ASSOSIATION OF ENDOGENOUS CARDIOTONIC STEROIDS WITH SALT-SENSITIVITY OF BLOOD PRESSURE IN GEORGIAN POPULATION.

This investigation differentiates types of essential hypertension in a Georgian population as well as describes endogenous cardiotonic steroids in salt-sensitive and salt-resistant subjects. This case control study included 185 subjects: 94 cases with stage 1 essential hypertension (JNC7) naïve to antihypertensive treatment, and 91 controls. A salt-sensitivity test was used to dichotomize case and control groups into salt-sensitive and salt-resistant subgroups. Blood and urine samples were obtained to categorize participants as consuming high and low salt diets. Endogenous cardiotonic steroids, sodium and plasma-renin activity (PRA) were measured in both samples at the different sodium conditions. Determinants of circulating levels of endogenous sodium pump inhibitors were carried out using the ELISA and RIA methods; PRA was assessed by radioimmunoassay. Descriptive statistics were used to analyze the data. Differences in variables between sodium conditions were assessed using paired t-tests. Salt-sensitivity was found in 60.5% of the total population investigated, with a higher proportion in females. A statistically significant positive correlation was found between salt-sensitivity and age in females (r=0.262, p<0.01), and with 24-hour urine sodium concentration changes (r=0.334, p<0.01). A significant negative correlation was found between salt-sensitivity and PRA. At the high sodium condition, endogenous MBG and OU were high in salt-sensitive subjects compared to those who were salt-resistant. These compounds decreased with a low-salt diet in both salt-sensitive cases and controls but remained the same in salt-resistant individuals. The MBG and OU levels positively correlated with systolic blood pressure in salt-sensitive individuals but no variability was evident among salt-resistant subjects. Our results show that MBG and OU levels start to increase at the normotensive stage and sustained high concentrations can lead to elevated systolic blood pressure, a risk factor for arterial hypertension in salt-sensitive subjects.

Ca2+ entry mode of Na+/Ca2+ exchanger as a new therapeutic target for heart failure with preserved ejection fraction.

AIMS: Left ventricular (LV) fibrosis and stiffening play crucial roles in the development of heart failure with preserved ejection fraction (HFPEF). Plasma level of digitalis-like factors (DLFs) is increased in patients with hypertension, a principal underlying cardiovascular disease of HFPEF. Digitalis-like factors inhibit ion-pumping function of Na(+)/K(+)-ATPase and activate the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger (NCX). Digitalis-like factors are known to promote collagen production in fibroblasts. The aim of this study was to explore whether the pharmacological inhibition of the NCX entry mode is effective in the prevention of LV fibrosis and in the development of HFPEF. METHODS AND RESULTS: (i) Dahl salt-sensitive rats fed 8% NaCl diet from age 6 weeks served as hypertensive HFPEF model. In this model, 24 h urine excretion of DLFs was greater than that in the age-matched control at compensatory hypertrophic and heart failure stages. (ii) Continuous administration of ouabain for 14 weeks developed LV fibrosis without affecting blood pressure in Sprague-Dawley rats. (iii) Ouabain elevated intracellular Ca(2+) concentration through the entry of extracellular Ca(2+), increased the phosphorylation level of p42/44 mitogen-activated protein kinases, and enhanced (3)H-proline incorporation in cardiac fibroblast; and SEA0400, the inhibitor of the NCX entry mode, suppressed these effects. (iv) In the HFPEF model, administration of SEA0400 at subdepressor dose improved the survival rate in association with the attenuation of LV fibrosis and stiffening. CONCLUSION: Digitalis-like factors and the subsequently activated NCX entry mode may play an important role in the development of hypertensive HFPEF, and the blockade of the NCX entry mode may be a new therapeutic strategy for this phenotype of heart failure.

Effects of mono- and dual blockade of the renin-angiotensin system on markers of cardiovascular status in hypertensive patients with mild and moderate renal failure.

