The ability of a number of
calcium antagonistic drugs including
nitrendipine,
D600, and D890 to block
contractures in single skinned (sarcolemma removed) muscle fibers of the frog Rana pipiens has been characterized.
Contractures were initiated by ionic substitution, which is thought to depolarize resealed transverse tubules in this preparation. Depolarization of the transverse tubules is the physiological trigger for the release of
calcium ion from the sarcoplasmic reticulum and thus of
contractile protein activation. Since the transverse tubular membrane potential cannot be measured in this preparation, tension development is used as a measure of activation. Once stimulated, fibers become inactivated and do not respond to a second stimulus unless allowed to recover or reprime (Fill and Best, 1988). Fibers exposed to
calcium antagonists while fully inactivated do not recover from inactivation (became blocked or paralyzed). The extent of
drug-induced block was quantified by comparing the height of individual
contractures. Reprimed fibers were significantly less sensitive to block by both
nitrendipine (10 degrees C) and
D600 (10 and 22 degrees C) than were inactivated fibers. Addition of
D600 to fibers recovering from inactivation stopped further recovery, confirming preferential interaction of the
drug with the inactivated state. A concerted model that assumed coupled transitions of independent
drug-binding sites from the reprimed to the inactivated state adequately described the data obtained from reprimed fibers. Photoreversal of
drug action left fibers inactivated even though the
drug was initially added to fibers in the reprimed state. This result is consistent with the prediction from the model. The estimated KI for
D600 (
at 10 degrees and 22 degrees C) and for D890 (
at 10 degrees C) was approximately 10 microM. The estimated KI for
nitrendipine paralysis of inactivated fibers
at 10 degrees C was 16 nM. The sensitivity of reprimed fibers to
paralysis by
D600 and D890 was similar. However, inactivated fibers were significantly less sensitive to the membrane-impermeant derivative (D890) than to the permeant species (
D600), which suggests a change in the
drug-binding site or its environment during the inactivation process. The enantomeric
dihydropyridines (+) and (-)
202-791, reported to be
calcium channel agonists and antagonists, respectively, both caused
paralysis, which suggests that blockade of a transverse tubular membrane
calcium flux is not the mechanism responsible for antagonist-induced
paralysis. The data support a model of excitation-contraction coupling involving transverse tubular
proteins that bind
calcium antagonists.