Anti-ouabain Antibodies Research Articles

Reduction in endogenous cardiac steroids protects the brain from oxidative stress in a mouse model of mania induced by amphetamine

ObjectivesBipolar disorder (BD) is a severe mental illness characterized by episodes of mania and depression. Numerous studies have implicated the involvement of endogenous cardiac steroids (CS), and their receptor, Na+, K+ -ATPase, in BD. The aim of the present study was to examine the role of brain oxidative stress in the CS-induced behavioral effects in mice. MethodsAmphetamine (AMPH)-induced hyperactivity, assessed in the open-field test, served as a model for manic-like behavior in mice. A reduction in brain CS was obtained by specific and sensitive anti-ouabain antibodies. The level of oxidative stress was tested in the hippocampus and frontal cortex by measuring the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the levels of antioxidant non-protein thiols (NPSH) and oxidative damage biomarkers thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC). ResultsAMPH administration resulted in a marked hyperactivity and increased oxidative stress, as manifested by increased SOD activity, decreased activities of CAT and GPx, reduced levels of NPSH and increased levels of TBARS and PC. The administration of anti-ouabain antibodies, which reduced the AMPH-induced hyperactivity, protected against the concomitant oxidative stress in the brain. ConclusionsOur results demonstrate that oxidative stress participates in the effects of endogenous CS on manic-like behavior induced by AMPH. These finding support the notion that CS and oxidative stress may be associated with the pathophysiology of mania and BD.

Endogenous cardiac steroids in animal models of mania.

Bipolar disorder (BD) is a complex psychiatricdisordercharacterized by mania and depression. Alterations in brain Na(+) , K(+) -ATPase and cardiac steroids (CSs) have been detected in BD, raising the hypothesis of their involvement in this pathology. The present study investigated the behavioral and biochemical consequences of a reduction in endogenous brain CS activity in animal models of mania. Amphetamine (AMPH)-induced hyperactivity in BALB/c and black Swiss mice served as a model of mania. Behavior was evaluated in the open-field test in naïve mice or in mice treated with anti-ouabain antibodies. CS levels were determined by enzyme-linked immunosorbent assay (ELISA), using sensitive and specific anti-ouabain antibodies. Extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) phosphorylation levels in the frontal cortex were determined by western blot analysis. Administration of AMPH to BALB/c and black Swiss mice resulted in a marked increase in locomotor activity, accompanied by a threefold increase in brain CSs. The lowering of brain CSs by the administration of anti-ouabain antibodies prevented the hyperactivity and the increase in brain CS levels. AMPH caused an increase in phosphorylated ERK (p-ERK) and phosphorylated Akt (p-Akt) levels in the frontal cortex, which was significantly reduced by administration of the antibodies. A synthetic 'functional antagonist' of CSs, 4-(3'α-15'β-dihydroxy-5'β-estran-17'β-yl) furan-2-methyl alcohol, also resulted in attenuation of AMPH-induced hyperactivity. These results are in accordance with the notion that malfunctioning of the Na(+) , K(+) -ATPase/CS system may be involved in the manifestation of mania and identify this system as a potential new target for drug development.

Reduction in Maternal Circulating Ouabain Impairs Offspring Growth and Kidney Development

Ouabain, a steroid present in the circulation and in various tissues, was shown to affect the growth and viability of various cells in culture. To test for the possible influence of this steroid on growth and viability in vivo, we investigated the involvement of maternal circulating ouabain in the regulation of fetal growth and organ development. We show that intraperitoneal administration of anti-ouabain antibodies to pregnant mice resulted in a >80% decline in the circulating ouabain level. This reduction caused a significant decrease in offspring body weight, accompanied by enlargement of the offspring heart and inhibition of kidney and liver growth. Kidney growth inhibition was manifested by a decrease in the size and number of nephrons. After the reduction in maternal circulating ouabain, kidney expression of cyclin D1 was reduced and the expression of the α1 isoform of the Na(+), K(+)-ATPase was increased. In addition, the elevation of proliferation signals including ERK1/2, p-90RSK, Akt, PCNA, and Ki-67, and a reduction in apoptotic factors such as Bax, caspase-3, and TUNEL were detected. During human pregnancy, the circulating maternal ouabain level increased and the highest concentration of the steroid was found in the placenta. Furthermore, circulating ouabain levels in women with small-for-gestational age neonates were significantly lower than the levels in women with normal-for-gestational age newborns. These results support the notion that ouabain is a growth factor and suggest that a reduction in the concentration of this hormone during pregnancy may increase the risk of impaired growth and kidney development.

Endogenous ouabain regulates cell viability

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.

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.