BACKGROUND/AIMS: Dual renin-angiotensin system (RAS) blockade has no more efficiency to decrease cardiovascular mortality than mono-blockade. Our goal was to explore differences between other cardiovascular markers in patients with RAS blockade. METHODS: We analyzed two groups of patients treated with a long-term ACE inhibitor (MONO-group, n = 20) and an ACE inhibitor and angiotensin II receptor blocker (DUAL-group, n = 15). Ambulatory blood pressure monitoring, echocardiography, arterial stiffness and levels of catecholamine, endogenous ouabain (EO), pro-brain natriuretic peptide and more types of urinary albumin measurements were performed. RESULTS: In the DUAL-group, we found significantly better cardiac parameters, but the levels of EO and urinary albumins were similar in both groups. The level of EO correlates with nighttime mean arterial blood pressure (R = 0.556, p = 0.032) and arterial ß-stiffness (R = 0.512, p = 0.042). Urinary immuno-unreactive albumin showed a relationship with diastolic dysfunction of the heart (R = -0.508, p = 0.045) diurnal index of diastolic blood pressure (R = -0.569, p = 0.021) in the MONO-group. CONCLUSION: Cardiac parameters were more prosperous in the DUAL-group, but the levels of EO did not differ between groups. The level of EO correlated with blood pressure and arterial stiffness markers in the MONO-group only. The urinary immuno-unreactive albumin may be a new marker of cardiovascular conditions.

Selective association of endogenous ouabain with subclinical organ damage in treated hypertensive patients.

According to previous studies endogenous ouabain (EO) closely correlates with high blood pressure, congestive heart failure and kidney disease in humans. Our aims were to analyse associations between plasma, urinary EO level and various markers of cardiovascular damage in treated hypertensive patients. Forty-one adult patients with hypertension and/or diabetes mellitus (DM) and/or chronic kidney disease (CKD) were studied. We assessed plasma and urinary EO, pro-brain natriuretic peptide and catecholamines, profile of ambulatory blood pressure monitor and cardiovascular status by echocardiography and echo-tracking. The highest level of plasma EO (19.7±9.5 pmol l⁻¹) was measured in hypertensive patients with DM and CKD. The nighttime mean arterial blood pressure independently correlated with the level of plasma EO (P=0.004), while independent predictor of the ß-stiffness of carotid artery was the urinary EO (P=0.011). Elevated level of EO was associated with nighttime blood pressure and subclinical organ damage in treated hypertensive patients, suggesting possible role of EO in the pathogenesis of impaired diurnal blood pressure rhythm and arterial stiffness.

Endogenous sodium pump inhibitors and age-associated increases in salt sensitivity of blood pressure in normotensives.

Factors that mediate increases in salt sensitivity of blood pressure with age remain to be clarified. The present study investigated 1) the effects of high-NaCl intake on two Na pump inhibitors, endogenous ouabain (EO) and marinobufagenin (MBG), in middle-aged and older normotensive Caucasian women; and 2) whether individual differences in EO and MBG are linked to variations in sodium excretion or salt sensitivity. A change from 6 days of a lower (0.7 mmol.kg(-1).day(-1))- to 6 days of a higher (4 mmol.kg(-1).day(-1))-NaCl diet elicited a sustained increase in MBG excretion that directly correlated with an increase in the fractional Na excretion and was inversely related to age and to an age-dependent increase in salt sensitivity. In contrast, EO excretion increased only transiently in response to NaCl loading and did not vary with age or correlate with fractional Na excretion or salt sensitivity. A positive correlation of both plasma and urine levels of EO and MBG during salt loading may indicate a casual link between two Na pump inhibitors in response to NaCl loading, as observed in animal models. A linear mixed-effects model demonstrated that age, dietary NaCl, renal MBG excretion, and body mass index were each independently associated with systolic blood pressure. Thus, a sustained increase in MBG in response to acutely elevated dietary NaCl is inversely linked to salt sensitivity in normotensive middle-aged and older women, and a relative failure of MBG elaboration by these older persons may be involved in the increased salt sensitivity with advancing age.

