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...

Elevated circulating levels of an endogenous ouabain (EO) have been associated with essential hypertension. To investigate structure-activity relationships relevant to blood pressure, we infused either ouabain, ouabagenin, digoxin or digitoxin at 30 ,Mg/kg/day in normal Sprague Dawley rats. After five weeks, the ouabain and ouabagenin infused rats were hypertensive, whereas blood pressures declined below their vehicle controls in rats infused with digoxin or digitoxin. In a second study, mean blood pressures were 118.5 +1.7 mmHg in rats infused with ouabain (15 ,ug/kg/day) on day 35 vs. 98.3+1.8 and 100.3±1.1 mmHg in the digoxin (30 ,ag/kg/day) and vehicle infused groups (both p<0.005 vs. ouabain), respectively. Plasma and kidney levels of ouabain immunoreactivity were increased 4-8 fold in ouabain infused rats while blood pressure and plasma levels of ouabain returned to normal one week following discontinuation of the steroid infusion. In rats with ouabain-dependent hypertension, secondary infusions of digoxin or digitoxin (30 ,ag/kglday) normalized blood pressure even though circulating ouabain remained elevated. In digoxin infused rats, neither blood pressure nor kidney digoxin immunoreactivity was raised whereas plasma digoxin was increased. Collectively, the results show that the hemodynamic effects of these sodium pump inhibitors differ dramatically during prolonged administration and that tissue rather than circulating levels of these agents appear to better explain their effects on blood pressure. These studies suggest that sodium pump inhibition is not the exclusive mediator of the hemodynamic effects of these cardiac glycosides and demonstrate the presence of structure-specific mechanisms that regulate their tissue levels and effects on long-term blood pressure. (Hypertens Res 2000; 23 Suppl: S77-S85)

Ouabain has recently been identified as an endogenous Na + -K + pump inhibitor. We administered ouabain chronically to normotensive rats with varying degrees of reduced renal mass (RRM) and to normal two-kidney rats to see whether hypertension could be produced. Normal male Wistar rats and rats with 25%, 60%, and 70% RRM received ouabain (13.9 Mg/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 figJkg per day IP) for 3 to 4 more weeks. Respective control animals received vehicle only. Blood pressure was recorded weekly by tail plethysmography. Animals received tap water and standard rat chow, except for 70% RRM rats, which received distilled water and sodium-free chow. After 6 to 8 weeks of treatment, with rats under thiobutabarbital anesthesia, direct blood pressure was determined. Plasma, tissue, and urinary ouabain levels were measured with a specific radioimmunoassay. Animals receiving ouabain developed significant increases in mean blood pressure compared with control animals (70% RRM, 147±4 vs 116±4 mm Hg; 60% RRM, 140±4 vs 107±3 mm Hg; 25% RRM, 131 ±5 vs 100±2 mm Hg; no RRM, 116±4 vs 98±5 mm Hg). Plasma ouabain levels measured 24 hours after the last ouabain dose were not different in animals receiving ouabain vs those receiving vehicle. However, kidney tissue ouabain levels were significantly greater (6J9±1.17 vs 2.36±0. 52 fig/kg, P<.05) in animals receiving ouabain. In conclusion, ouabain, given chronically, is associated with the development of hypertension in RRM rats as well as in normal rats. Blood pressure was greater in animals with greater degrees of RRM for a given ouabain dose. (Hypertension 1993^2:178-187)

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.

Abstract-The basic requirement for declaring an association study positive is that the "hypertension-favoring" allele is more frequent in hypertensive cases than in normotensive controls. However, both positive and negative associations with hypertension have been found for the same polymorphism when studied in different populations. In the present study, we addressed the question of the possible cause(s) of this discrepancy among populations by using the ␣-adducin polymorphism as a paradigm. Four hundred ninety hypertensives and 176 normotensives enrolled in Sassari, Italy, and 468 hypertensives and 181 normotensives enrolled in Milano, Italy, were genotyped for the ␣-adducin Gly460Trp polymorphism. The blood pressure response to 2 months of hydrochlorothiazide therapy could be evaluated in 143 (85 in Sassari and 58 in Milano) hypertensives with and without the 460Trp ␣-adducin allele. The ␣-adducin 460Trp allele was not significantly more frequent in hypertensives in the Sassari population but was more frequent in hypertensives than in normotensives in Milano (Pϭ0.019). Basal plasma renin activity was lower and blood pressure fall after diuretic therapy more pronounced (PϽ0.01) in hypertensives carrying at least one 460Trp allele than in Gly460Gly homozygotes, irrespective of their membership in the Sassari or Milano cohort. The effect of ␣-adducin genotype in predicting basal plasma renin activity and blood pressure decrease with diuretic treatment is similar in Sassari and Milano, despite the lack of association of the ␣-adducin genotype with hypertension in Sassari. (Hypertension. 1999;34:649-654.)

