Studies on the localization of the cardiac glycoside receptor

Abstract

A B S T R A C T The purpose of this study was to see whether the receptor for cardiac glycosides might be localized upon or within the plasma membrane of digitalis-sensitive cells. Ouabain and digoxin were joined covalently to several large protein molecules. These macromolecular conjugates are too large to enter intact cells; consequently, any pharmacologic or biochemical effects which they display should arise from interaction with a cell surface receptor. Conjugates were tested in several cardiac glycoside-sensitive systems: (a), contractility response of isolated cardiac muscle; (b), active 'Rb+ uptake by red cells; (c), enzymatic activity of isolated myocardial microsomal (Na+ + K+) -activated adenosine triphosphatase (ATPase); and (d), enzymatic activity of solubilized red cell (Na+ + K+) -activated ATPase. Results demonstrated that in all of these systems, the macromolecular-glycoside conjugates were 100-to 1000-fold less active than the free glycosides. Careful chromatographic examination of the various conjugates revealed that they contained a small but persistent free cardiac glycoside contaminant. The amount of this species ranged from 0.1 to 1.0% of the total macromolecule-bound glycoside, and its presence fully explains the levels of biologic activity observed with the conjugates.To try to minimize steric factors which could interfere with glycoside-receptor interaction, digoxin and ouabain were also coupled to macromolecule via long, flexible polyamide side-chains. These extended chain

Inotropic effects and changes in sodium and calcium contents associated with inhibition of monovalent cation active transport by ouabain in cultured myocardial cells.

“…To validate the use of Rb + as a K + analog, we performed studies of the 42 + § simultaneous uptake by cultured heart cells of K (total [K ] 4.0 raM) and ~Rb + (total [Rb +] 0.1 raM). As shown in Table I, the experimentally determined ratios of both active and passive uptake of K+: Rb + approximated 40:1, consistent with our previous experience with other myocardial cells (Smith et al, 1972;Hougen and Smith, 1978) and indicating that Rb + at 0.1mM concentration is a suitable K + analog for the study of active and assive transport. In other studies, K + uptake by cultures in media containing K + as the only radioisotope was compared to K + uptake by cultures in Coverslips with attached heart cell monolayers labeled with [aH]leucine were exposed to medium containing ~Rb + and 42K+ (5 ~Ci/ml ~Rb +, 0.…”

“…In 1938, Cattell and Gold demonstrated that digitalis exerts a direct positive inotropic effect on cardiac muscle. Since then, substantial evidence has accumulated indicating that the membrane-bound monovalent cation transport enzyme complex known as sodium, potassium-activated adenosine triphosphatase (NaK-ATPase) is the pharmacologic receptor for this effect of cardiac glycosides (Smith et al, 1972;Schwartz, 1976). Allen and Blinks (1978) have demonstrated that exposure of cardiac muscle to cardioactive steroid results in augmentation of the increase in intracellular free ionized calcium concentration, [Ca2+]i, that occurs during an action potential.…”

Effects of Digoxin-Specific Antibodies on Accumulation and Binding of Digoxin by Human Erythrocytes

“…8) is attributable to the antibodies forming a complex with the ['H]digoxin which was released from the cells during the initial portion of the incubation period and thereby, preventing this [3H] digoxin from binding to the erythrocyte membrane. Additional evidence against the possibility that digoxin-specific antibodies can directly facilitate the release of bound digoxin molecules from the erythrocyte membrane can be obtained from the studies of Smith et al mentioned previously (20). Since digoxin-specific antibodies are presumably larger than the glycoside-protein molecules used by Smith et al, they should likewise be unable to reach the glycoside receptor.…”

The Molecular Organization of Membranes

“…[There are also antibodies known which can. inactivate this enzyme (203)(204)(205), but because there are many possible explanations for such inactivation, this tells nothing definitive about the transport mechanism.] These results suggest that further investigation of fixed pore mechanisms in transport would be justified.…”