Diverse potentiators of contraction have basically identical, activestate mechanical effects, but act by different membrane-mediated electromechanical coupling mechanisms. The falling phase of the action potential is greatly prolonged by Zn(2+) (0.1 mM) and UO(2)(2+) (0.5 to 1 microM), neither of which affects the mechanical threshold. Caffeine (1 mM), like the lyotropic anions, acts conversely. Thus changes in the duration and mechanical threshold of the action potential determine independent electromechanical coupling processes which can act individually, or conjointly in the action of other potentiators, in determining the duration of the active state and thus the potentiation of twitch tension.

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ~Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ~Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on 4~Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ~Ca by normally polarized muscle. However, there are variable effects of quinine upon ~Ca uptake in potassium-depolarized muscle. Quinine (2 raM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 raM) and quinine (2 mM) act to release ~Ca from muscles that have been washed in Ringer's solution from which Ca was omitted and to which EDTA (5 inM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca ~+ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K + or an action potential. In respect to the myoplasmic Ca 2+ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.Caffeine and quinine affect frog skeletal muscle similarly in some respects. In low concentrations (caffeine, 1 mM; quinine, 0.1 raM) they increase the tension output of the twitch; i.e., they are twitch potentiators (for review, see , whereas in somewhat higher concentrations they not only potentiate the twitch but also engender contracture, though with considerable variability (caffeine: Axelsson

Zn ++ potentiates the twitch tension of the frog's sartorius muscle by as much as two to three times, and prolongs twitch time parameters. Tetanus tension is unchanged, but fusion frequency is reduced. Thus, the basic mechanical effect of Zn ++ is prolongation, but not intensification, of the active state. Threshold effects appear at about 0.005 m~,i Zn ++, and maximal changes at 0.05 raM. In 0.05 m~t, potentiation begins after a delay of about 1 min. and develops with half-time of 6 rain.; full changes reverse in pure Ringer's with halftime of 60 min. Diffusion theory analysis of these kinetics indicates that the potentiating action of zinc involves special features not found in potentiation by anions: a primary membrane action is not excluded, but Zn ++ may have to penetrate to and act at sarcoplasmic reticular or myoplasmic sites. Zn ++ does induce excitatory membrane effects: the fall of the action potential is slowed and the refractory period is increased about four times; but excitability as such is not changed. Unique chemical features of Zn ++ are discussed in relation to possible mechanisms of its action on muscle fibers.We have developed the research of this paper in consequence of an unexpected observation made while studying certain photodynamic effects of basic dyes on muscle. The genesis of this observation, and preliminary results of the present work, are discussed in detail in a previous report (Sandow and Isaacson, 1960). In brief, we found that acridine orange caused not only photodynamic effects, but also a very large potentiation of the twitch tension of the directly stimulated frog's sartorius muscle without having any significant effect on the tetanus output. Our acridine orange contained zinc on a mole to mole basis (Beers, 1960), and special control tests proved that the twitch augmentation depended merely on the presence of the metal in the dye and it could be evoked in muscles treated simply with 1 mM ZnCI~ in Ringer's solution.Quite a large number of chemically diverse substances are known to potentiate the twitch (e.g., bromide, nitrate, and iodide (Kahn and Sandow, 1950, 1955; Hill and Macpherson, 1954); quinine and quinidine (Harvey, 1939;

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In addition to the previously studied Zn ~+, low concentrations (about 0.5 mM) of Be g+, Ba ~+, Cd 2+, Ni ~+, Cu 2+, Pt 4+, and, outstandingly, 0.5 /~ of ~+ UOa , potentiate the twitch of frog sartorius and toe muscles by prolonging the active state of contraction. The degree of potentiation is a roughly S-shaped function of p(metal2+), suggesting that each metal binds to a ligand of the muscle fiber, representative apparent affinity constants being: UO~ +, 5 X 106; Zn ~'+, 2.8 X 105; and Cd 2+, 2 X 104. UO~ + potentiation effects are rapidly reversed by PO4, and Zn 2+ and Cd 2+ effects by EDTA, PO4, and cysteine. The rapidity of these reversals by the nonpenetrating EDTA and PO~, and the fact that heavy metal ions evidently potentiate by prolonging the action potential, indicate that the metal potentiators exert their primary action at readily accessible (i.e. plasma and T tubular) membrane sites. The relatively slow kinetics of development of potentiation, and the even slower reversal of it in pure Ringer's solution, indicate that the metal ions are bound to connective tissue, as well as to muscle fibers. The binding effects at the readily accessible membrane sites evidently impairs delayed rectification and thus modifies the action potential and excitation-contraction coupling so as to cause potentiation. SH is excluded, and PO4 and imidazole are possibilities, as the membrane ligand binding the potentiating metal ions.Contraction of skeletal muscle is potentiated by many chemically diverse substances which include a group of divalent heavy metals, exemplified especially by zinc ; for general review of potentiation, see . All these agents typically augment the isometric twitch by as much as 2 to 3 times, but do not significantly change the peak tetanus tension (for certain exceptions see Brust, 1965). In our previous work we attempted to locate the site of action of Zn by using techniques which involved the kinetics of development and reversal of twitch potentiation and located the primary site of action of the anionic potentiators NO~-and I -at either the plasma, or T tubular, membrane (Kahn and Sandow, 1950, 1955;Hill