Endogenous bufadienolide mediates pressor response to ethanol withdrawal in rats.

An endogenous natriuretic and vasoconstrictor Na/K-ATPase inhibitor, marinobufagenin (MBG), is implicated in NaCl-induced hypertension and in ethanol addiction. In rats, MBG suppresses voluntary alcohol intake, while immunization against MBG induces alcohol-seeking behavior. Since alcohol withdrawal is associated with elevation of blood pressure (BP) and renal sodium retention, we hypothesized that MBG mediates pressor response to ethanol withdrawal. In male Sprague-Dawley rats, forced ethanol intake (20% v/v, 2.8+/-0.2 g/day for 7 days) did not affect BP and MBG excretion. Ethanol withdrawal was associated with a 21 mm Hg increase in BP, a 10% decrease in hematocrit, and a three-fold increase in renal MBG excretion. In vivo administration of anti-MBG antibody to rats prevented withdrawal-induced BP elevation. Therefore, MBG mediates pressor response to ethanol withdrawal, and may link mechanisms of ethanol dependence and hypertension.

Salt intake and depletion increase circulating levels of endogenous ouabain in normal men.

High-salt diets elevate circulating Na+ pump inhibitors, vascular resistance, and blood pressure. Ouabain induces a form of hypertension mediated via the alpha2-Na+ pump isoform and the calcium influx mode of the vascular sodium calcium exchanger (NCX). Whereas elevated levels of an endogenous ouabain (EO) and NCX have been implicated in salt-sensitive hypertension, acute changes in sodium balance do not affect plasma EO. This study investigated the impact of longer-term alterations in sodium balance on the circulating levels and renal clearance of EO in normal humans. Thirteen normal men consumed a normal diet, high-salt diet, and hydrochlorothiazide (HCTZ), each for 5-day periods to alter sodium balance. EO and other humoral and urinary variables were determined daily. On a normal diet, urinary sodium excretion (140 +/- 16 meq/day), plasma EO (0.43 +/- 0.08 nmol/l) and urinary EO excretion (1.04 +/- 0.13 nmol/day) were at steady state. On the 3rd day of a high-salt diet, urine sodium excretion (315 +/- 28 meq/day), plasma EO (5.8 +/- 2.2 nmol/l), and the urinary EO excretion (1.69 +/- 0.27 nmol/day) were significantly increased, while plasma renin activity and aldosterone levels were suppressed. The salt-evoked increase in plasma EO was greater in older individuals, in subjects whose baseline circulating EO was higher, and in those with low renal clearance. During HCTZ, body weight decreased and plasma renin activity, aldosterone, and EO (1.71 +/- 0.77 nmol/l) rose, while urinary EO excretion remained within the normal range (1.44 +/- 0.31 nmol/day). Blood pressure fell in one subject during HCTZ. HPLC of the plasma extracts showed one primary peak of EO immunoreactivity with a retention time equivalent to ouabain. High-salt diets and HCTZ raise plasma EO by stimulating EO secretion, and a J-shaped curve relates sodium balance and EO in healthy men. Under normal dietary conditions, approximately 98% of the filtered load of EO is reabsorbed by the kidney, and differences in the circulating levels of EO are strongly influenced by secretion and urinary excretion of EO. The dramatic impact of high-salt diets on plasma EO is consistent with its proposed role as a humoral vasoconstrictor that links salt intake with vascular function in hypertension.

Brain ouabain stimulates peripheral marinobufagenin via angiotensin II signalling in NaCl-loaded Dahl-S rats.

