Endogenous ouabain and cardiomyopathy in dialysis patients.

BACKGROUND AND METHODS: Endogenous ouabain (EO) is markedly raised in patients with chronic renal failure. As high EO induces myocardial cell hypertrophy in vitro and it is associated with left ventricular hypertrophy (LVH) in essential hypertensives and in patients with heart failure we investigated the relationship between plasma EO and LV mass and geometry in 156 end-stage renal disease (ESRD) patients. EO was measured by a specific radioimmunoassay and by mass spectrometry. RESULTS: On univariate analysis, plasma EO was directly related to LV mass (r = 0.26, P = 0.001) and LV end diastolic volume (r = 0.25, P = 0.002) and these relationships held true in multiple linear regression models including a series of potential confounders. Patients with eccentric LVH (n = 41, i.e. 26%) had the highest plasma levels of EO when compared to patients with other patterns of LV geometry (P = 0.001). Furthermore, plasma EO had diagnostic value for eccentric LVH because the area under the corresponding ROC curve (68%) was significantly greater (P = 0.002) than the threshold of diagnostic indifference. In this analysis, the sensitivity was 91% and the specificity was 36%. The positive predictive value was 33% but EO had a remarkably high negative predictive value (92%) for the exclusion of eccentric hypertrophy. CONCLUSIONS: In ESRD patients, plasma EO is independently associated with LV mass, LV volume and eccentric LVH. The results of this study are compatible with the hypothesis that EO is involved in alterations of LV mass in ESRD.

Ouabain is secreted by bovine adrenocortical cells.

Ouabain is a specific inhibitor of the sodium pump. This steroid has been found in the mammalian circulation in significant amounts and may be of adrenal origin. Secretion of ouabain from adrenal cells has been little studied and the purpose of the present work was to determine the adrenal distribution of ouabain, aldosterone and cortisol, and to characterize the effects of ACTH and angiotensin II on the secretion of these steroids in primary cultures of bovine adrenocortical cells. In fresh bovine adrenals, the cortical to medullary ratios for aldosterone, cortisol and ouabain were 14, 4.25 and 2.5, respectively. All three steroids were detected in elevated amounts in the conditioned medium of primary cultures of adrenocortical cells. Reverse phase HPLC of the secreted ouabain immunoreactivity showed it was isopolar with commercial ouabain. In the presence of 10 nM ACTH or angiotensin II, the secretion of all three steroids increased significantly with similar time courses. The stimulated secretion of ouabain exceeded the intracellular content of this steroid in either control or activated cells by 3-5 fold. The amount of angiotensin II stimulated ouabain secretion was greater from cells incubated in larger volumes. These results show that ouabain is enriched in the bovine adrenal cortex, and is secreted by primary cultures of these cells. The secretion of ouabain is increased by ACTH and angiotensin II, is due to either de novo synthesis or transformation of an intracellular precursor that is not overtly immunoreactive, and is feedback regulated by either ouabain itself or a cosecreted factor. These cells may be useful to study stimulus-secretion coupling and the biosynthetic pathway of ouabain.

Purification and characterization of digitalislike factors from human plasma.

Increased levels of a humoral inhibitor of active sodium transport have been associated with the response to acute and chronic hypervolemia and various forms of experimental as well as human essential hypertension. In this report, we describe the purification of inhibitors of Na+, K+-adenosine triphosphatase (ATPase) from the plasma of volume-expanded individuals. Of the two amphipathic materials obtained, only one of the factors when present in high concentrations showed the slow time-dependent component of inactivation similar to that of the cardiac glycosides. Inhibition was reduced in the presence of plasma proteins and was freely reversible. Both factors inhibited potassium-dependent p-nitrophenylphosphatase activity and specific [3H]ouabain binding in a manner similar to the cardiac glycosides. In contrast to ouabain and vanadate, however, high concentrations of potassium or norepinephrine, respectively, did not affect the magnitude or kinetic characteristics of inhibition. Structural analysis by mass spectroscopy showed a mass of 444 for factor 1, whereas factor 2 was conclusively identified as lysophosphatidylcholine-gamma-palmitoyl. These factors probably inhibit Na+, K+-ATPase by a nonspecific mechanism involving reversible perturbation of lipid-enzyme interactions required for normal catalytic activity. The significance of these factors as modulators of sodium transport may be limited to pathological states associated with abnormalities in plasma protein binding or lipid metabolism. They do not appear to be directly related to the humorally mediated disturbance of cellular sodium transport in hypertension.

Effects of angiotensin II on sodium potassium pumps, endogenous ouabain, and aldosterone in bovine zona glomerulosa cells.

