Anthopleurin-A

Background:Previous studies have showed that the interval between the peak and the end of the T wave (Tp-e) is a marker of transmural dispersion of ventricular repolarization.Objective:We studied the relationship between (a) the Tp-e on local pseudo transmural electrograms (pseudo transmural Tp-e) or limb leads of body surface electrocardiogram (surface Tp-e) and (b) the intracardiac left ventricular (LV) repolarization during a drug-induced QT-interval prolongation.Methods:Using open-chested canine intact hearts treated by anthopleurin-A, transmural LV electrograms were recorded via needle electrodes placed in the basoanterior, midanterior, apicoanterior, basolateral, midlateral, and apicolateral LV wall. Recovery time (RT) was calculated as an index of local repolarization at each transmural unipolar electrode.Results:This model showed slower heart rate-dependent heterogeneous distribution of ventricular repolarization both along the basal to apical axis and along the transmural axis. RT was longer at the LV apex than at the base and longer in the lateral than in the anterior wall during the slower heart rate. A high correlation was found between surface Tp-e and total LV dispersion. In contrast, pseudo transmural Tp-e correlated with transmural RT dispersion. The shortest RT in the heart roughly corresponded to the peak, as did the longest RT with the end of the T wave on the surface electrocardiogram.Conclusion:During drug-induced QT-interval prolongation with a large apicobasal and anterolateral dispersion of ventricular repolarization, the Tp-e in the limb leads expresses spatial (total) distribution of repolarization in the whole left ventricle.

Anthopleurin-A stimulated the initial rate of 201thallium uptake by isolated adult rat heart cells by a factor of 3.41 +/- 0.56, and induced a unique pattern of spontaneous beating activity. Ouabain inhibited the basal uptake rate by 58 +/- 11% and all the anthopleurin-A stimulated rate. The Km for thallium uptake was 0.95 +/- 0.26 mM, and was not changed by anthopleurin-A. Accumulated thallium was quickly released from cells by EDTA addition. Such release was inhibited 87 +/- 10% by verapamil. Thallium reuptake was initiated by restoration of magnesium to the medium. Reuptake was mostly inhibited by ouabain, but the residual ouabain-insensitive uptake remained. The ouabain-insensitive uptake was inhibited by ATP depletion. Anthopleurin-A stimulated the rate of 22Na entry into cells by a factor of 3.17 +/- 1.65, and EDTA stimulated the rate of entry by a factor of 29.5 +/- 13.0. The EDTA-induced 22Na entry was inhibited 86 +/- 11% by verapamil. From this we draw three conclusions: The major pathway for thallium uptake is the Na-K pump. The rate of uptake by this route, like the rate of K+ uptake, is governed by the rate of cellular sodium influx; A residual ouabain-insensitive uptake route also exists which appears to require ATP but not a monovalent ion gradient; Removal of Mg and Ca induces a verapamil-sensitive monovalent channel activity which is both massive and reversible.

Effects of Anthopleurin-A (AP-A, polypeptide from sea anemone) were studied on electrophysiological properties of isolated canine Purkinje and ventricular muscle fibers. Ap-A (in concentrations above 20 micrograms/l) produced a dose-dependent increase in action potential duration (APD) and the refractory period (RFP) in electrically driven Purkinje fibers, but had no effect on other parameters. Similar but less prominent change in APD and RFP was observed in ventricular muscle fibers. AP-A in high concentrations (200 micrograms/l or higher) did alter the spontaneous firing rate of Purkinje fibers. Since AP-A in low concentrations will increase the refractory period of conducting fibers without affecting a conduction velocity, it may abolish some re-entrant arrhythmias.

The sea anemone polypeptide anthopleurin-A (AP-A) at nanomolar concentrations enhances myocardial contractility without affecting automaticity. It has a therapeutic index higher than that of the digitalis glycosides, and may serve as a molecular model for designing a new class of inotropic drugs acting on the myocardial Na channel at site 3. AP-A is a 49 residue peptide crosslinked by three disulfide bonds; its tertiary structure has been determined by NMR. Here we report the solid-phase synthesis of this polypeptide. Synthetic AP-A displayed CD and NMR spectra identical with those of the natural toxin; it possessed 94 +/- 15% of the inotropic activity of natural AP-A. Therefore, it is feasible to prepare various type 1 sea anemone toxin analogs by solid-phase chemical synthesis in order to identify side chains important for peptide folding and interaction with sodium channels.