Generation and application of anti-ouabain IgY antibodies

Ouabain is a bioactive hapten and is very difficult to be accurately quantified because of the lack of useful reagents. Furthermore, where ouabain is produced in the adrenal glands has not been identified. In this study, ouabain-BSA was generated for immunizing the laying hens to generate ouabain-specific IgY antibodies in chicken eggs. The anti-ouabain IgY antibodies were detected in eggs 1 week after the last immunization and their concentrations increased with time. The highest concentrations of anti-ouabain IgY antibodies reached at 1:10,240 for ELISA 5 weeks after immunization and maintained for 4 weeks in chicken eggs. Following PEG precipitation, an average of 8.5 mg of anti-ouabain IgY antibodies with a purity of 87.6% was achieved from a single egg. Further analysis revealed that the anti-ouabain IgY antibodies had little immunoreactivity to hydrocortisone, dexamethasone, cedilanid, and digoxin, indicating their high specificity, and the purified IgY antibodies effectively detected endogenous ouabain in the cytoplasm of cells predominately in the zona reticularis of rat and human adrenal glands, indicating their high immunoreactivity. Given that IgY has an unique structure and bioactive features, the generated anti-ouabain IgY antibodies may be used as a new reagent for accurately quantifying ouabain in biological studies.

Neutralization of endogenous digitalis-like compounds alters catecholamines metabolism in the brain and elicits anti-depressive behavior

Depressive disorders are among the world's greatest public health problems. Na +, K +-ATPase is the established receptor for the steroidal digitalis-like compounds (DLC). Alteration in brain Na +, K +-ATPase and DLC have been detected in depressive disorders raising the hypothesis of their involvement in these pathology. The present study was designed to further elaborate this hypothesis by investigating the behavioral and biochemical consequences of neutralization in brain DLC activity attained by anti-ouabain antibodies administrations, in normal Sprague–Dawley (SD) and in the Flinders Sensitive Line (FSL) of genetically depressed rats. Chronic i.c.v. administration of anti-ouabain antibodies to FSL rats elicited anti-depressive behavior. Administration of anti-ouabain antibodies intracerebroventriculary (i.c.v.) to SD rats significantly changed the levels of catecholamines and their metabolites in the hippocampus, ventral tegmentum and nucleus accumbence. These results are in accordance with the notion that endogenous DLC may be involved in the manifestation of depressive disorders and suggests that alteration in their levels may be of significant therapeutic value.

An ouabain-like factor is secreted from immortalized hypothalamic cells in an aldosterone-dependent manner

Ouabain-like factor (OLF) modulates blood pressure via sodium pump inhibition in the central nervous system and in the peripheral circulation. Ouabain-like factor (OLF) is thought to be produced in the adrenal gland and hypothalamus, and it may relate locally to the renin–angiotensin–aldosterone system. However, the evidence for the latter was obtained from in vivo experiments using animals. In the present study, we investigated ouabain production in the immortalized hypothalamic cell line N1. First, cell culture supernatant was collected from the immortalized hypothalamic cell line N1 at 0.5, 4, 8, and 24h. A newly developed enzyme-linked immunosorbent assay (ELISA) that used anti-ouabain antibody showed that immunoreactivity in the supernatant was increased significantly at 24 vs. 0.5h (0.01±0.004 vs. 0.16±0.033pmol/mg protein, p<0.01). A combination of HPLC and ELISA was used to characterize N1 cell-derived OLI, showing that the highest peak of OLI had the same retention time as authentic ouabain. Thereafter, N1 cells were cultured with (1–10μM) aldosterone, and supernatant was collected after 24h of culture. In addition, N1 cells were cultured with 5μM eplerenone, a mineralocorticoid receptor blocker, plus aldosterone. OLI was significantly increased in the supernatant of the cells cultured with 10μM aldosterone (0.40±0.078pmol/mg protein), and this increase was abolished by the addition of the aldosterone antagonist eplerenone (0.12±0.030pmol/mg protein). These data suggest that the immortalized hypothalamic N1 cells secrete OLF and that aldosterone stimulates its secretion via mineralocorticoid receptors.

Highlights From The Literature

Highlights From The Literature

Endogenous ouabain-like compounds in locus coeruleus modulate rapid eye movement sleep in rats

