Endogenous Ouabain-like Substance Research Articles

Hypertension from chronic central sodium chloride in mice is mediated by the ouabain-binding site on the Na,K-ATPase α2-isoform

A chronic increase in the concentration of sodium chloride in the cerebrospinal fluid (CSF) (↑CSF [NaCl]) appears to be critically important for the development of salt-dependent hypertension. In agreement with this concept, increasing CSF [NaCl] chronically by intracerebroventricular (icv) infusion of NaCl-rich artificial CSF (aCSF-HiNaCl) in rats produces hypertension by the same mechanisms (i.e., aldosterone-ouabain pathway in the brain) as that produced by dietary sodium in salt-sensitive strains. We first demonstrate here that icv aCSF-HiNaCl for 10 days also causes hypertension in wild-type (WT) mice. We then used both WT and gene-targeted mice to explore the mechanisms. In WT mice with a ouabain-sensitive Na,K-ATPase α(2)-isoform (α2(S/S)), mean arterial pressure rose by ~25 mmHg within 2 days of starting aCSF-HiNaCl (0.6 nmol Na/min) and remained elevated throughout the study. Ouabain (171 pmol/day icv) increased blood pressure to a similar extent. aCSF-HiNaCl or ouabain given at the same rates subcutaneously instead of intracerebroventricularly had no effect on blood pressure. The pressor response to icv aCSF-HiNaCl was abolished by an anti-ouabain antibody given intracerebroventricularly but not subcutaneously, indicating that it is mediated by an endogenous ouabain-like substance in the brain. We compared the effects of icv aCSF-HiNaCl or icv ouabain on blood pressure in α2(S/S) versus knockout/knockin mice with a ouabain-resistant endogenous α(2)-subunit (α2(R/R)). In α2(R/R), there was no pressor response to icv aCSF-HiNaCl in contrast to WT mice. The α2(R/R) genotype also lacked a pressor response to icv ouabain. These data demonstrate that chronic ↑CSF [NaCl] causes hypertension in mice and that the blood pressure response is mediated by the ouabain-like substance in the brain, specifically by its binding to the α(2)-isoform of the Na,K-ATPase.

Enhanced pressor response to increased CSF sodium concentration and to central ANG I in heterozygous α2Na+-K+-ATPase knockout mice

Intracerebroventricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain Na(+)-K(+)-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The Na(+)-K(+)-ATPase alpha (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the Na(+)-K(+)-ATPase alpha(2)- and/or alpha(3)-subunits). We hypothesize that OLS-induced inhibition of the alpha(2)-subunit mediates this response. Therefore, a chronic reduction in alpha(2) expression via a heterozygous gene knockout (alpha(2) +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both wild-type (+/+) and alpha(2) +/- mice, but to a greater extent in alpha(2) +/-. Likewise, the pressor response to ICV ouabain was enhanced in alpha(2) +/- mice, demonstrating enhanced sensitivity to brain Na(+)-K(+)-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in alpha(2) +/- vs. alpha(2)+/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of Na(+)-K(+)-ATPase alpha(2)-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain alpha(2)-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension.

Endogenous brain Na pumps, brain ouabain-like substance and the α2 isoform in salt-dependent hypertension

