|Home | About | Journals | Submit | Contact Us | Français|
The Na+ pump and its Endogenous modulator Ouabain (EO) can be considered as an ancestral enzymatic system, conserved among species ranging from Drosophila to humans, related to Na handling. In this review, we examine how EO is linked with vascular function in hypertension and if it impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium–calcium exchanger duet. Biosynthesis of EO occurs in adrenal glands and is under the control of angiotensin II, ACTH and epinephrine. Elevated concentrations of EO and in the sub-nanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure. Experimental data suggest that the Na/K-ATPase α2-catalytic subunit causes EO-induced vasoconstriction. Finally, maneuvers that promote Na depletion, as diuretic therapy or reduced Na intake, raise the EO levels. Taken together, these findings suggest a key role for EO in body Na homeostasis.
For several million years the evolutionary ancestors of humans ate a diet that contained 1 g salt/day . This implies that present-day humans are genetically programmed to a salt intake of that amount. The deliberate addition of salt to food only began ~5000–10,000 years ago with the beginning of agriculture so that the present consumption of ~10 g/day on average is, in evolutionary terms, relatively recent. Hypertension is the major risk factor for cardiovascular disease. High dietary salt is implicated in hypertension; however, the sensitivity of blood pressure to salt among individuals is variable, and the mechanisms of salt-sensitive hypertension are speculative . Others have presumed a causal interrelationship between salt intake, total body Na+, fluid balance and blood pressure . According to this model, kidney controls total body Na+ and thereby extracellular volume homeostasis. Reduced renal Na+ excretion results in extracellular volume expansion and augmented blood flow. Traditionally, the Na+ cation is thought to be largely restricted to the extracellular compartment, while K+ is stored intracellularly. This balance is maintained by the activity of the Na/K-ATPase in the cell membrane. The Na/K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, that is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, this site has only recently been shown to have a natural function in blood pressure control . The Na/K-ATPase can be considered as an ancestral enzymatic system related to Na+ handling that has adapted its function during evolution. In this review, we examine how sodium and endogenous ouabain influence blood pressure and some related disorders. We examine the evidence for EO biosynthesis in humans, the role of EO in primary hypertension and in renal Na handling, and in Ménière’s Syndrome. Finally, the trophic effect of EO on cardiovascular function will be mentioned.
Since the original report published in 1991, EO has been isolated and detected by a variety of independent laboratories on different continents. HPLC and immunoassay methods show that EO is present in bovine  and human adrenal glands , bovine hypothalamus [7,8], rat adrenomedullary cells  and biological fluids [10–13]. Mass spectrometry, NMR studies and the cochromatography of EO with ouabain in all tested chromatography systems confirm the presence of EO beyond doubt and show that the mammalian compound is identical to plant ouabain [7–9,14–17].
A list of experimental evidences suggests that the adrenal is the main source of EO in humans and rats:
The molecular basis for the elevated plasma EO in Milan Hypertensive Strains [10,25] was probed in the hypothalamus and adrenal using bioinformatics and genomic techniques. Elevated transcripts for cholesterol side-chain cleavage (also known as cytochrome P450scc, CYP11A1) and β-hydroxysteroid dehydrogenase/δ5-4 isomerase genes (HSD3B) were detected in hypothalamus . Other studies have suggested that, as with aldosterone (Aldo), the biosynthesis of cardiac glycosides by the adrenal gland likely involves cholesterol side-chain cleavage to form pregnenolone with further metabolism of progesterone [27–29]. Recently, we observed marked increases in circulating EO in uremic patients . These data imply that, in addition to secretion, renal clearance is one of the major determinants of plasma EO. The biosynthesis of EO involves cholesterol side-chain cleavage (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B) with sequential metabolism of pregnenolone and progesterone. Furthermore, the renal excretion of cardiac glycosides is mediated, in part by the organic anion transporter (SLCO4C1)  at the basolateral membrane and in part by the P-glycoprotein (PGP, encoded by MDR1)  at the apical membrane of the nephron. A single-nucleotide polymorphisms (SNPs) and haplotype-based association study was performed with a total of 26 informative SNPs in a large cohort of hypertensive patients. Among patients with essential hypertension, CYP11A1 and MDR1 loci were found associated to circulating EO and Diastolic Blood Pressure (DBP), most likely by influencing EO synthesis and transmembrane transport, respectively .
Human kidneys are poised to conserve sodium and excrete potassium. Prehistoric humans, who consumed a sodium-poor and potassium-rich diet, were well served by this mechanism . With such a diet, sodium excretion is negligible and potassium excretion is high, matching potassium intake. In the contemporary dietary environment where high intakes of salt are common, the kidney must meet the obligation to excrete excess salt. Accordingly, it is of considerable interest that the reabsorption of filtered sodium by the renal tubules is increased in primary hypertension . The augmented reabsorption appears to be mediated by the stimulation of several sodium transporters located at the luminal membrane, as well as the sodium pump, which is localized to the basolateral membrane and provides the energy for such transport. The circulating levels of EO change in response to variation of Na balance.
