Activation of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by analogues of sulmazole.
1. The effect of sulmazole and several structurally related analogues on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel gating and on [3H]-ryanodine binding to isolated SR membrane vesicles has been investigated. 2. The optical isomers, (+)- and (-)-sulmazole, increased the open probability (Po) of single Ca(2+)-release channels incorporated into phospholipid bilayers held under voltage clamp by increasing the frequency and duration of open events. The respective EC50s were 423 microM and 465 microM at 10 microM activating cytosolic Ca2+ and the Hill coefficients for activation were approximately two, suggesting that at least two molecules of either enantiomer are required to bind for channel activation. 3. Similarly the related enantiomers, (+)- and (-)-isomazole, which differ from sulmazole in the position of the pyridine nitrogen (4.5b for sulmazole; 4.5c for isomazole), were approximately as potent as each other and as potent as the isomers of sulmazole with EC50s of approximately 445 microM. 4. In contrast, EMD 46512 and EMD 41000, which are sulmazole and isomazole analogues respectively, each with the methylsulphinyl oxygen removed, increased single-channel Po with EC50s of 42 microM and 40 microM. The open and closed lifetime distributions were similar to those of the less potent analogues and the Hill coefficients were the same, suggesting that these compounds act at the sulmazole site on the Ca(2+)-release channel. 5. All of the compounds tested were able to increase the Po of channels in the absence of activating Ca2+ but were less potent than in the presence of Ca2+. The drugs were effective only when added to the cytosolic face of the channel. None of the drugs could fully activate the channel in the absence of Ca2+,partly due to only one drug molecule binding in the absence of Ca2+, which is in contrast to the situation when activating Ca2+ is present. This suggests a synergistic action of these drugs and Ca2+ in Ca2+-release channel activation.6. EMD 46512 and EMD 41000 increased [3H]-ryanodine binding to HSR vesicles with Hill coefficients of approximately two and EC50s of 25 MicroM and 28 MicroM, respectively, at 10 MicroM Ca2+. These drugs also increased [3H]-ryanodine binding to HSR vesicles at PM Ca2+ but with Hill slopes of only one and EC50s of 112 and 133 MicroM for EMD 46152 and EMD 41000, respectively. In addition, maximal binding was reduced at PM Ca2+ in comparison to 10 MicroM Ca2+.7. These data show that analogues of sulmazole increase the PO of the cardiac SR Ca2+-release channel and this occurs as the result of an increase in the frequency and duration of open events. They also demonstrate that the activation of the channel by these drugs is not stereoselective and therefore the configuration of the oxygen atom or methyl group attached to the sulphur atom does not affect their ability to elicit their effect. Similarly, the results show that the nitrogen in the 4, 5b or 4, Sc position of the pyridine ring does not affect Ca2+-dependent or Ca2+-independent activation of the Ca2+-release channel. However, removal of the methylsulphinyl oxygen in sulmazole and isomazole results in two drugs which display a ten fold increase in potency over their respective parent compound in the activation of the Ca2+-release channel. It is apparent that minor modifications of the sulmazole or isomazole molecules around the terminal sulphur atom dramatically affect potency but not maximal attainable effect, suggesting that the area around the sulphur atom may be critically involved in channel activation.
Department of Cardiac Medicine, National Heart and Lung Institute, University of London.
S J
SJ
McGarry
A J
AJ
Williams
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D066298:In Vitro Techniques; D009206:Myocardium / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012756:Sheep; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D002316:Cardiotonic Agents; D007093:Imidazoles; D012433:Ryanodine; D002110:Caffeine; C029758:sulmazole