OBJECTIVE: In NaCl-loaded Dahl salt-sensitive (DS) rats the transient stimulation of brain endogenous ouabain (EO) precedes the increase in renal excretion of marinobufagenin (MBG), a vasoconstrictor and natriuretic. In hypertensive DS rats, EO raises blood pressure (BP) via an ATII-sensitive pathway. We hypothesized that an NaCl-induced increase in MBG is linked to the EO-stimulated ATII pathway. METHODS: We studied the effects of 3 h of NaCl loading (17 mmol/kg, intraperitoneally) in male DS rats treated with antibodies to MBG or ouabain, or with losartan (25 mg/kg). RESULTS: NaCl loading alone induced a transient stimulation of pituitary EO (22.4 +/- 1.8 versus 12.2 +/- 1.3 pmol/g) and ATII (39.4 +/- 2.8 versus 18.4 +/- 3.2 ng/g), a sustained increase in MBG excretion (5.2 +/- 0.6 versus 1.1 +/- 0.2 pmol/h), a 40% inhibition of the renal sodium pump, a natriuretic response, a 35 mmHg increase in systolic BP, and an increase in adrenocortical ATII and MBG levels and in plasma norepinephrine. The anti-MBG antibody reduced the natriuresis (36%) and BP (40 mmHg), and restored renal sodium pump activity. The anti-ouabain antibody prevented the increase in pituitary ATII, reduced MBG excretion, natriuresis and BP, increased sodium pump activity, and prevented increases in plasma norepinephrine, pituitary and adrenocortical ATII, and adrenocortical MBG. Losartan mimicked the effects of the anti-ouabain antibody, but did not affect the excretion of EO. In adrenocortical cells of DS rats, ATII stimulated MBG secretion, and losartan blocked this effect. CONCLUSIONS: In response to NaCl loading, brain EO, via an AT1 receptor pathway and probably via sympathetic activation, stimulates adrenocortical MBG, which inhibits the renal sodium pump and elevates BP.

Marinobufagenin, an endogenous alpha-1 sodium pump ligand, in hypertensive Dahl salt-sensitive rats.

Dahl salt-sensitive rats (DS), which have a mutation in the alpha-1 subunit of Na(+)/K(+)-ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na(+) and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na(+)/K(+)-ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the alpha-1 than alpha-3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162+/-9 mm Hg at week 2 versus 110+/-2 mm Hg in baseline, P<0.01), and increased less in DR (124+/-3 mm Hg at week 2 versus 112+/-2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9+/-7.6 pmol versus 9.1+/-1.3 pmol in baseline, P<0.01) in DS, but by only 25% in DR (13.2+/-0.9 pmol versus 10.3+/-0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na(+)/K(+)-ATPase from rat kidney (which expresses only alpha-1 Na(+)/K(+)-ATPase isoform). Unlike ouabain (IC(50)=248 micromol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na(+)/K(+)-ATPase (IC(50)=70 and 78 nmol/L, respectively). Our results suggest that an alpha-1 Na(+)/K(+)-ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant alpha-1 Na(+)/K(+)-ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.

Involvement of endogenous digitalis-like factors in voluntary selection of alcohol by rats.

This study tested the hypothesis that endogenous digitalis-like factor (DLF) is involved in the development of alcohol dependence in rats. In 33 male Wistar rats in conditioned place preference (CPP) experiment, ethanol evoked increase in time spent in the ethanol-associated compartment (702+/-82 in ethanol-treated vs. 426+/-86 sec in the controls). Digoxin pretreatment (125 microg/kg, i/p) did not affect the time spent in the water-associated compartment (476+/-80 sec), but prevented the acquisition of ethanol CPP (385+/-112 sec in ethanol-paired side, P<0.05). In a two bottle choice test, where rats (n=6 per group) chose between drinking water and 9% ethanol, immunization against two putative DLFs, marinobufagenin and ouabain (MBG and OLC) resulted in a 60% increase of ethanol consumption. Acute intragastric administration of 9% ethanol to the rats was associated with increased OLC in cerebrospinal fluid, and stimulated urinary excretion of MBG and OLC. Thus, in rats, digoxin, which mimics the effects of DLFs, suppresses the free choice of alcohol, while immunization against DLFs is associated with alcohol seeking behavior.

