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 ...

BACKGROUND AND PURPOSECalcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca 2+ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. EXPERIMENTAL APPROACHTo study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. KEY RESULTSWe showed that istaroxime enhances SERCA2a activity, Ca 2+ uptake and the Ca 2+ -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca 2+ cycling. CONCLUSIONS AND IMPLICATIONSIstaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca 2+ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way. LINKED ARTICLE AbbreviationsAHF, acute heart failure; CPA, cyclopiazonic acid; +dP/dt, rate of LV pressure rise; −dP/dt, rate of LV pressure decay; LV, left ventricle; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca 2+

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose.Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension.

PST 2238 is a new antihypertensive compound that is able to correct the molecular and functional alterations of the renal Na-K pump and the pressor effect associated with either alpha-adducin mutations or high circulating levels of endogenous ouabain (EO) in genetic and experimental rat models. Due to the close relationship between renal Na-K pump function and tubular Na reabsorption, PST 2238 was investigated to determine whether it is endowed with diuretic activity and consequently might trigger alterations of the renin-aldosterone system and the carbohydrate and lipid metabolism often associated with chronic diuretic therapy. In Milan hypertensive (MHS) rats, in which hypertension is genetically associated with alpha-adducin mutation, increased tubular Na reabsorption, and hyperactivation of the renal Na-K pump. PST 2238 reduced blood pressure and normalized the renal Na-K pump activity at oral doses of micro g/kg, but did not induce, either acutely or chronically, any diuretic activity or hormonal or metabolic alterations. In contrast, HCTZ, given to MHS rats orally at 40 mg/kg, although it displayed diuretic activity and reduced the renal Na-K pump activity, did not lower blood pressure and caused hyperactivation of the renin-aldosterone system, hypokaliemia, and hyperglycemia. The findings lead to the conclusion that PST 2238 is a new antihypertensive compound that normalizes the altered function of the renal Na-K pump associated with hypertension in rat models, but that it is devoid of diuretic activity and does not induce the diuretic-associated side effects.