An endogenous ouabain-like substance (OLS) plays a critical role in the etiology of experimental models of human hypertension induced by a high salt diet. Early on, evidence for a role of this Na, K-ATPase inhibitor in blood pressure regulation was provided mainly by correlations of blood pressure with the levels of circulating Na, K-ATPase inhibitor. However, over the past decade, numerous studies have shown that endogenous Na pump inhibitors in the brain mediate salt-dependent hypertension in a variety of experimental models, including Dahl salt-sensitive (Dahl-S) and spontaneously hypertensive (SHR) rats on a high-salt diet. Other forms of hypertension that are known to be mediated by endogenous ouabain-like substances include steroid/salt- (e.g., DOCA-salt) and ACTH-induced hypertension. Even when exogenous ouabain is peripherally administered and/or the plasma ouabain/OLS level is increased in rats, the resulting hypertension is of CNS origin. After peripheral ouabain administration, ouabain levels increase in the plasma and the inhibitor subsequently accumulates in the brain. The ensuing hypertension is abolished by the intracerebroventricular (icv) administration of an anti-ouabain antibody (but not by the same antibody dose given iv), by discrete excitotoxic lesions in the brain or by ganglionic blockade, demonstrating that the response is neurally mediated. The pressor response to stimuli that increase the brain OLS (high salt diet, icv sodium) or to icv ouabain is abolished by icv losartan, demonstrating that the brain OLS activates the brain renin-angiotensin system (RAS) downstream. There are three isoforms of the catalytic α subunit of the Na, K-ATPase in the brain and cardiovascular system (α1, α2 and α3), but it is not known which brain isoform(s) mediate the hypertensive effects of circulating/CNS ouabain. Preliminary studies in gene-targeted mice suggest that the α2 isoform plays a critical role.

Rapid eye movement sleep deprivation induces changes in the high-affinity binding of [ 3H]-ouabain to the rat cortical membranes

Rapid eye movement sleep (REMS) suppresses seizures. On the other hand, REMS deprivation (REMSD) increases brain susceptibility to seizures. Sodium–postassium/ATPase is involved in the control of brain excitability. Ouabain, a cardiotonic glycoside, binds to a regulatory extracellular allosteric site in the sodium–potassium/ATPase inhibiting/stimulating its activity depending on its concentration. Endogenous ouabain-like substances exist in the brain; therefore, changes in the ouabain binding site may be involved in the increased brain excitability induced by REMSD. Adult, Wistar male rats were deprived of REMS for 96 hours by the flower-pot method (REMSD). A stress control group was kept in the same environment on a larger platform (LP). A third group of rats was kept in the same room in their home-cages (CONTROL). After REMSD all rats were sacrificed by decapitation and their cerebral cortex dissected. High-affinity [ 3H]-ouabain binding was carried out in cortical crude membrane preparation using 8 concentrations of [ 3H]-ouabain (1–24 nM). The results show a statistically significant increase of KD in the REMSD rats compared to both CONTROL and LP groups. There were no statistically significant differences in th B max among the experimental groups. There was also no change either in cortical activity of K + stimulated p-nitrophenylphosphatase, the dephosphorylation reaction of phosphorylated sodium–potassium/ATPase or in Mg 2+-stimulated p-nitrophenylphosphatase. An increase in the KD of [ 3H]-ouabain binding to the sodium–potassium/ATPase in REMSD rats indicates a lower affinity to the endogenous inhibitors/stimulators of the enzyme. Therefore, this decreased affinity of the endogenous ouabain-like substances may be involved in the increased excitability induced by REMSD.

Modulation of Aspartate Release by Ascorbic Acid and Endobain E, an Endogenous Na+, K+-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na+, K+ -ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM D-[3H]aspartate (15 min at 37 degrees C), centrifuged, washed, incubated in the presence of additions (60 s at 37 degrees C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5-5.0 mM ascorbic acid, D-[3H]aspartate release was roughly 135-215% or 110-150%, with or without 40 mM KCI, respectively. The endogenous Na+, K+ -ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5-10.0 mM commercial ouabain enhanced roughly 100% D-[3H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50-60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na+, K+ -ATPase, may well modulate neurotransmitter release at synapses.

Furosemide: progress in understanding its diuretic, anti-inflammatory, and bronchodilating mechanism of action, and use in the treatment of respiratory tract diseases.