Effect of carbidopa on the excretion of sodium, dopamine, and ouabain-like substance in the rat.

The effect of carbidopa, a competitive inhibitor of dopamine synthesis on the excretion of sodium, dopamine (DA), and endogenous sodium transport inhibitor (ESTI), was examined in rats on normal and high salt diets for 7 days. ESTI activity was measured (1) as ouabain-like substance (OLS) by radioimmunoassay using an antibody raised against ouabain, (2) by inhibition of purified Na+,K+-ATPase activity (ATPI), and (3) as digoxin-like substance (DLS) using a commercial digoxin assay. The OLS immunoassay was validated against rubidium transport studies. High salt diet caused an increase in OLS and ATPI but not DLS. Sodium excretion in rats on normal sodium intake was not affected by carbidopa treatment but was significantly lower in the high sodium diet group during the first 5 days of the study. In the low salt group, carbidopa treatment caused significant increases in OLS. We conclude that ESTI (measured as OLS) and DA are important in the natriuresis of salt loading.

Urinary excretion of endogenous ouabain-like substance is reduced in NaCl supplemented premature infants.

Ouabain or an isomer has been identified as endogenous ouabain-like substance (EOLS). The role of EOLS in the adaptation of premature infants to alterations of sodium balance was investigated by measuring urinary ouabain excretion serially in 9 low birth weight premature infants with (group S, mean birth weight 1,578 g, mean gestational age 30.4 weeks) and without (group NS, mean birth weight 1,537 g, mean gestational age 30.8 weeks) NaCl supplementation. The study was performed on the 7th day and weekly thereafter until the 5th week of life. NaCl supplementation was given in a dose of 3-5 and 1.5-2.5 mmol/kg/day at the postnatal ages of 8-21 and 22-35 days, respectively. Prior to NaCl supplementation, urinary ouabain excretion was similar in the two groups (146.2 +/- 16.8 pg/kg/h in group S versus 180.0 +/- 9.6 pg/kg/h in group NS) and remained at about the same level throughout the study when supplemental NaCl was provided. In infants of group NS, urinary ouabain excretion increased significantly by the 3rd week (p < 0.01) and no consistent change occurred later on. As a result, the differences in urinary ouabain excretion between the two groups proved to be significant during weeks 2-5 (p < 0.001). Essentially the same pattern of ouabain excretion was seen when it was expressed in terms of pg/mg creatinine. In infants receiving high sodium diet there was a significant positive correlation between urinary sodium and ouabain excretion. It is concluded that premature infants receiving low sodium intake have elevated EOLS excretion by the 3rd week of life. Although the relationship between ouabain and sodium excretion in supplemented premature infants suggests some physiological significance for sodium excretion, ouabain does not appear to be regulated by extracellular volume.

Urinary excretion of endogenous ouabain-like substance in furosemide-treated neonates: its relation to renal excretory pattern and endocrine factors.