Although the detailed mechanism of spontaneous generation and regulation of rapid eye movement sleep (REMS) is yet unknown, it has been reported that noradrenergic REM-OFF neurons in the locus coeruleus (LC) cease firing during REMS and, if they are kept active, REMS is significantly reduced. On the other hand, the activity as well as expression of Na-K ATPase has been shown to increase in the LC following REMS deprivation. Ouabain is a specific inhibitor of Na-K ATPase, and endogenous ouabain-like compounds are present in the brain. These findings led us to propose that a decrease in the level of ouabain-like compounds spontaneously available in and around the LC would stimulate and increase the REM-OFF neuronal activities in this region and thus would reduce REMS. To test this hypothesis, we generated anti-ouabain antibodies and then microinjected it bilaterally into the LC in freely moving chronically prepared rats and recorded electrophysiological signals for evaluation of sleep-wakefulness states; suitable control experiments were also conducted. Injection of anti-ouabain antibodies into the LC, but not into adjacent brain areas, significantly reduced percent REMS (mean +/- SEM) from 7.12 (+/-0.74) to 3.63 (+/-0.65). The decrease in REMS was due to reduction in the mean frequency of REMS episode, which is likely due to increased excitation of the LC REM-OFF neurons. Control microinjections of normal IgG did not elicit this effect. These results support our hypothesis that interactions of naturally available endogenous ouabain-like compounds with the Na-K ATPase in the LC modulate spontaneous REMS.

Physiological roles of endogenous ouabain in normal rats

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.

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.

Intrahippocampal microinjection of an exquisitely low dose of ouabain mimics NaCl loading and stimulates a bufadienolide Na/K-ATPase inhibitor

Brain endogenous ouabain (EOU) raises blood pressure (BP) via an angiotensin II (ATII)-sensitive pathway in NaCl-loaded Dahl salt-sensitive rats (DSS). Brain EOU activates central and adrenocortical renin-angiotensin systems, and stimulates marinobufagenin, a vasoconstrictor and natriuretic inhibitor of sodium pump. We studied effects of acute NaCl loading (17 mmol/kg NaCl, intraperitoneally) on levels of EOU and marinobufagenin in several brain areas in DSS. We then studied effects of intrahippocampal administration of very-low-dose ouabain (60 pg) on EOU, marinobufagenin, BP, sodium excretion, and sodium-pump activity in the aorta and renal medulla in the absence and presence of anti-marinobufagenin and anti-ouabain antibodies, and losartan. NaCl loading of DSS induced transient increases of EOU in the hippocampus and amygdala (15 min; 300%), supraoptical nucleus of hypothalamus (SON) (30 min, 230%) and pituitary (30 min; 85%), and ATII elevation in the SON (30 min). Intrahippocampal administration of ouabain (60 pg) stimulated ATII in the SON, produced natriuresis, 40 mmHg rise in BP, inhibition of sodium-pump in the renal medulla (19.6%) and aorta (25%), and a two-fold increase in renal marinobufagenin excretion. Pretreatment of rats with anti-marinobufagenin antibody prevented ouabain-induced pressor and natriuretic responses and sodium-pump inhibition. Pressor responses to ouabain were also prevented by losartan (intravenously) and by administration of anti-ouabain antibody into the SON. NaCl loading of DSS induces a cascade of events, triggered by brain EOU and ATII. Intrahippocampal administration of a low-dose ouabain mimics effects of NaCl loading and stimulates marinobufagenin, which produces natriuresis, and inhibits the vascular sodium-pump, inducing an increase in BP.

Endogenous Na/K-ATPase inhibitors in patients with preeclampsia.

Although preeclampsia (PE) is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Digitalis-like cardiotonic steroids (CTS) are believed to be involved in the pathophysiology of PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody which binds CTS, lowers blood pressure in PE. Recently we reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor CTS, are increased fourfold in patients with severe PE. In the present study, we tested whether anti-MBG, or anti-ouabain antibodies, or DIGIBIND can reverse inhibition of erythrocyte Na/K-ATPase (NKA) from patients with mild PE (blood pressure, 149 +/- 3/93 +/- 3 mm Hg; age, 28 +/- 2 years; gestational age, 37 +/- 1 weeks). Development of PE was associated with twofold rise in plasma MBG levels (1.58 +/- 0.15 vs. 0.80 +/- 0.11 nmol/L; P<0.01). The activity of erythrocyte NKA in 12 patients with PE was lower than in 6 normotensive gestational age-matched subjects (1.56 +/- 0.18 vs. 3.11 +/- 0.16 micromol Pi/ml/hr; P<0.001). In vitro treatment of erythrocytes from PE patients with anti-MBG antibody fully restored the NKA activity (3.26 +/- 0.41 micromol Pi/ml/hr; P<0.01). The effects of DIGIBIND was marginally significant (2.53 +/- 0.32 micromol Pi/ml/hr), while the anti-ouabain antibody was not effective (2.25 +/- 0.25 micromol Pi/ml/hr, P>0.5). The present observations provide evidence for a role for MBG in the pathogenesis of PE, and suggest that antibodies against MBG may be useful in the treatment of this syndrome.

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

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. 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). 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. 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.

Endogenous ligand of alpha(1) sodium pump, marinobufagenin, is a novel mediator of sodium chloride--dependent hypertension.