The effects of anthopleurin-A (AP-A, 1 x 10(-8) M) on the membrane action potential and contraction of isolated rabbit ventricular muscle were compared with those of ouabain (5 x 10(-7) M). Under control conditions, AP-A and ouabain showed submaximal (about 80% of maximal) positive inotropic effects without any toxic manifestations. AP-A caused a marked prolongation of action potential duration (APD) without affecting any other parameters of the action potential, whereas ouabain caused a shortening of APD and a slight decrease in maximum diastolic potential (MDP), overshoot (OS), and upstroke velocity (dV/dtmax) of the action potential. The positive inotropic effect of AP-A was relatively well maintained even under hypoxia or in glucose-free medium. Under these abnormal experimental conditions, AP-A caused a prolongation of APD only at the late stage of repolarization (APD80), whereas APD at the early stage of repolarization (APD30) was further shortened. Other parameters of action potential and resting tension (RT) were not influenced by AP-A. In contrast, under similar experimental conditions, ouabain caused no apparent positive inotropic action, but resulted in a marked increase in RT (contracture). In addition, after exposure to ouabain, a progressive shortening of APD and marked decreases in MDP, OS, and dV/dtmax were observed. These results indicate the AP-A has pharmacological properties quite different from those of ouabain and suggest possible advantages of this newly developed cardiotonic agent over cardiac glycosides when acting on the energy-depleted myocardium.

Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution. Using 1H- and 13C-NMR spectroscopy, we have now shown that this conformational heterogeneity arises from cis-trans isomerization about the Gly 40-Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts a cis conformation. Furthermore, the increased sensitivity afforded by higher-field NMR has allowed identification of additional minor conformations of AP-A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high-field NMR of previously unobserved minor conformations of proteins in solution.

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na+ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was -140 mV and test potentials ranged from -100 to +40 mV (pulse duration 100 or 1000 ms). AP-A (50-100 nM) markedly slowed the rate of decay of Na+ current (INa) and increased peak INa conductance (gNa) by 38 +/- 5.5% (mean +/- SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the gNa/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state INa availability (h infinity) demonstrated an increase in slope factor from 5.9 +/- 0.8 mV in control to 8.0 +/- 0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified INa showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (-15 +/- 2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (-3 +/- 1%). This use-dependent effect was probably the result of a decrease in the recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1 +/- 0.3 ms in control to 6.0 +/- 1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2 +/- 6.5 ms in control to 188.9 +/- 36.4 ms (P < 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca2+ or Mg2+) to 10 mM abolished the effects of AP-A on the rate of INa decay. These results demonstrate that modification of cardiac Na+ channels by AP-A markedly slowed INa inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in gNa presumably by increasing the number of channels open at peak INa. AP-A slows the rate of recovery of INa from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

1 Anthopleurin-B (AP-B, greater than 3 x 10(-9) M), a newly isolated polypeptide from sea anemone (Anthopleura xanthogrammica), caused powerful rhythmic contractions in the guinea-pig isolated vas deferens. The other polypeptides anthopleurin-A from A. xanthogrammica and anthopleurin-C from A. elegantissima, elicited similar effects but in higher concentrations ( less than 5 x 10(-8) M). Toxin II (10(-6) M) isolated from the sea anemone, Anemonia sulcata, had no effect. 2 The rhythmic contractions induced by AP-B were inhibited by phentolamine, bretylium, guanethidine, reserpine, 6-hydroxydopamine, tetrodotoxin (TTX) and verapamil. Mecamylamine, atropine, methysergide, chlorpheniramine, and indomethacin had no effect. 3 AP-B (10(-8) M approximately 10(-5) M) caused a dose-dependent increase in the amount of endogenous noradrenaline (NA) released from the vas deferens. AP-B (10-5M) increased the amount of NA released to approximately 310 times (12 micrograms/g tissue) that of untreated tissues. 4 The AP-B-induced release of NA was inhibited or abolished by TTX, verapamil, or incubation in Ca-free medium. 5 These results suggest that the AP-B-induced rhythmic contraction of the vas deferens is mediated through the release of NA from adrenergic nerve endings; AP-B is one of the most potent substances in stimulating NA release from the vas deferens.

The positive inotropic effect of anthopleurin-A (AP-A) was studied in vitro on isolated cat heart papillary muscles and in vivo in anesthetized and conscious dogs. In vitro, in low Ca2+ solution (1.27 mM), AP-A increased the force of contractions of isolated cat heart papillary muscles at concentrations from 0.2 x 10(-8) M and higher; on a molar basis, AP-A was more than 200 times as potent as digoxin and on a weight basis, 33 times as potent. In vivo in anesthetized dogs, AP-A at 0.2 microgram/kg/min i.v. increased myocardial contractile force; the geometric mean dose of AP-A required to increase the contractile force by 25% was 2.6 micrograms/kg; the corresponding dose of digoxin (infused at 2.8 micrograms/kg/min) was 107.4 micrograms/kg. The geometric mean lethal dose of AP-A for 8 dogs was 19.3 and that of digoxin 263.2 micrograms/kg i.v. The therapeutic index of AP-A was significantly higher than that of digoxin. All animals that received either AP-A or digoxin died in ventricular fibrillation. The reversal of t-wave was typical for AP-A. As measured by left ventricular pressure telemetry, AP-A, 2 micrograms/kg i.v. single dose, increased LV dp/dt max in conscious dogs for longer than 2 hr.

Background:The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains.Results:AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures.Conclusions:We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.