Cardiotonic steroids encompass a group of compounds that share the capacity to bind to the extracellular surface of the major cellular ion transport protein, the membrane-inserted Na+-K+-ATPase (Na pump). Members of this group of compounds include the endogenous ouabain and the aglycone steroid marinobufagenin (MBG). Both compounds have been purified from mammals and mass spectral analysis reveals that they are structurally identical, respectively, to the plant derived ouabain  and to MBG  found in toads, Bufo marinus. Until now, contrasting results regarding the role of these two endogenous glycosides in Na and volume-expanded states have been published. In this issue of the journal, Fedorova et al.  report on their experiments on Dahl salt-sensitive rats (DS), which were chronically fed on a high salt diet and developed hypertension and cardiac remodelling. The authors show that the transition from a compensatory left ventricle hypertrophy to a decompensate congestive heart failure in DS rats was associated with changes in the plasma levels of MBG or endogenous ouabain linked to the modulation of the myocardial Na+-K+-ATPase isoform expression. These findings greatly help in understanding the growing body of experimental evidence supporting the hypothesis that both endogenous ouabain and MBG are present in the mammal circulation and play distinct pathophysiological roles.
Three major points sustain this proposal, as outlined below.
MBG and endogenous ouabain differ with respect to their molecular target, the Na+-K+-ATPase
The catalytic subunit of this ion pump, which contains the binding sites for cardiac glycosides, has three isoforms showing species and tissue-dependent distribution: the α1 is particularly enriched in renal tubules, vascular tissue and adult rat myocardium; the α2 and α3 are mainly localized to skeletal muscle, neural sarcolemma, nerve endings and cardiac conduction system . The expression of these isoforms may be physiologically modulated during postnatal development  or may change under different pathological conditions associated with alterations of salt and water homeostasis, such as hypertension and cardiac disease [6,7]. Noticeably, in rats, the Na+-K+-ATPase isoforms differ in ouabain and MBG sensitivity: the α1 is highly resistant to ouabain but shows a great affinity for MBG whereas the α3 is highly sensitive to ouabain but less to MBG . It is worth noting that, although structurally very similar, endogenous ouabain shows three-fold greater affinity than plant ouabain for rat renal α1 Na+-K+-ATPase, implying that endogenous ouabain, differently from plant ouabain, might be able to interact and modulate the renal α1 Na+-K+-ATPase at physiological concentrations .
Collectively, these data provide important evidence that different Na+-K+-ATPase isoforms might be functionally modulated by selective endogenous inhibitors which may act as specific regulators in particular cellular districts.
Additional evidence has been collected over recent years further clarifying the complex pathophysiological action of the endogenous ouabain. Several studies performed either in vivo or in cultured rat renal cells have shown that subnanomolar concentrations of ouabain may stimulate, and not inhibit, renal Na+-K+-ATPase, and that this effect might be associated with the development of hypertension . These data appear to be inconsistent with the original ‘natriuretic hypothesis’  suggesting that the ‘Na+-K+-ATPase inhibitor', which is increased following a condition of extracellular volume expansion, might be able to promote natriuresis and diuresis through inhibition of renal Na+-K+-ATPase. It appears that it is possible to reconcile the old and new data by proposing, as previously demonstrated , that ouabain/endogenous ouabain shows a biphasic action on Na+-K+-ATPase: low concentrations of ouabain/endogenous ouabain stimulate the Na+-K+-ATPase while higher concentrations are needed to inhibit the enzyme.
Furthermore, it has been shown that ouabain, in addition to behaving like an ion modulator of the Na+-K+ pump, might activate the expression of growth-related genes and induce cellular hypertrophy . Ouabain may initiate this intracellular signalling cascade through the interaction with a pool of Na+-K+-ATPase restricted within specialized plasma membrane subdomains. However, an additional and novel receptor, distinct from the Na+-K+-ATPase, with high affinity for ouabain (Kd approximately 15 nmol/l) has been identified by Hamlyn et al.  in the adrenal cortex. Although its functional role has not been completely elucidated, this new receptor might be able to bind endogenous ouabain at concentration 10–9 mol, close to the plasma values found in humans.
These data suggest that ouabain/endogenous ouabain may play a novel role as a hypertrophic hormone and thus affect cardiovascular function and structure, being responsible for cardiac remodelling that contributes to an increased risk of morbid events. In view of these recent findings on the ouabain/endogenous ouabain molecular mechanism, the sequence of the intracellular events activated by MBG that lead to cardiac hypertrophy, as shown by Fedorova et al. , remain to be elucidated.
Elevated levels of plasma MBG
Elevated levels of plasma MBG have been shown in several volume-expanded hypertensive states, including hypertension induced by high NaCl intake in DS . Enhanced MBG production occurs in volume expanded dogs and rats and pathological states associated with fluid retention. Moreover, a natriuretic effect of MBG has been demonstrated in this experimental form of high blood pressure . However, plasma endogenous ouabain in hypertensive patients rises after acute or chronic sodium depletion  in keeping with the evidence that ouabain/endogenous ouabain is involved in the activation of renal Na+-K+-ATPase. No significant change in circulating endogenous ouabain was observed after a 2-h saline infusion or controlled Na+ intake (2 weeks). Moreover, Wang et al.  investigated the distribution of plasma ouabain in the general population in relation to blood pressure and other determinants of sodium homeostasis. Plasma endogenous ouabain behaves similarly to a blood pressure-modulating factor, possibly released in response to potassium, and either inhibiting the pressor effect of excessive salt intake or counteracting the depressor action of sodium depletion. It appears that a complex inter-relationship exists between endogenous ouabain and salt in the homeostatic regulation of blood pressure.
Together, these findings clearly indicate a differential mechanism of action between MBG and ouabain/ endogenous ouabain because MBG behaves like a natriuretic compound while endogenous ouabain rises in response to a sodium depletion condition.
What is the relevance of these two cardiac glycosides in the pathogenesis of human heart failure?
As reported in the present study by Fedorova et al.  in DS rats, the development of congestive heart failure (CHF) in humans is associated with a down-regulation of α1 isoform of Na+-K+-ATPase in left ventricular myocardium  and reduced sensitivity to MBG and with up-regulation of α3 Na+-K+-ATPase and enhanced sensitivity to ouabain . These data support the involvement of ouabain in cardiac remodelling and in the transition to heart failure. It emerges that the different Na+-K+-ATPase isoforms expressed during left ventricular hypertrophy and heart failure may be directly involved in the cardiac response to MBG or endogenous ouabain.
Recently, the same group showed that, in hypertensive patients with CHF, MBG increases progressively and correlates with left ventricular systolic function . By contrast to the results obtained with DS rats, patients with advanced CHF did not exhibit any increase in endogenous ouabain. As suggested by the authors, this may depend on the fact that their patients with CHF NYHA stage 4 had relatively high values of left ventricular ejection fraction (LVEF) (40%), which can be expected from hypertensive patients with diastolic dysfunction. Moreover, in agreement with the present experimental findings and the earlier data of Gottlieb et al.  who reported elevated levels of endogenous ouabain in patients with CHF and low LVEF, Fedorova et al.  reported preliminary results (unpublished data) that patients with an LVEF less than 30% exhibited greater renal excretion of endogenous ouabain than patients with a greater LVEF (30–55%). Because no uniform results have been collected from the data so far available, further studies will be required to better understand the role of the two endogenous glycosides in patients with congestive heart failure.
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