Angiotensin (Ang) II stimulates secretions of aldosterone and an endogenous ouabain-like steroid (EO) from bovine adrenal zona glomerulosa (BAG) cells. The BAG cell sodium pump, a possible target of EO, affects aldosterone secretion although little is known about this pump. Here, we describe the effects of Ang II on the characteristics of this transporter and steroid secretions. Under serum-free conditions, 3H-ouabain bound to a single class of sites on BAG cells. Binding of label was time and concentration dependent, was sensitive to extracellular potassium ions, and was displaced by ouabain and digoxin with EC50 of approximately 218 and approximately 232 nmol/L, respectively. Sodium pump-mediated 86Rb uptake was inhibited by ouabain (EC50 approximately 301 nmol/L). Ang II dose dependently augmented secretions of EO and aldosterone, increased ouabain-sensitive 86Rb uptake and 3H-ouabain binding, and increased the affinity for 3H-ouabain binding (Kd, from 205 to 80 nmol/L) with no change in the maximal number of sodium pumps (5.45x10(6)) per cell. Losartan blocked all effects of Ang II except EO secretion, which was inhibited by PD123319. We conclude that BAG cells express sodium pumps in high density and bind ouabain to a single class of low-affinity sites. The characteristics of the sodium pumps protect BAG cells from EO autotoxicity but may exclude them from mediating feedback inhibition of EO secretion. The effects of Ang II on sodium pump activity, ouabain binding affinity, and aldosterone secretion are mediated via Ang II type 1 receptors, whereas Ang II type 2 receptors augment EO secretion. The role of the Ang II-mediated increase in the ouabain sensitivity of BAG cell sodium pumps in the secretions of aldosterone and EO remains to be elucidated.

Different signaling pathways mediate stimulated secretions of endogenous ouabain and aldosterone from bovine adrenocortical cells.

Angiotensin II stimulates secretion of corticosteroids and an ouabain-like compound from adrenocortical cells. The angiotensin AT1 and AT2 receptor subtypes have been linked with stimulated secretion of aldosterone and endogenous ouabain, respectively, but the second messenger mechanisms involved in the latter secretion are not known. Accordingly, we investigated the effects of several pharmacological agents that affect signaling pathways on the basal and stimulated secretions of aldosterone and endogenous ouabain from primary cell cultures of bovine adrenocortical cells. The AT2 receptor antagonist, PD 123319, blocked the effects of angiotensin II on secretion of endogenous ouabain but not aldosterone. Treatment of the cells with either dibutyryl cAMP, a membrane permeant analog, or the phorbol ester tetradecanoyl phorbol acetate stimulated aldosterone secretion but had no effect on the secretion of endogenous ouabain. On the other hand, the membrane permeant analog, 8BcGMP, maximally activated secretion of endogenous ouabain whereas incubation of cells with sodium orthovanadate blocked angiotensin II stimulated secretion of endogenous ouabain. Neither 8BcGMP nor sodium orthovanadate affected the basal or stimulated components of aldosterone secretion. These results show that the secretions of aldosterone and endogenous ouabain from bovine adrenocortical cells are mediated by different intracellular signaling mechanisms and provide evidence that the adrenal secretions of these steroids are regulated differently.

Angiotensin II stimulates secretion of endogenous ouabain from bovine adrenocortical cells via angiotensin type 2 receptors.

Angiotensin II stimulates secretion of corticosteroids and ouabain-like activity from adrenocortical cells. Distinct adrenocortical angiotensin II receptor subtypes (AT1, AT2) have been described, and the present studies investigated their roles in steroid secretion. Using primary bovine adrenocortical cell cultures under serum free conditions, angiotensin II stimulated the secretions of aldosterone, cortisol, and endogenous ouabain as verified by high-performance chromatography. The dose-response curves for stimulated steroid secretion were parallel with unitary slopes while the half-maximally effective concentrations of angiotensin II were 0.31 to 0.38 nmol/L for secretions of aldosterone and cortisol and 2.3 nmol/L for endogenous ouabain. The nonselective mammalian antagonist (Sar1-Ile8) angiotensin II blocked stimulated secretion of all three steroids without affecting basal output. In the presence of the AT1 antagonist DuP753, angiotensin II-stimulated secretions of aldosterone and cortisol were blocked while secretion of endogenous ouabain was unaffected. In the presence of the AT2 antagonist PD123319, both basal and angiotensin II-stimulated secretions of aldosterone and cortisol were normal while stimulated secretion of endogenous ouabain was inhibited. The secretion of endogenous ouabain was activated maximally by the AT2 agonist CGP42112 under conditions in which aldosterone secretion was unaffected. These results demonstrate that AT2 receptors stimulate secretion of endogenous ouabain from bovine adrenocortical cells. The specificity of AT1 and AT2 receptor stimulation indicates that separate signaling mechanisms having minimal cross talk control the adrenocortical secretions of corticosteroids and cardiac-active steroids. Adrenocortical AT2 receptors may be important in the adaptation to low salt diets and other conditions in which angiotensin II is increased.

Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays.

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Isolation and characterization of a sodium pump inhibitor from human plasma.

