Molecular Pharmacology, Vol 19, 68-77, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Effect of the Cardioactive Drug AR-L 57 on Free Intracellular Calcium and Ouabain-Insensitive Ion Fluxes in the Squid Giant Axon

¹ Institut für Phrarmakologie und Toxikologie der Technischen Universität München, D-8000 München 40, West Germany, and Department of Physiology, King’s College, University of London, London WC2R 2LS, England

The cardioactive drug AR-L 57 [6-(2,4-dimethoxyphenyl)-imidazo-(4,5-)-pyridine] causes a rapid and reversible reduction of calcium efflux and light output from axons of Loligo forbesi which received microinjections of both ⁴⁵Ca and the calcium-sensitive photoprotein aequorin. The light output from aequorin injected into extruded axoplasm is also reduced by AR-L 57, but the drug does not inhibit the light from an aqueous buffer solution containing aequorin. The effect on calcium efflux is prevented if the axon is injected with enough calcium-ethylene glycol bis(-aminoethyl ether)-N,N'-tetraacetic acid to buffer the intracellular ionized calcium close to the physiological level. The large Na₀- and Ca₀-dependent calcium efflux from axons poisoned with cyanide or carbonyl-cyanide p-trifluoromethoxyphenyl-hydrazone is insensitive to AR-L 57. These results indicate that AR-L 57 inhibits the calcium efflux from unpoisoned axons by increasing intracellular calcium binding. AR-L 57 has no noticeable direct effect on the axolemmal calcium pump of unpoisoned axons or on the calcium efflux system operating in poisoned axons. AR-L 57 inhibits the calcium influx from lithium-artificial seawater and the Ca₀-dependent sodium efflux from axons injected with ²²Na and superfused with sodium-poor (lithium or potassium) seawater. The light response evoked in axons injected with aequorin by a potassium depolarization in the presence of a high external calcium concentration is reversibly reduced by AR-L 57. It is concluded that AR-L 57 inhibits calcium entry both via sodium-calcium exchange and through the voltage-sensitive late calcium channel. AR-L 57 does not affect the ⁸⁶Rb efflux at 10 mmoles per liter [K]₀ but reversibly inhibits the increased rubidium efflux from axons depolarized by 410 mmoles of potassium per liter. The outward (potassium) current is inhibited by the drug both with time and voltage during a voltage-clamp depolarization, whereas the transient inward (sodium) current is unaffected. However, AR-L 57 does inhibit the maintained tetrodotoxin-sensitive ²²Na efflux through sodium channels kept open by veratridine. The normal sodium inactivation mechanism appears to protect the sodium channels from blockade by AR-L 57. None of the effects on the axolemma reported here, if applied to the sarcolemma of myocardial cells, provides an obvious explanation for the cardiotonic action of AR-L 57. Although AR-L 57 has been shown previously to inhibit the sodium pump, the present results show that the drug affects a number of ion transport processes that are insensitive to cardiac glycosides.

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ACKNOWLEDGMENTS We wish to thank the director and staff of the Laboratory of the Marine Biological Association, Plymouth, England, for providing material and facilities for this work. The authors are grateful to Dr. G. C. Malachowski and Dr. D. van Helden for performing the voltage-clamp experiments.