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Three phases in the inotropic response of acetyl strophanthidin (AcS) on the electromechnical activity of the frog ventricular myocardium were identified and studied using a single sucrose voltage-clamp technique and other conventional electrophysiological methods. 2. the positive inotropic response of the drug was accompanied by a shift in tension-voltage relation, so that more tension developed with every depolarization step above the mechanical threshold (-50mV). Only at higher drug concentrations or with long exposure times did the mechanical threshold shift to more negative membrane potentials (-60 to-70 mV). 3. In tetrodotoxin-treated muscles AcS produced marked potentiation of twitch tension and an appropriate shift in the tension-voltage relation. 4. the positive inotropic response of the drug was not related to the magnitude of the direction of the fast or slow Na current. 5. in tetrodotoxin-treated ventricular strips the direction or the magnitude of the secondary inward current (ICa or INa) were not related to the inotropic effect of AcS. 6. AcS shortens the action potential markedly during the later stages of its positive inotropic response. When Ca2+ is omitted from the bathing solution AcS not only fails to shorten the action potential, but often prolongs it. 7. The shortening of the action potential in the presence of AcS is accompanied by an increase in the "instantaneous" membrane conductance both at rest and during the time course of the plateau. 8. The decline in the positive inotropic response of the drug was accompanied by the shortening of the action potential. Electrical or chemical prolongation of the action potential restored the full positive inotropic response if the membrane had not depolarized. 9. Membrane depolarization and the development of diastolic tension always occurred at later stages of drug action. Elevation of [Mg+2]degrees to 5 or 10 mM prevented or suppressed the membrane depolarization and the diastolic tension. 10. KCl-induced contractures were potentiated throughout the duration of drug exposure. The tonic component of the contracture tension was markedly elevated especially at later stages of drug action. 11. The experimental evidence suggests that no unitary mechanism could account for multiple actions of acetyl strophanthidin. However, the contributions of the Na pump, the Ca+2 sequestering system, and the K-efflux system to the various stages of drug action are discussed.