On the sarcolemmal site of action of cardiac glycosides

An electron microscopic study of the guinea pig myocardial cell was conducted. By the use of LaC13, two morphologically distinct tubular systems could be demonstrated. The larger one represents the transverse tubular system which originates from the plasma membrane and is continous with the extracellular space (ECS). In the heart muscle cell it forms a three-dimensional network and it closely apposed to the myofilaments at the level of the Z and I bands. A comparatively small and sparsely developed tubular system which is not accessible from the ECS represents the sarcoplasmic reticulum (SR). By means of sucrose-density centrifugation in a discontinuous gradient two main microsomal fractions were obtained (F1 and F3). The pre-existing structure of F1 could be identified as the transverse tubular system, whereas F3 originates from the SR. The morphological features (e. g., an enclosed basement membrane) and the fact that after perfusion of hearts with [3H] inulin and preparation of microsomes the ECS marker is almost completely retained in F1 indicate that upon fractionation and vesiculation inside-out vesicles are formed which trap the former extracellular fluid. Moreover, the former extracellular surface is hidden and no longer accessible for membrane-impermeable compounds. After perfusion of hearts with [3H] ouabain and fractionation and centrifugation the radioactive material was found to accumulate in F1, the kinetics being identical with that of binding to the intact tissue and with the time course of the inotropic action of ouabain. The inside-out vesicles derived from the plasma membrane are characterized by the presence of a (Na+-K+)-ATPase activity, an outward Ca pump, a high binding capacity for Ca, and a low Ca pereability. Since the (Na+-K+)-ATPase is known to react with cardiac glycosides (CG) at the outer cell surface it was not surprising to find a complete lack of inhibition of the enzyme by the membrane-impermeable CG ouabain if administered to the intact sarcolemmal microsome. After disruption of the microsomes, however, a considerable inhibition of the (Na+-K+)-ATPase became demonstrable. Similarly, the Ca permeability of the plasma membranes could only be enhanced by CG if interacting with the former outside. Although ouabain again failed to exert any action, the membrane permeable digoxin increased the Ca pereability of the intact vesicular membrane. The rate of Ca transport as an indicator of the Ca pump activity remained unaffected by CG provided from either side of the membrane. The passive Ca binding to the plasma membrane, which results in a concentration up to 5 mM in this structure, is thought to represent the Ca pool essential for excitation-contraction coupling. The CG are thought to alter the Ca binding in this pool, thus rendering depolarization more effective in releasing Ca++.