Accumulated experimental and clinical data suggest that adrenocorticosteroids and/or endogenous ouabain-like substances may play an important role in the mechanism of furosemide diuretic action. It was reported that the drug is highly bound in the adrenals, lungs, kidney, spleen, and liver. In patients with liver cirrhosis, furosemide exerted a markedly decreased natriuretic effect compared with normal subjects, and the plasma levels of circulating endothelin and atrial natriuretic factor (ANF) were significantly elevated. In neonates, after administration of furosemide, the urinary excretion of endothelin-1 and aldosterone increased markedly, and it is known that endothelin may release ANF and aldosterone in a dose-dependent manner. Furosemide was used to stimulate zona glomerulosa, whereas ANF decreased the production of steroids in zona glomerulosa and fasciculata cell culture owing to stimulation by various factors. Because the concomitant use of ANF and furosemide appeared to be diuretically effective in newborns after cardiac surgery, one may suggest that furosemide competes with ANF for its effects on the adrenals. Furosemide administered by inhalation exerted a protective effect on allergic and perennial nonallergic rhinitis and was effective in preventing the postsurgical recurrence of nasal polyposis. The drug can also be used as an antiasthmatic agent. In preterm ventilator-dependent infants with chronic lung disease, aerosolized furosemide improved pulmonary function with no marked effect on diuresis. In adults and children with asthma, furosemide exerted a protective effect against bronchoconstriction induced by several indirect stimuli similar to that of disodium cromoglycate or nedocromil. Aerosolized furosemide had a preventive effect also on bronchoconstriction induced by inhaled lysine acetylsalicylate in patients with aspirin-sensitive asthma. In high-dose beclomethasone-dependent asthma, inhaled lysine acetylsalicylate and furosemide exerted a mutually potentiating antiasthmatic activity, allowing considerable sparing of the inhaled steroid. It is proposed that this effect may be explained by the corticosteroid-sparing action of lysine released from the lysine acetylsalicylate molecule because similar beneficial effects were also obtained after the concomitant use of epsilon-aminocaproic acid (whose chemical structure is almost the same as that of lysine) and prednisone. Furosemide exhibited an anti-inflammatory effect through inhibition of production and release of cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha from peripheral mononuclear cells, which may have a beneficial effect on local inflamed tissue imbalance in the ratio of different cytokines, thus improving the sensitivity of target cells to endogenous glucocorticosteroids.

Exercise, A Measure to Lower Blood Pressure and Reduce Other Risks

By 1990, exercise had been proven to lower blood pressure, and subsequently the intensity of exercise recommended was lowered from moderate to mild (lactic threshold or 50% maximum oxygen uptake). Exercise is now recommended as a useful measure to lower blood pressure in many guidelines for the management of hypertension. The antihypertensive mechanism is multifactorial involving sympathicolytic as well as diuretic actions through activation of relevant metabolic pathways; that is, decrease in endogenous ouabain-like substance, increase in s-taurine and prostaglandin E and urinary dopamine and kallikrein excretion, etc. Other metabolic changes seem to operate simultaneously, and other risk factors, such as sugar and lipid metabolism and insulin resistance are improved. Prospective epidemiologic study has suggested that a physically active lifestyle will prevent cardiovascular complications.

Endogenous ouabain secretion in man is not regulated by ACTH

It has been suggested that endogenous ouabain-like substance (OLS) is of adrenal origin and the secretion of OLS may be ACTH dependent. To determine if OLS is influenced by the pituitary–adrenal axis, we studied the effect of adrenal stimulation (0.25 mg Synacthen) and suppression (1 mg Dexamethasone) on two separate groups of nine subjects. Serum OLS was measured by a radioimmunoassay (RIA) developed in our lab, and cortisol and ACTH were measured by commercial assay kits. Dexamethasone significantly ( P<0.001) suppressed serum cortisol and ACTH concentrations, without effecting endogenous OLS concentration (0.64±0.17 vs 0.85±0.18 nmol/l). Synacthen increased the concentration of cortisol in serum ( p<0.001) over the test period; OLS concentration, again, remained unchanged (0.45±0.04 vs 0.43±0.05 nmol/l). In further studies, serum concentrations of cortisol and OLS were compared between left (LAV) and right (RAV) adrenal veins with that from the inferior vena cava (IVC). Concentration of cortisol in the LAV and RAV was five-fold greater than that in IVC. However, there was no difference in OLS concentration at the corresponding sites. In addition, serum OLS concentrations in patients having undergone bilateral adrenalectomy or diagnosed with Addison's disease (0.62±0.19 nmol/l) were similar to concentrations in healthy subjects (0.67±0.21 nmol/l). Examination of bovine adrenal, liver, kidney, heart and human placenta demonstrated that OLS content of bovine adrenal was comparable with other tissues analysed. HPLC studies of human serum and bovine adrenal gland produced identical elution profiles, resolving a single peak which coincided with the retention time observed for standard ouabain. We conclude that the adrenal is unlikely to be the source of endogenous OLS, the secretion of which appears to be independent of ACTH.