The present study was undertaken to define the possible role of endogenous ouabain-like substance (EOLS) in controlling renal excretory pattern, in mediating the renal responses to furosemide and in inducing endocrine reactions in furosemide-treated neonates. Ten newborn infants with mean birthweight of 2,752 g and mean gestational age of 37.1 weeks were given furosemide in a dose of 1 mg/kg. Prior to and following furosemide therapy, urine was collected for a period of 12 h and analyzed for creatinine, osmolality, sodium and potassium, as well as for EOLS, arginine vasopressin (AVP), aldosterone and endothelin-1 (ET-1). In response to furosemide administration, urine flow rate and urinary osmolar, sodium and potassium excretion increased significantly, whereas creatinine excretion remained unchanged. Furthermore, following furosemide therapy, urinary excretion of EOLS (148.7 +/- 70.8 vs. 200.1 +/- 98.1 pg/kg/h) and AVP (19.5 +/- 5.4 vs. 27.8 +/- 7.8 pg/kg/h) tended to increase, whereas ET-1 (36.0 +/- 5.6 vs. 61.4 +/- 8.7 fmol/kg/h, p < 0.01) and aldosterone (507 +/- 120 vs. 751 +/- 203 ng/kg/h, p < 0.05) increased significantly. Prior to furosemide, urinary EOLS excretion was found to correlate positively with diuresis (r = 0.80, p < 0.01), sodium (r = 0.91, p < 0.001), creatinine (r = 0.75, p < 0.001), osmolar (r = 0.96, p < 0.001), ET-1 (r = 0.90, p < 0.001) and AVP (r = 0.92, p < 0.001) excretion. After furosemide, urinary EOLS excretion significantly correlated only with diuresis (r = 0.69, p < 0.05), ET-1 (r = 0.77, p < 0.01) and AVP (r = 0.92, p < 0.001). Urinary EOLS proved to be independent of aldosterone excretion irrespective of furosemide administration. It is concluded that urinary EOLS excretion is closely related to neonatal renal excretory pattern and it may have a role in controlling diuresis and natriuresis. The renal response to furosemide appears to be independent of EOLS, although interdependent endocrine reactions can be induced by furosemide which may modulate the production rate and urinary excretion of EOLS.

Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA.

Recent studies suggest that ouabain or a ouabain-like substance (OLS) may be present endogenously in humans. We developed a RIA for ouabain with antisera raised in goat against ouabain conjugated to keyhole limpet hemocyanin and ovalbumin. The antiserum was of high antibody titer (200,000) and was specific for ouabain, with little cross-reactivity with common steroids and structurally related compounds such as ouabagenin (4%), strophanthidin (4%), and dihydroouabain (2%). The RIA had a working range of 0.06-2.0 nmol/L, and the intra- and interassay CV was 6.5% at a concentration of 1.7 nmol/L. With this assay the effect of salt loading on urinary excretion of OLS was examined in 10 healthy volunteers (ages 18-22 years) who increased their salt intake (sodium) for 5 days and reduced it for the next 5 days. Urine was collected and OLS concentration was measured by RIA after solid-phase extraction with a Bond Elut C18 column. Excretion of OLS and sodium were maximal on day 5 and lowest on days 9 and 10. Urine excretion of OLS on day 5 (2.66 +/- 1.22 nmol/24 h) was significantly higher (P < 0.0001) than on day 10 (1.47 +/- 0.69 nmol/24 h). We conclude that (a) the assay developed has sufficient sensitivity and specificity to detect endogenous OLS present in biological fluids, and (b) salt intake increases the excretion of OLS.

Ouabainlike compound in hypertension associated with ectopic corticotropin syndrome.

Molecular mechanisms related to sodium retention have been implicated in the pathogenesis of hypertension. It is unclear how sodium retention leads to a rise in blood pressure, but ouabainlike compound may act as a final common pathway in sodium-induced hypertension. In ectopic corticotropin syndrome, hypertension has been attributed to cortisol inactivation overload, giving rise to mineralocorticoid-type hypertension. We sequentially measured plasma and urinary levels of ouabainlike compound over 2 months to evaluate its role in the hypertensive mechanisms in a 64-year-old man with this syndrome caused by lung cancer. His data included hypokalemia and increased cortisol concentrations, corticotropin levels, and urinary 17-hydroxycorticosteroid excretion. Plasma renin activity was suppressed. Plasma and urinary levels of ouabainlike compound were markedly increased concomitantly with high blood pressure. The maximum plasma level was 40-fold the normal range of the subject. After chemotherapy, ouabainlike compound levels gradually decreased in parallel with the decline in blood pressure and rise in potassium concentration. A correlation was observed between plasma and urinary levels of ouabainlike compound (P < .05). Plasma and urinary levels of ouabainlike compound correlated with systolic (P < .01) and diastolic (P < .05) pressures, respectively. The peak of ouabainlike compound in plasma and urine coincided with that of authentic ouabain on high-performance liquid chromatography. Ouabainlike compound derived from urine inhibited [3H]ouabain binding to human erythrocytes. These findings suggest that ouabainlike compound with biological activity could partly account for hypertension in ectopic corticotropin syndrome.