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.

Obsessive-compulsive disorder (OCD) and Tourette Syndrome (TS) are highly heritable neurodevelopmental disorders that are thought to share genetic risk factors. However, the identification of definitive susceptibility genes for these etiologically complex disorders remains elusive. Here, we report a combined genome-wide association study (GWAS) of TS and OCD in 2723 cases (1310 with OCD, 834 with TS, 579 with OCD plus TS/chronic tics (CT)), 5667 ancestry-matched controls, and 290 OCD parent-child trios. Although no individual single nucleotide polymorphisms (SNPs) achieved genome-wide significance, the GWAS signals were enriched for SNPs strongly associated with variations in brain gene expression levels, i.e. expression quantitative loci (eQTLs), suggesting the presence of true functional variants that contribute to risk of these disorders. Polygenic score analyses identified a significant polygenic component for OCD (p=2×10−4), predicting 3.2% of the phenotypic variance in an independent data set. In contrast, TS had a smaller, non-significant polygenic component, predicting only 0.6% of the phenotypic variance (p=0.06). No significant polygenic signal was detected across the two disorders, although the sample is likely underpowered to detect a modest shared signal. Furthermore, the OCD polygenic signal was significantly attenuated when cases with both OCD and TS/CT were included in the analysis (p=0.01). Previous work has shown that TS and OCD have some degree of shared genetic variation. However, the data from this study suggest that there are also distinct components to the genetic architectures of TS and OCD. Furthermore, OCD with co-occurring TS/CT may have different underlying genetic susceptibility compared to OCD alone.

Abstract-Abnormalities in renal sodium reabsorption may be involved in the development and maintenance of experimental and clinical hypertension. Adducin polymorphism is thought to regulate ion transport in the renal tubule. It has recently been shown that there is a significant linkage of ␣-adducin locus to essential hypertension and that the 460Trp allele is associated with hypertension. Patients with this allele display larger blood pressure changes with body sodium variation. The aim of this study was to test whether ␣-adducin polymorphism is involved in abnormalities of renal function. Because proximal tubular reabsorption has been shown to be tightly coupled to renal perfusion pressure, this segmental tubular function was investigated in 54 (29 Gly/Gly and 25 Gly/Trp) untreated hypertensive patients in basal conditions with the use of endogenous lithium concentration and uric acid. Fractional excretions of lithium and uric acid were significantly decreased in the Gly/Trp hypertensive patients compared with the Gly/Gly hypertensives. The contribution of ␣-adducin to fractional excretion of lithium was investigated by multiple regression analysis. Adducin genotype was significantly (R 2 ϭ0.11, Fϭ6.5; PϽ0.01) and directly related to fraction excretion of lithium; gender, age, urinary Na ϩ , urinary uric acid, mean blood pressure, and plasma renin activity were not related. In conclusion, the adducin gene can be considered to be a 'renal hypertensive gene' that modulates the capacity of tubular epithelial cells to transport Na ϩ and hence contributes to the level of blood pressure. (Hypertension. 1999;33:694-697.) Key Words: lithium Ⅲ genes Ⅲ human Ⅲ renal function Ⅲ blood pressure Ⅲ adducin Ⅲ hypertension, genetic C ross-transplantation studies in rat models of primary hypertension have shown that at least a portion of hypertension follows the kidney. This finding has been obtained in all strains in which such an experiment has been carried out, suggesting that the kidney contributes significantly to hypertension. 1 Data consistent with those in rats have also been obtained in humans. 2 Many abnormalities in kidney function and cell membrane ion transport have been described in hypertensive rats and humans. 3 The logical sequence of events going from a genetic-molecular abnormality to a cellular abnormality that causes hypertension via a modification of kidney function is difficult to prove. In Milan hypertensive (MHS) rats we showed that (1) hypertension develops because of a primary increase in renal tubular sodium reabsorption 4 ; (2) a significant portion of blood pressure difference between MHS and normotensive animals (MNS) is due to a functional point mutation within the gene coding for ␣-adducin 5 ; (3) transfection of either MHS or MNS ␣-adducin cDNA into rat renal epithelial cells showed that cells expressing MHS adducin had a significantly greater Na ϩ pump activity at Vmax and a larger number of Na ϩ pump units expressed on the cell surface 6 ; and (4) studies in a cell-free system have shown that...