Cardiotonic Steroid Ouabain Research Articles

Sodium pump and steroid hormone receptor. Na(+)/K(+)-ATPase.

Sodium ions have two main functions in biological systems: they act as signal transducers of hormones, producing action potentials, and energize the active transport of energy substrates in epithelia. It is the opposing function of the sodium pump of the plasma membrane to restore the uneven distribution of Na+ and K+ ions across the cell membrane and to create thereby a membrane potential. To understand the biological function of the sodium pump, research has focussed for a long while on the structure and function of this Na+/K+-ATPase, its gene structure and subunit composition, the folding of its subunit proteins in the membrane, and its catalytic function with the hope of bringing to light how chemical energy stored in ATP is converted into a vectorial transport process of ions in opposite directions. Many of these questions are now starting to be answered on a molecular level, and even the ionophoric structures of the pump are close to being defined. Beginning, however, with the unexpected finding of the existence of isoforms of the catalytic α subunit of the sodium pump and their specific localization within cells and tissues, new questions concerning the biological role of such isoforms have arisen. The demonstration of differing affinities of the various α isoforms for cardiac glycosides, specific inhibitors of the sodium pump, and the observation that α subunits are substrates of protein kinases and hence under the control of hormones, has led to the possibility that in certain tissues, a change in the activity of the sodium pump may be part of a hormonal signaling cascade. This aspect has been supported in recent years by the finding that some members of the well-known group of cardiotonic steroids, which previously were considered to be toxins of plants and some amphibians, are in fact mammalian hormones released by the adrenal gland and the hypothalamus. Additionally, it became evident that, in contrast to previous assumptions, the sodium pump itself may act as a hormone receptor, transducing the signal of an interaction with the cardiotonic steroid ouabain via protein–protein interaction within the plasma membrane and with cytosolic proteins via at least three different signaling routes, to produce responses such as increased contractility of the heart and arteries, arterial hypertension, and altered cell growth and differentiation. Because the machinery of signal transduction used by ouabain is also used by other hormones, considerable cross-talk between signaling pathways is expected to take place in individual cells. Hence, local alteration of the intracellular Na+ concentration in cells is also mediated by hormones that affect the sodium pump. The following three Minireviews not only summarize the present knowledge in the field of the sodium pump but aim to make readers from outside the field aware of the opening of new avenues of research dealing with the regulation of sodium metabolism via the sodium pump.

Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR.

Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes ((13)C, (2)H, and (19)F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na(+)/K(+)-ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of (2)H-labeled inhibitors revealed that the sugar group was only loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K(+)-channel region. Crosspolarization magic-angle spinning NMR showed that chemical shifts of inhibitors (13)C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A (19)F, (13)C- rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surface of the Na(+)/K(+)-ATPase alpha-subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.

Cardiotonic steroid in humans is not ouabain

A compound found in human plasma that has properties similar to the cardiotonic steroid ouabain is not, in fact, identical to ouabain. This finding by researchers at four institutions, presented at...

The new adrenal steroid hormone ouabain

It has been proposed that an unknown circulating factor inhibits the plasma membrane sodium-potassium pumps and thereby plays an important role as a natriuretic or vasoconstrictive agent in the regulation of volume and pathogenesis of low-renin hypertension. The sodium pumps have specific high-affinity receptors for the digitalis glyrosides. With the analogy of endogenous opioids and nitric oxide, this has fostered speculation that an endogenous ligand exists that is “digitalis-like.” Recently, an endogenous factor has been extracted from human plasma, has been purified, and has been structurally identified by mass spectroscopy. This factor binds with high affinity to the sodium pump receptor and is indistinguishable from the cardiotonic steroid ouabain. Considerable evidence suggests that ouabain is synthesized in the adrenal cortex. It has a basic steroid nucleus, it is detected in high concentration in adrenocortical tissue, a continuous venous-arterial gradient across the adrenal can be demonstrated in the dog, and after bilateral adrenalectomy, plasma ouabain levels fall significantly. Additionally, adrenocortical cells in culture synthesize ouabain de novo. A modest elevation in plasma ouabain has been demonstrated in various forms of hypertension in humans, and the chronic infusion of ouabain to raise the plasma level to a comparably modest degree produces reversible normal renin hypertension in the rat. Circulating ouabain may also prove to be important in heart failure and in the pathogenesis and evolution of primary aldosteronism. To date, the precise site in the adrenal cortex and pathways for the biosynthesis of ouabain have not been determined. This information and the clarification of the pressor mechanism and the significance of ouabain and other possible sodium transport inhibitors in disorders involving volume regulation are major areas for future research. Nevertheless, the presence of ouabain in the circulation and tissues suggests that it could prove to be of widespread biologic significance.

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10 −7 M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K + in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.