The Milan hypertensive strain of rats (MHS) develops a genetic form of renal hypertension that, when compared to its normotensive control (MNS), shows renal dysfunction similar to that of a subset of human patients with primary hypertension. MHS and MNS were shown to be homozygous by multilocus miisatellite analysis and monolocus microsatellite markers. We show here that one point mutation in each of two genes coding for the membrane skeleton protein adducin is associated with blood pressure in the Milan strain of rats. Adducin is a heterodimer formed by a and 13 subunits that promotes the assembly of actin with spectrin. MHS and MNS differ, respectively, by the amino acids Y and F at position 316 of the a subunit. In the ,&adducin locus, MRS is always homozygous for R at position 529 while in MNS either R or Q occurs in that position. The R/Q heterozygotes showed lower blood pressure than any of the homozygotes. In vitro phosphorylation studies suggest that both of these amino acid substitutions occur within protein kinase recognition sites. Analysis of an F2 generation demonstrated that Y alleles segregated with a significant increment in blood pressure. This effect is modulated by the presence of the R allele of the 1 subunit. Taken together, these dings strongly support a role for adducin polymorphisms in causing variation of blood pressure in the Milan strain of rats.

In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 g/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nM and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (؉11%, p < 0.05), and kidney weight (؉9%, p < 0.01). These effects in vivo are associated with a significant enrichment of ␣1, ␤1, ␥a Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat ␣1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 g/kg/day in vivo or 10 ؊10 -10 ؊8 M in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.Until recently, the main, if not unique, function ascribed to the integral membrane protein Na-K ATPase is the maintenance and regulation of the electrochemical gradient across the cell membrane in all tissues (1). Ouabain and other steroidal cardenolides (2) or bufadienolides (3) are considered to be the specific inhibitors of the Na-K ATPase activity. However, in recent years, several studies have indicated that Na-K ATPase can also act as a signal transducer in response to the interaction with its natural ligand ouabain (4). This finding originates mainly from studies carried out on cultured rat cardiomyocytes or renal tubular cells based on effects on cell growth and hypertrophy of ouabain in the micromolar range. At these rather high concentrations, which, however, do not seem to affect the bulk intracellular Na ϩ and Ca 2ϩ concentrations (5), ouabain activates: (a) tyrosine phosphorylation of the epidermal growth factor receptor (EGFr), 1 Src, and p42/44 mitogenactivated protein kinase (MAPKs) in both neonatal rat cardiac myocytes and A7r5 cells (4, 6); (b) the same signaling pathway within the cellular membrane microdomain of caveolae in isolated perfused rat heart (7); and (c) slow intracellular Ca 2ϩ oscillations in rat tubular cells that favor the association of Na-K ATPase with the inositol 1,4,5-trisphosphate receptor (InsP 3 ...

Abstract-Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased (PϽ0.0001) in patients with EH (377Ϯ19 pmol/L) versus normotensive (253Ϯ53 pmol/L) subjects. The distribution of plasma OLF was unimodal in normotensives, whereas it was bimodal in EH. Pϭ0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that Ϸ50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events. Key Words: sodium Ⅲ sodium-potassium pump Ⅲ cardiac glycosides Ⅲ digoxin Ⅲ human Ⅲ hypertension, essential Ⅲ heart A rterial hypertension is a risk factor for sudden cardiac death. Moreover, among hypertensives, this risk is increased further in those with left ventricular hypertrophy. 1,2 The molecular background of cardiac hypertrophy has been associated with changes in myocardial gene expression as well as activation of the tissue renin-angiotensin system and the sympathetic nervous system. The sodium pump is of major importance for active ion transport across the sarcolemma and contributes to the electrical and contractile function of the myocardium. Low concentrations of ouabain, via partial inhibition of the Na pump, cause a small increase in intracellular Na, affect sarcolemmal Na-Ca exchange, and lead to an increase in intracellular Ca and contractility. The rise in cell Ca 2ϩ stimulates the signal transduction pathways that regulate the expression of growth-related genes in heart. 3 It has recently been shown that the incubation of cultured rat neonatal cardiac myocytes with nontoxic concentrations of ouabain induces the transcription of some cardiac earlyresponse proto-oncogenes and late-response fetal genes, 4,5 which have been implicated as markers of myocyte hypertrophic growth. In contrast with these findings, other investigators have demonstrated that the acute inotropic effect of ouabain is associated with the inhibition of protein synthesis in papillary muscle of adult rats. 6 Evaluation of the controversy between these 2 types of findings should take int...

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Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

The genetic and environmental heterogeneity of essential hypertension is responsible for the individual variability of antihypertensive therapy. An understanding of the molecular mechanisms underlying hypertension and related organ complications is a key aspect for developing new, effective, and safe antihypertensive agents able to cure the cause of the disease. Two mechanisms, among others, are involved in determining the abnormalities of tubular Na+ reabsorption observed in essential hypertension: the polymorphism of the cytoskeletal protein alpha-adducin and the increased circulating levels of endogenous ouabain (EO). Both lead to increased activity and expression of the renal Na+-K+ pump, the driving force for tubular Na transport. Morphological and functional vascular alterations have also been associated with EO. Rostafuroxin (PST 2238) is a new oral antihypertensive agent able to selectively antagonize EO, adducin pressor, and molecular effects. It is endowed with high potency and efficacy in reducing blood pressure and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, Rostafuroxin antagonizes EO triggering of the Src-epidermal growth factor receptor (EGFr)-dependent signaling pathway leading to renal Na+-K+ pump, and ERK tyrosine phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by nanomolar ouabain. A very high safety ratio and an absence of interaction with other mechanisms involved in blood pressure regulation, together with initial evidence of high tolerability and efficacy in hypertensive patients, indicate Rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and EO-hypertensive mechanisms.

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.