Effect of high salt intake on plasma and tissue concentration of endogenous ouabain-like substance in the rat

The effect of high salt intake on serum concentration and tissue distribution of ouabain-like substance (OLS) was examined in rats. Sprague-Dawley rats (n = 8) were placed on a high salt diet by the inclusion of 1.8% sodium chloride in drinking water for 7 days and a ‘control’ group (n = 8) was maintained on normal drinking water during the study period. Serum and tissue OLS was measured by radioimmunoassay after solid phase extraction. High salt intake significantly increased serum OLS concentration (1.43 ± 0.06 vs 1.14 ± 0.05 nmol/L; mean ± SEM, p = 0.002). In both groups, the adrenal snowed significantly (p < 0.001) higher OLS content compared to liver, kidney, heart and brain. HPLC of rat serum extract resolved a major peak with a retention time identical to that of standard ouabain, further confirming the nature of OLS. We conclude that high salt intake increases endogenous production of OLS, which appears to originate from the adrenal gland in the rat.

Endogenous Ouabain-Like Substance (Eols) in Neonatal Urine

The present study was undertaken to define the possible role of EOLS in controlling renal functions. in mediating the renal responses to furosemide(F) and in inducing endocrine reactions in F-treated neonates. Ten newborn infants with mean birthweight of 2752 g and mean gestational age of 37.1 weeks were given F in a dose of 1 mg/kg. Prior to and following F therapy urine was collected for a period of 12 hours and analyzed for creatinine, osmolality, Na and K, as well as for EOLS, AVP, aldosterone and ET-1. In response to F urine flow rate and urinary osmolar, sodium and potassium excretion increased significantly, whereas creatinine excretion remained unchanged. Furthermore, following F therapy there was an increase in EOLS (148.7±70.8 vs 200.1±98.1 pg/kg/h, p<0.01), ET-1 (36.0±5.6 vs 61.4±8.7 fmol/kg/h. p<0.001), AVP (19.5±5.4 vs 27.8±7.8 pg/kg/h, p<0.05) and aldosterone (507±120 vs 751±203 ng/kg/h, p<0.05) excretion, respectively. Prior to F urinary EOLS excretion was found to correlate positively with diuresis, Na, creatinine, osmolar, ET-1 and AVP excretion. After F urinary EOLS excretion significantly correlated only with diuresis ET-1 and AVP. Urinary EOLS proved to be independent of aldosterone excretion irrespective of F administration. It is concluded that urinary EOLS excretion is closely related to neonatal renal functions and it may have a role in controlling diuresis and natriuresis.

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.

Plasma potassium may protect sodium pumps of toad hearts from an endogenous inhibitor.