Characterization of ouabain-like immunoreactivity in human urine.

OBJECTIVE: To characterize ouabain-like immunoreactivity in human urine. METHODS: Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. RESULTS: Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. CONCLUSION: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.

Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs.

OBJECTIVES: This study investigated effects of acute plasma volume expansion on plasma levels and urinary output of two endogenous Na,K-ATPase inhibitors, marinobufagenin-like and ouabain-like immunoreactive substances. METHODS: Plasma volume was expanded for 3 h via intravenous saline infusion in three groups of anesthetized dogs--nontreated (n = 5); pretreated with rabbit antidigoxin (n = 5); and pretreated with rabbit antimouse (control) antibody (n = 4). RESULTS: Plasma marinobufagenin-like immunoreactivity increased to 11.87 +/- 3.16 nmol.l-1 (vs. 0.30 +/- 0.16 nmol.l-1) within 10 min of volume expansion, in parallel with a 15% increase in LVdP/dt, then decreased to 2.21 +/- 0.59 nmol.l-1, and in 90 min increased to 11.8 +/- 2.8 nmol.l-1, in parallel with the maximal natriuretic response. Plasma concentrations of ouabain-like immunoreactive material were increased after 90 min of saline infusion (0.019 +/- 0.004 nmol.l-1 vs. 0.139 +/- 0.056 nmol.l-1). Pretreatment of the animals with antidigoxin antibody blocked the positive inotropic and reduced natriuretic response to volume expansion, and decreased the urinary release of marinobufagenin-like, but not ouabain-like, material. CONCLUSIONS: These results show the presence of marinobufagenin-like immunoreactive substance in dog plasma and suggest that mammalian EDLF may have a bufodienolide nature. Endogenous marinobufagenin-like immunoreactive substance, which is likely to cross-react with antidigoxin antibody, is involved in the natriuretic and positive inotropic responses to plasma volume expansion.

Endogenous marinobufagenin-like immunoreactive factor and Na+, K+ ATPase inhibition during voluntary hypoventilation.

In previous studies investigators found that conditioned hypoventilatory breathing potentiated a sodium-sensitive form of hypertension in dogs that was not mediated by sympathetic nervous system arousal. Our study investigated effects of 30 minutes of voluntary hypoventilation, maintained by a respiratory gas monitor and feedback procedure, in 16 normotensive humans of both sexes on (1) plasma concentrations of endogenous digitalis-like factors (ouabain-like and marinobufagenin-like immunoreactivity), (2) activity of erythrocyte Na+, K+ -ATPase, (3) inhibitory activity of plasma Na+, K+ -ATPase, and (4) blood pressure. Increased end tidal PCO2 (41 +/- 0.78 mm Hg versus 37.6 +/- 1.03 mm Hg) was associated with (1) an increase in plasma marinobufagenin-like immunoreactivity (1.23 +/- 0.47 versus 4.96 +/- 1.19 nmol/L), (2) an inhibition of Na+, K+ -ATPase in red blood cells (3.68 +/- 0.22 versus 2.15 +/- 0.25 mmol Pi/mL-1/h-1; P < .01), (3) increase in plasma Na+, K+ -ATPase inhibitory activity (34.9 +/- 4.0% versus 48.8 +/- 2.1%, P < .02), and (4) increases in systolic (112.4 +/- 2.6 versus 107.6 +/- 1.8 mm Hg) and diastolic (73.5 +/- 2.1 versus 68.8 +/- 2.1 mm Hg) blood pressures. Plasma levels of ouabain-like immunoreactivity did not increase significantly. Incubation of erythrocytes obtained during hypoventilation with antidigoxin antibody restored the Na+, K+ -ATPase activity (3.99 +/- 0.34 mmol Pi/mL-1/h-1). Cessation of hypoventilation was associated with decreases in diastolic blood pressure (70.5 +/- 2.2 mm Hg) and restoration of Na+, K+ -ATPase activity in erythrocytes (2.99 +/- 0.43 mmol Pi/mL-1/h1).(ABSTRACT TRUNCATED AT 250 WORDS)

