied either during Ca²⁺ loading or during exposure to caffeine. TMB-8 had no substantial influence on the Ca⁺-tension of skinned fibres without functional SR. In isolated papillary muscle preparations, TMB-8 prolonged the action potential duration and decreased the maximum rate of rise of potential, leading to abolition of contraction at 100 M. In conclusion, TMB-8 may be a useful pharmacological tool for inhibiting Ca²⁺ release from SR, but only in skinned myocardial preparations.     

Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel.

In this paper, we study the modulation of the rabbit fast twitch skeletal muscle calcium release channel by assaying the kinetics of [3H]ryanodine binding, 45Ca2+ flux, and single-channel activity. The effects of modulators of the Ca2+ release channel (confirmed here with both flux and single-channel data) were examined for effects on [3H]ryanodine binding to terminal cisternae vesicles. We find that activators of the release channel, such as adenine nucleotides (1 mM) and caffeine (1 mM), enhance the rate of association of [3H]ryanodine, whereas inhibitors, such as Mg2+ (1 mM) and ruthenium red (100 nM), decrease the rate of association. High concentrations of either ryanodine or ruthenium red, which close the channel, slow the dissociation of [3H]ryanodine, suggesting that at these concentrations the inhibitory effects of both ryanodine and ruthenium red occur as the result of binding at a site distinct from but interacting cooperatively with the high affinity site. Our data are consistent with a model in which the high affinity ryanodine binding site is within a conformationally sensitive area of the channel, such that conditions that open the channel (ATP, caffeine, etc.) enhance the rate at which [3H]ryanodine reaches its binding site and other conditions that close the channel (the binding of ryanodine and ruthenium red to a low affinity site) slow the dissociation of [3H]ryanodine from the high affinity site. Some conditions that inhibit channel activity (high concentrations of Mg2+ and Ca2+) slow association but do not affect dissociation of bound [3H]ryanodine, suggesting a completely different state of the channel from that which is inactive in the presence of high concentrations of ryanodine or ruthenium red. In summary, the functional state of the fast twitch skeletal muscle calcium release channel can be characterized by the changes in the kinetics of [3H]ryanodine binding. Different modulators (activators/inhibitors) affect different aspects of ryanodine binding (association/dissociation).     

Effect of R56865 on cardiac sarcoplasmic reticulum function and its role as an antagonist of digoxin at the sarcoplasmic reticulum calcium release channel.

1. The effect of R56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl-2- benzothiazolamine) on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel function was investigated. The effect of R56865 on [3H]-ryanodine and [3H]-digoxin binding to SR vesicles and its effect on the ATP-stimulated 45Ca2+ uptake into SR vesicles was also studied. 2. R56865 (0.5-50 microM) had no effect on single-channel open probability (Po) when added to native cardiac SR Ca(2+)-release channels, incorporated into planar phospholipid bilayers, that had previously been activated by 10 microM Ca2+. The single-channel conductance (93 pS) and the Ca2+/Tris+ permeability ratio (12.5) were also unaffected by R56865. 3. R56865 failed to affect the rapid Ca(2+)-induced efflux of 45Ca2+ from cardiac SR vesicles. The initial efflux rate at an extravesicular [Ca2+] of 0.1 microM was 176 +/- 33 nmol 45Ca2+ mg-1 protein s-1 (n = 5). Addition of 0.5-50 microM R56865 to the efflux solution did not affect the initial efflux rate or the total amount of 45Ca2+ released from the vesicles. 4. The specific binding of [3H]-ryanodine to SR vesicles can be viewed as a marker for SR Ca(2+)-release channel activation. R56865 (0.05-50 microM) did not change the amount of specific [3H]-ryanodine bound at 10 microM activating Ca2+. Taken together these data (points 2, 3 and 4) suggest that R56865 does not affect the Ca2+ activation of the cardiac SR Ca(2+)-release channel. 5. R56865 (0.5-50 microM) decreased the ATP-stimulated uptake of 45Ca2+ into cardiac SR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)