Resibufogenin (3-hydroxy-14,15-epoxy-20,22-dienolide glycoside) is a potent sodium pump inhibitor present in toad toxin. It is present in the skin of the cane toad (Bufo marinus) at a concentration equivalent to ouabain of approximately 1 mM. Because toads, like other amphibians, have permeable skin, resibufogenin is also found in high concentrations in the blood. In the cane toad the blood concentration is estimated to be 1 microM (D. Lichtstein, S. Kachalsky, and J. Deutsch. Life Sci. 38: 1261-1270, 1986; D. Lichtstein, S. Samuelov, J. Deutsch, H. Xu, R. A. Lutz, S. S. Chernick and S. S. Chernick. Klin. Wochenschr. 65, Suppl. 8: 40-48, 1987), a concentration thousands of times that required to produce toxicity in humans (J. S. Flier, E. Matatos-Flier, J. A. Pallotta, and D. McIsaac. Nature Lond. 279: 341-343, 1979). In examining how the cane toad avoids inhibiting its own sodium pumps, work on the heart showed that 1) cane toads possess a similar number of cardiac sodium pumps as other vertebrates, and 2) normal plasma K+ levels completely prevent ouabain, and presumably resibufogenin, from binding to cardiac sodium pumps of the cane toad. Other species, i.e., rat (Rattus norvegicus) and salamander (Ambystoma mexicanum), did not show K+ protection of their cardiac ouabain binding sites up to normal plasma K+ levels. These species do not possess the high level of endogenous ouabain-like substance found in the toad. K+ demonstrated a capacity to protect the enzymatic activity of the toad heart sodium pumps from the inhibitory effects of ouabain.

Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome.

The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.

Hypertension and exercise.

A sedentary lifestyle may be a risk for hypertension, according to the results of both cross sectional and longitudinal studies. However, exercise may reverse the adverse effects of lack of activity. Many controlled studies have shown that exercise lowers systolic/diastolic blood pressure by at least 10/5 mmHg. Exercise not only improves blood pressure, but also attenuates other risk factors for cardiovascular complications. Dynamic isotonic exercise (e.g., weight lifting). Milder (e.g., brisk walking for 30-60 minutes/day) rather than moderate to severe exercise (e.g., running) is also recommended because of similar effectiveness and better compliance. The underlying mechanism of action of exercise on blood pressure seems to be multifactorial involving a decrease in pressor factors such as plasma norepinephrine, the serum Na/K ratio, endogenous ouabain-like substance and erythrocyte mean corpuscular volume, as well as an increase in depressor factors such as plasma prostaglandin E, serum taurine and urinary dopamine excretion.

Purification and properties of endogenous ouabain-like substances from hemofiltrate and adrenal glands.

Chick embryo heart cells in culture were found to contain two cell types differing in their [Ca2+]i. Elevation of [Ca2+]i in cells with 99.4 +/- 7.1 nM [Ca2+]i was induced half-maximally at 2 x 10(-9) M ouabain and of cells with 27.9 +/- 4.4 nM [Ca2+]i at 4 x 10(-8) M ouabain. The localization of a sodium pump with differing sensitivity for ouabain in different cell types of the heart raised the question of the existence of endogenously formed ouabain. From 6,000 I hemofiltrate of uremic patients, a substance of 582 Da cross-reacting with ouabain antibodies was purified 42,000-fold. The substance was slightly more hydrophobic on a C-18 reversed-phase high-performance liquid chromatography (HPLC) than ouabain was. It inhibited 86Rb+ uptake into red blood cells and raised like ouabain [Ca2+]i in both types of chick heart cells. From pig adrenal glands, a substance inhibiting 86Rb+ uptake into erythrocytes was purified by affinity chromatography on a column containing antibodies against ouabain. A major part of the eluted substances chromatographed on a C-18-HPLC column as ouabain did, but a minor part was somewhat more hydrophobic. It is presumed that more than a single endogenous ouabain-like factor exists in mammals.

Purification and Characterization of Specific Endogenous Ouabainlike Substance from Bovine Adrenal

Endogenous inhibitors of Na, K-ATPase have been implicated in the pathogenesis of salt-induced hypertension. Despite an intensive search, the inhibitor(s) have long remained elusive. We have been able to purify such an inhibitor from methanol extracts of bovine adrenal glands by multiple steps of high-performance liquid chromatography (HPLC). This compound showed striking similarity to the cardiac glycoside ouabain in its dose dependency in the inhibition of Na, K-ATPase and Na-pump activity, competitive binding to the ouabain-binding site, and dependence of these effects on K+ concentration. These results indicate that vertebrate animals contain a regulator of Na, K-ATPase.