Digitalis-like sodium pump ligands (SPLs) effect natriuresis via inhibition of renal tubular Na(+),K(+)-ATPase but may induce vasoconstriction. The present study investigated the potential roles of 2 putative endogenous SPLs, an ouabain-like compound (OLC) and an alpha(1) Na(+),K(+)-ATPase inhibitor, marinobufagenin (MBG), in regulating natriuresis and blood pressure (BP) responses to sustained and acute NaCl loading in Dahl salt-sensitive rats (DS). During 4 weeks of an 8% NaCl diet, DS exhibited a progressive increase in MBG renal excretion (66 +/-13 pmol/24 hours at week 4 versus 11 +/- 1 pmol/24 hours at baseline, n=48), which paralleled an increase in systolic BP (174 +/- 10 mm Hg at week 4 versus 110 +/- 2 mm Hg at baseline). By contrast, OLC excretion peaked at week 1 and returned to baseline levels. Administration of an anti-MBG, but not anti-ouabain antibody, to DS after 3 weeks of a high NaCl diet lowered BP (139 +/- 7 versus 175 +/- 5 mm Hg, P<0.001, n=5). Acute NaCl loading (2 hours) of DS (n=5) increased MBG and OLC excretion and natriuresis. Pretreatment of acutely NaCl-loaded DS with an anti-MBG antibody (n=5) reduced the excretion of sodium and MBG but not that of OLC. An anti-ouabain antibody (n=5) reduced sodium excretion and both OLC and MBG. An initial transient stimulation of OLC induced by NaCl loading of DS precedes an MBG response. A sustained increase in MBG production in DS contributes to the chronic BP elevation induced by a sustained high NaCl intake.

Effects of Endogenous Ouabain on the Development of Hypertension in 1k1c Hypertensive Rats

This study was designed to evaluate the role of endogenous ouabain (EO) in the development of hypertension in 1ktc (one kidney, one clip) hypertensive rats. First, the EO content of the serum of 1k1c hypertensive rats and normal Sprague-Dawley (SD) rats was detected by the enzyme linked immunosorbent assay method (ELISA). Second, blood pressure changes in the 1k1c rats were recorded directly after the 1k1c rats were injected randomly with anti-ouabain antibody, normal rabbit IgG, and normal saline, respectively. The results showed that EO levels in the serum of 1k1c hypertensive rats were significantly higher than those of normal SD rats (2.25 +/-0.92 microg/l vs. 1.12 +/- 0.17 microg/l, p< 0.01), and correlated significantly with systolic blood pressure (r= 0.59, p< 0.05). Anti-ouabain antibody was able to significantly decrease the blood pressure of 1k1c hypertensive rats in a dose-dependent manner, while normal rabbit IgG or normal saline was not. These results indicate that endogenous ouabain might play an important role in the development of hypertension in 1k1c hypertensive rats.

Detection of digitalis compounds using a surface plasmon resonance-based biosensor

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC 50 value of 5×10 −10 M, 7.0×10 −10 M and 2×10 −8 M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC 50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

Vasodepressor Effects of Exercise are Accompanied by Reduced Circulating Ouabainlike Immunoreactivity and Normalization of Nitric Oxide Synthesis

Our object was to evaluate the effects of regular mild exercise on blood pressure and on circulating level of ouabainlike factors (OLF) and of nitrate anion, an endproduct of nitric oxide (NO) in humans. We measured plasma ouabainlike immunoreactivity (OLI) and nitrate ions (NO3) before and after mild exercise for 3 months' duration in 16 patients with essential hypertension, diabetes mellitus, obesity, or hyperlipidemia. Plasma OLI was measured using an amplified ELISA system with anti-ouabain antibody and biotinyl-tyramide. Serum NO3 was measured with high-performance liquid chromatography (HPLC) with an anion-exchange column. With the reverse phase HPLC system with an octa decylsilyl silicagel column, the elution volume of plasma OLI of a healthy volunteer matched that of authentic ouabain in a gradient elution system of acetoNtriIe/HzO. PlasmaOLI levels decreased significantly by about 34% after mild exercise, and NO3′ levels tended to be within the reference interval in normal volunteers. Body weight, diastolic and systolic blood pressure, serum triglyceiide and acetylcholine esterase (a marker of the fatty liver) were significantly decreased (p< 0.01) after 3 months of regular mild exercise. The plasma OLI level was significantly correlated with plasma N03-: there was a trend toward a correlation with diastolic blood pressure (pa. 06) before and after regular exercise. Regular mild exercise led to a decrease in plasma levels of OLI, and acetylcholine esterase activity and blood pressure in adult patients. Results suggest that changes in OLF production contribute to the blood pressure regulation seen in patients who exercise regularly.

Endogenous marinobufagenin-like immunoreactive substance: A possible endogenous Na,K-ATPase inhibitor with vasoconstrictor activity

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.