Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets

Abstract

Endogenous cardiotonic steroids (CTS), also called digitalis-like factors, have been postulated to play important roles in health and disease for nearly half a century. Recent discoveries, which include the specific identification of endogenous cardenolide (endogenous ouabain) and bufadienolide (marinobufagenin) CTS in humans along with the delineation of an alternative mechanism by which CTS can signal through the Na(+)/K(+)-ATPase, have increased the interest in this field substantially. Although CTS were first considered important in the regulation of renal sodium transport and arterial pressure, more recent work implicates these hormones in the regulation of cell growth, differentiation, apoptosis, and fibrosis, the modulation of immunity and of carbohydrate metabolism, and the control of various central nervous functions and even behavior. This review focuses on the physiological interactions between CTS and other regulatory systems that may be important in the pathophysiology of essential hypertension, preeclampsia, end-stage renal disease, congestive heart failure, and diabetes mellitus. Based on our increasing understanding of the regulation of CTS as well as the molecular mechanisms of these hormone increases, we also discuss potential therapeutic strategies.

Fig. 1.

Structure of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase consists of two α and β polypeptides in equimolar ratios. The α catalytic subunit has 10 transmembrane segments, schematically presented in an “unfolded” disposition; in reality, there is a bungle around M4, M5, and M6 transmembrane segments. The extracellular segments of α subunit form a binding site for CTS (shown in red), which include TM1–TM2, TM5–TM6, and TM7–TM8 loops and several amino acids from the transmembrane regions M4, M6, and M10 (see the explanation in the text). The binding site for ATP is located on the intracellular loop TM4–TM5 (shown in blue), which forms the “pocket” for this nucleotide. The phosphorylation domain (P; shown in orange) located on the proximal and distal parts of intracellular loop TM4–TM5; phosphate of ATP is transiently transferred on the aspartyl residue 376 of DKTGT motif. The actuator domain, specifically its TGES motif, is responsible for the dephosphorylation step; it is constituted by the cytoplasmic NH₂-terminal and TM2–TM3 intracellular loop (shown in green). The regulatory β glycoprotein subunit is a single-transmembrane protein with a several glycosylation sites (two are shown). The extracellular part of β subunit interacts with a conserved motif SYGQ on the extracellular loop TM7–TM8 of α subunit. The αβ-subunit complex of Na⁺/K⁺-ATPase associates with third subunit, which contains the conserved motif FXYD identical for all seven proteins from this family. FXYD2 protein is known as the earlier described γ subunit of Na⁺/K⁺-ATPase. These proteins, including the γ subunit, are not an integral part of sodium pump, but they are associated with specific domains of αβ-subunit complex and modulate catalytic properties of the Na⁺/K⁺-ATPase.

Fig. 2.

Chemical structures of cardiotonic steroids: cardenolides (ouabain and digoxin) and bufadienolides (marinobufagenin and proscillaridin A). Rostafuroxin, a digitoxin derivative, is an antihypertension compound that opposes the prohypertensive effects of endogenous ouabain.

Fig. 3.

Prohypertensive effects of endogenous ouabain. In the kidney, in the presence of adducin polymorphism, EO increases sodium reabsorption via interaction with a pool of ouabain-sensitive α1 Na⁺/K⁺-ATPase located in the caveolae of renotubular cells. The resultant activation Src-EGFR-dependent tyrosine phosphorylation pathway reduces internalization of the Na⁺/K⁺-ATPase and increases the net sodium pump activity (left column). EO may also increase vascular tone via inhibition of ouabain-sensitive α2 Na⁺/K⁺-ATPase in vascular sarcolemma and resultant activation of Na⁺/Ca⁺² exchange (right column) and, in addition, directly induces hypertrophic signaling in cardiovascular tissues. Rostafuroxin exhibits its antihypertensive effects via antagonism of the interaction of EO and adducin on vascular and renal Na⁺/K⁺-ATPase. MAPK, mitogen-activated protein kinase.

Fig. 4.

Interaction between brain endogenous ouabain, the renin-angiotensin system, and marinobufagenin in pathogenesis of salt-sensitive hypertension. In NaCl-loaded Dahl salt-sensitive rats, the impairment of renal sodium transport causes sodium retention, which stimulates brain endogenous ouabain in hippocampus, hypothalamus, and pituitary gland. Brain endogenous ouabain stimulates the brain RAS in the hypothalamus and pituitary and activates sympathetic nervous system (SNS), which activates adrenocortical RAS. Angiotensin II activates adrenocortical production of MBG with a primary adaptive aim to induce natriuresis via inhibition of renotubular Na⁺/K⁺-ATPase. Excessive MBG production, however, induces a maladaptive effect by inhibiting the Na⁺/K⁺-ATPase in vascular smooth muscle cells and by potentiating vasoconstriction.

Fig. 5.

Schematic diagram contrasting “classic” (“ionic”) versus “signaling” pathways for CTS effects. In the classic pathway, any signaling through the Na⁺/K⁺-ATPase requires inhibition of the Na⁺/K⁺-ATPase pumping activity, which in turn is accompanied by changes in cytosolic [Na⁺] and [K⁺]. As discussed in the text, some authors feel that the caveolar Na⁺/K⁺-ATPase may be more sensitive to CTS in terms of enzymatic function. The increases in [Na⁺] and decreases in [K⁺] then induce an increase in cytosolic [Ca²⁺], which, in turn, activates a variety of pathways that have combinations of genomic and nongenomic effects. In contrast, the signaling pathway involves the association of Src with the Na⁺/K⁺-ATPase in a caveolar domain. Binding of the CTS to the Na⁺/K⁺-ATPase activates Src, which, in turn, transactivates the EGFR and phospholipase C (PLC). This leads to a cascade that involves generation of ROS, activation of mitogen-activated protein kinase (ERK) through activation of its mitogen-activated protein kinase kinase (MEK), activation of PI(3)K, stimulation of endocytosis and activation of Akt as well as activation of protein kinase C. This signaling construct proposes that these steps induce increases in cytosolic [Ca²⁺] and induce the combinations of genomic and nongenomic effects. Note that whereas both the classic and signaling pathways allow for intervention at the level of CTS binding to the Na⁺/K⁺-ATPase [(a), immunoneutralization or pharmacological neutralization], the signaling pathway presents a number of potential targets such as (b) interference with Src activation and EGFR transactivation, (c) PLC activation, (d) MEK activation, (e) ROS generation or scavenging, or (f) PI(3)K activation. Modulation of the signaling pathways at the level of PKC (g), ERK, and Akt might also be possible.