ryanodine and 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 o

Topics: Animals; Caffeine; Calcium Channels; Cardiotonic Agents; Imidazoles; In Vitro Techniques; Myocardium; Ryanodine; Sarcoplasmic Reticulum; Sheep; Stereoisomerism; Structure-Activity Relationship

The properties of calcium-release channels of sheep cardiac muscle junctional sarcoplasmic reticulum (SR), have been investigated under voltage-clamp conditions following the fusion of isolated membrane vesicles with planar phospholipid bilayers. In the presence of activating calcium on the cytosolic side of the membrane, additions of the benzimidazole derivative sulmazole (AR-L 115BS) increased the open probability (Po) of the channel reaching saturating values of 1.0 at 3 mM sulmazole. The drug did not affect single-channel conductance and activation was readily reversible. Analysis of channel open and closed lifetimes suggested that low concentrations of sulmazole (0.1 mM) may sensitize the channel to activating calcium, while at higher concentrations (1 mM and above), calcium and sulmazole act synergistically to produce a unique gating scheme for the channel. Millimolar concentrations of sulmazole also stimulate a degree of channel opening at subactivating (60 pM) calcium concentrations. Openings occurring under these conditions show very different kinetics to those of the calcium-activated channel but have an identical single-channel conductance and are modified by ATP, magnesium, ruthenium red and ryanodine in a similar manner to the calcium-activated channel. The release of calcium from the SR following the activation of the calcium-release channel by sulmazole may contribute to the positive inotropic action of this drug on mammalian cardiac muscle.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Cardiotonic Agents; Imidazoles; Magnesium; Membrane Potentials; Myocardium; Ruthenium Red; Ryanodine; Sarcoplasmic Reticulum; Sheep

[3H]Ryanodine binding to a preparation of isolated cardiac sarcoplasmic reticulum has been investigated. A method is reported which produces a very high level of specific binding. Scatchard analysis of binding up to 50 nM ryanodine yields data which infer a single class of binding sites with a Kd of 1.4 nM and a Bmax of 9.7 pmol/mg protein. Micromolar calcium is the principal activating ligand and its effects on binding are modulated by ligands which similarly affect the activity of single calcium-release channels incorporated into artificial planar phospholipid bilayers. The benzimidazole drug, sulmazole, is able to stimulate ryanodine binding in the presence of sub-activating calcium concentrations. Ryanodine binds to the native channel only when it is in its open state and stimulation of maximal ryanodine binding is achieved by ligands which are insufficient to produce full single-channel activation. A model is proposed which relates the modulation of ryanodine binding to the behaviour of single channels.

Topics: Alkaloids; Animals; Binding Sites; Calcium; Calcium Channels; Cardiotonic Agents; Electric Conductivity; Imidazoles; Kinetics; Lipid Bilayers; Myocardium; Ryanodine; Sarcoplasmic Reticulum; Sheep

Ca2+ exchange has been measured in a suspension of rat ventricular myocytes treated with digitonin or saponin to render the sarcolemma permeable to small molecules and ions. Two fractions of exchange were identified, one that was attributed to the mitochondrial component of the cell and the other to a non-mitochondrial fraction. Mitochondrial Ca2+ uptake was blocked by sodium azide and depended on respiratory substrates whereas non-mitochondrial uptake occurred independently of these molecules but was dependent on ATP and creatine phosphate. Non-mitochondrial Ca2+ uptake could be induced at a Ca2+ concentration below 1 microM and the initial rate increased with concentration up to 100 microM. Uptake could be reversed by sulmazole (a caffeine-like substance) and this reversal in turn inhibited by ryanodine. These properties suggest that the major locus for non-mitochondrial Ca2+ exchange is at the sarcoplasmic reticulum. Ca2+ exchange could be modulated by a number of agents, including carnosine, but was unaffected by others, including Na+, inositol trisphosphate and cyclic AMP. A kinetic model of the data is presented, which incorporates similar data of Ca2+ uptake into the mitochondrial fraction. The rates of Ca2+ exchange measured in these experiments suggest that these two components of the cell can reduce the sarcoplasmic Ca2+ concentration rapidly enough to account for the observed transient nature of the isometric twitch. Furthermore, it is suggested that both non-mitochondrial and mitochondrial fractions of the cell could significantly contribute to tension relaxation in rat cardiac muscle.

Topics: Animals; Calcium; Cardiotonic Agents; Cells, Cultured; Cyclic AMP; Digitonin; Energy Metabolism; Ethylmaleimide; Heart Ventricles; Imidazoles; Kinetics; Mitochondria, Heart; Myocardium; Oxygen Consumption; Rats; Ryanodine; Sarcoplasmic Reticulum

[8]-Gingerol (gingerol), a component of ginger, produced a concentration-dependent positive inotropic effect on guinea pig isolated left atria at concentrations of 1 X 10(-6) to 3 X 10(-5) M. Gingerol also exhibited positive inotropic and chronotropic effects on guinea pig right atria. The gingerol-induced inotropic effect was abolished by ryanodine, but was little affected by propranolol, chlorpheniramine, cimetidine, tetrodotoxin, diltiazem or reserpine. The time to peak tension and relaxation time within a single contraction were shortened by gingerol (1 X 10(-5) M) as well as isoproterenol, whereas they were prolonged by BAY K 8644. In guinea pig isolated atrial cells, gingerol (3 X 10(-6) M) caused an increase in the degree and the rate of longitudinal contractions. In guinea pig left atria, gingerol (1 X 10(-6) to 3 X 10(-5) M) gave little influence on the action potential, although it increased the contractile force of the atria. The whole-cell patch-clamp experiments showed that the slow inward current was little affected by gingerol (1 X 10(-6) to 3 X 10(-5) M) in voltage-clamped guinea pig cardiac myocytes. The measurement of extravesicular Ca++ concentration using a Ca++ electrode indicated that gingerol (3 X 10(-6) to 3 X 10(-5) M) accelerated the Ca++ uptake of fragmented sarcoplasmic reticulum (SR) prepared from canine cardiac muscle in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium-Transporting ATPases; Cardiotonic Agents; Catechols; Electrophysiology; Fatty Alcohols; Guinea Pigs; Imidazoles; Male; Myocardial Contraction; Myocardium; Ryanodine; Sarcoplasmic Reticulum