In studies on the relationship between Na intake/Na excretion and EO in various human experimental conditions, we noted the following:
All together these findings suggest that EO has a double effect at renal level: normal circulating values (less than 250 pM) by stimulating the basolateral Na/K-ATP, induce a signaling cascade that lead to an increase of Na+ reabsorption, through the renal Na/Ca exchanger (NCX1). On the other hand, elevated plasma EO induce natriuresis. Indeed, recently we showed that hypertensive patients with elevated plasma EO (>323 pmol/l) showed increased FENa and increased Na tubular rejection fraction (P=0.007) after acute saline load [Manunta P. unpublised data]. Furthermore, the effect of renin angiotensin aldosterone system (RAAS) and EO have been investigated: RAAS reflects body sodium status and has primarily a compensatory role in the regulation of BP. Conversely, EO has a biphasic relationship with tubular reabsorption (favoring Na retention at low plasma levels and Na excretion at the higher levels) which is likely to affect total body Na and blood pressure. The rise in circulating EO is due either to the genetic or renal clearance background. It has been shown that among patients with essential hypertension, CYP11A1 and MDR1 loci are related to circulating EO and DBP, most likely by influencing EO synthesis and transmembrane transport, respectively .
Ménière’s disease (MD) is an inner ear disorder characterized by recurrent rotational vertigo and fluctuating sensorineural hearing loss. The pathogenetic mechanism is commonly accepted to be a raised endolymphatic pressure (hydrops). The peculiar ionic composition of endolymph, high K+ and low Na+ concentrations , is essential to mechano-electric transduction by hairy cells . Na/K-ATPase has been abundantly demonstrated in the inner ear [45,46]. High concentrations of ouabain inhibit Na/K-ATPase activity in guinea pig inner ear . Perilymphatic perfusion of ouabain decreases the endocochlear potential and induces ultrastructural changes in the stria vascularis . Moreover, round window application of ouabain has been found to produce degeneration of outer hair cells and spiral limbus fibrocytes in the cochlea in guinea pigs .
The plasma levels of EO in MD patients [50,51]according to ADD1 Gly460Trp polymorphism were not different compared to controls even if their genotypic frequencies differed significantly (P=0.0013, chi-square=5.29). As Ménière disease has a complex pathology, we suppose that ADD1 and plasma EO may act as independent factors in inner ear on the pump activity modulation. The small group of patients studied is not enough to detect the differences seen in larger group of hypertensives. This example of dysregulation of tissue sodium balance is in agreement with the observations done in hypertensive patients, where the ADD1 locus and plasma EO interact and are related with whole body sodium status.
EO activates signal transduction via the Src–epidermal growth factor receptor (EGFr)–extracellular signal-regulated kinase (ERK) pathway, and thereby triggers growth and proliferation of renal tubular cells and cardiomyocytes in vitro and in vivo, even at sub-nanomolar concentrations . We therefore investigated the association between left ventricular structure and function and plasma EO in different conditions:
The results summarized here emphasize the role of EO in normotensive and hypertensive subjects. First, the hypertensinogenic effects of EO are in keeping with the experimental evidence indicating that the α2-catalytic subunit causes hypertension and ACTH-induced high BP [61,62]. Furthermore, the mutation of the α2-Na+ pump ouabain-binding site abolishes the hypertensinogenic effect of ouabain and ACTH . Recently, it has been shown that the dynamic myogenic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and rise blood pressure in ouabain hypertensive rats . Accordingly, this vascular mechanism may be relevant in human primary hypertension where elevated plasma EO levels are correlated with blood pressure in 40–50% of patients [38–41].
Second, in addition to its direct influence on ion transport and vascular tone, ouabain is a growth factor that activates signaling cascades via a Src kinase pathway linked to the Na,K-ATPase [65,66]. The EO effect on structural and hyperthrophic cardiac myocytes has been discussed [40,52–55]. Third, the data presented demonstrate a concentration-dependent relationship of EO with Na metabolism: its effect on tubular reabsorption which is likely to directly affect total body Na and blood pressure . Recently, data in knock-out mice [62,63] suggest that the ouabain-binding site of the Na/K-ATPase affects the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands. However, maneuvers that promote Na depletion [37,39] (reduced Na intake, diuretic therapy) raise plasma EO (J curve, Fig. 1).
The experimental evidences are in keeping with our initial hypothesis that EO and the Na pump are ancestral systems related to Na handling and that their interaction evolved and was physiologically relevant under conditions where salt intake was low. The association of EO with essential hypertension and cardiac hypertrophy are likely to be recent phenomena that reflect the widespread introduction of salt into cooking and food preservation in the last few millennia.
The authors acknowledge the expert technical assistance of Cinzia Scotti.
Sources of funding
Supported in part by USPHS grants HL75584, HL04521 and HL0788705 (JH) and European Union grants: LSMH-CT-2006-037093 InGenious HyperCare and HEALTH-F4-2007-201550 HyperGenes (PM).
Conflict of interests
None of the authors has a con3ict of interest with regard to the data presented in this manuscript.