Blunted natriuretic response to human urine extracts with Na+,K(+)-ATPase inhibiting activity in experimental cirrhosis.

Human urine and plasma extracts contain a material that inhibits the enzyme Na+,K(+)-ATPase (the endogenous sodium pump) and produces natriuresis in the bioassay animal. This endogenous sodium pump inhibitor(s), also known as digitalis-like factor, is thought to be involved in sodium and extracellular fluid volume homeostasis. Increased urine and plasma sodium pump inhibiting activity have been reported in patients with cirrhosis and sodium retention. The aim of the study was to assess the renal response to i.v. administration (0.2 ml/min per kg bw for 10 min) of a human urine extract containing sodium pump inhibiting activity (28.5 nmol equivalent ouabain/ml) in eight conscious rats with cirrhosis and ascites and eight control rats. Baseline urinary excretion of Na+,K(+)-ATPase inhibiting activity was significantly higher in cirrhotic rats with ascites than in control rats (235 +/- 40 vs 91 +/- 16; p < 0.01). Human urine extract induced a significant (p < 0.05) increase in glomerular filtration rate in control (3.2 +/- 0.4 to 4.2 +/- 0.5 ml/min) and cirrhotic rats (3.0 +/- 0.3 to 4.0 +/- 0.5 ml/min). In control rats it also increased urinary sodium excretion (1.47 +/- 0.22 to 2.43 +/- 0.5 microEq/min, p < 0.01) and fractional sodium excretion (0.29 +/- 0.01 to 0.43 +/- 0.04%, p < 0.025). In contrast, in cirrhotic rats with ascites neither sodium excretion nor fractional sodium excretion was significantly affected. No changes were observed in plasma aldosterone and atrial natriuretic peptide concentrations in either group. These data suggest that in cirrhosis there is a renal resistance to the natriuretic effect of endogenous sodium pump inhibitor(s).

Comparison of low-molecular-weight plasma and urine Na-K-ATPase inhibitors/hypertensive factors.

Two highly purified low-molecular-weight (< 500 Da) Na-K-ATPase inhibitors, one originating from human plasma and the second from human urine, which both eluted in the identical locus from a C18 reversed-phase high-pressure liquid chromatography (HPLC) column, were compared with respect to (a) K effect on Na-K-ATPase inhibition; (b) displacement of [3H]ouabain from binding sites on purified hog brain Na-K-ATPase; (c) cross-reactivity with digoxin antibodies; and (d) vasoconstrictor effects in isolated rabbit femoral arteries. Inhibition of Na-K-ATPase by the plasma factor correlated inversely with K concentration, whereas inhibition by the urine factor correlated directly with K concentration. In the absence of K, the plasma factor displaced [3H]ouabain from both high- and low-affinity binding sites, whereas the urine factor displaced [3H]ouabain only from the low-affinity binding site. Neither factor possessed digoxin-like immunoreactivity. Both factors acted as direct vasoconstrictors, and potentiated the vasoconstrictor action of norepinephrine. The degree of vasoconstriction caused by the plasma factor diminished progressively with added K, indicating that the vasoconstrictor effect of this factor was mediated by the Na-K-ATPase pump. Thus, although both the plasma and urine Na-K-ATPase inhibitors are vasoconstrictors, their mechanisms of action are different.