An endogenous sodium pump inhibitor has been purified from human plasma. The purification scheme involved large scale dialysis, extraction of lyophilized dialysate by methanol followed by preparative and semipreparative scale reverse-phase high-performance chromatography. A single peak of biologically active material was obtained enriched by a factor of greater than 10 billion. This material showed high chromatographic polarity, was inactivated by charring, strong acid, or alkali, and was resistant to short-term boiling. The purified material had a molecular weight between 300 and 900 g/mol and was insensitive to type I esterase and a variety of proteolytic enzymes. The purified factor inhibited the ouabain-sensitive uptake of 86Rb by human erythrocytes, binding of [3H]ouabain, and activity of dog kidney Na,K-adenosine triphosphatase (ATPase) with high affinity (less than 0.3 nM) in a time- and dose-dependent manner. Maximally effective concentrations of the digitalislike factor showed no effect on either human red blood cell Mg- or Ca-ATPase, rabbit muscle sarcoplasmic reticulum Ca-ATPase, or guinea pig stomach H,K-ATPase. The purified material is a highly potent selective inhibitor of the ion transport, receptor, and hydrolytic functions of the sodium pump. The characteristic properties of this substance suggest it may be a mammalian endogenous digitalis and may be similar to the sodium transport inhibitor detected in the plasma of volume-sensitive forms of experimental and human hypertension.

Red cell cotransport activity and sodium content in black men. Relationship to essential hypertension.

Furosemide-sensitive sodium and potassium cotransport and intracellular sodium content ([Na]i) were measured in erythrocytes (red blood cells, RBCs) from a population of 90 adult black men with and without essential hypertension (EH). The mean values for sodium cotransport activity, expressed as furosemide-sensitive Na efflux (mmol/liter RBC/hr), were not significantly different among the EH patients and two control groups, normotensive subjects with a positive history (N+) and those with a negative family history (N-) for hypertensive disease (EH: 154 +/- 123, n = 53; N+: 167 +/- 93, n = 12; and N-: 207 +/- 142, n = 20; all values are means +/- SD). The mean [Na]i 9.66 +/- 3.02 mmol/liter RBC (n = 56) for the EH group was greater than the mean value for the N- control group (7.96 +/- 1.97, n = 20; p less than 0.05). The N+ group also displayed a higher mean [Na]i (10.38 +/- 3.18, n = 12; N+ vs N- p less than 0.01). Although there was substantial overlapping of [Na]i values between the groups and no clear dividing line, the distribution curve of the [Na]i values in EH was skewed toward higher concentrations than in N-. Nevertheless, we must conclude that erythrocyte cotransport and [Na]i are not clinically useful in the identification of EH in black men.

Red blood cell Na+,K+-ATPase in men with newly diagnosed or previously treated essential hypertension.

Alterations of cellular function of Na+,K+-adenosine triphosphatase (ATPase; Na+-K+ pump) have been implicated in the pathophysiology of essential hypertension. Therefore, this aspect of red blood cell (RBC) Na metabolism was studied in black men with newly diagnosed, untreated essential hypertension (NEH) and a normotensive control group. RBC Na content, Na+-K+ pump number (ouabain binding sites), and pump activity were measured. No statistically significant differences were found between the two groups for any of these three parameters. However, a group of previously treated essential hypertensive subjects (PEH) who had been withdrawn from therapy in the preceding 6 weeks were also studied. This group differed significantly from the NEH subjects in regard to all RBC Na+-K+ pump parameters. Their RBC Na content (10.27 +/- 3.23 vs 7.77 +/- 2.52 mmol Na/LRBC; p = 0.006) was higher, and their Na+-K+ pump activity (166 +/- 50 vs 221 +/- 87 nmol inorganic phosphate/mg membrane protein/hr; p = 0.03) and Na+-K+ pump number (213 +/- 40 vs 284 +/- 85 binding sites/RBC; p = 0.001) were lower compared with those in NEH subjects. Although the PEH subjects were older and somewhat less hypertensive than their NEH counterparts, these factors were not found to influence the Na+-K+ pump parameters. These results indicate that chronic diuretic therapy of patients with essential hypertension is associated with a reduced number of RBC Na+-K+ pumps. Since RBCs are not considered target cells for diuretics, the effects of these drugs on RBC electrolyte metabolism may occur at the time of erythropoiesis by the production of RBCs with fewer Na+-K+ pumps.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of an endogenous digitalislike factor from human plasma for structural analysis.

In previous reports, we described the isolation and characterization of an endogenous digitalislike factor (EDLF). In this report, we describe a unique combination of bioassay and large-scale purification methodology that made possible the purification of sufficient quantities of this inhibitor of Na+,K(+)-ATPase for structural analysis. Using an initial XAD-2 extraction and preparative high-performance liquid chromatography followed by a batch enzyme affinity extraction and two subsequent semipreparative chromatographic steps, 300 l of human plasma was processed, yielding 31 micrograms (53 nmol) of pure EDLF and representing purification on a dry weight basis in excess of 0.6 billionfold. Four divergent pieces of evidence, including chromatographic, mass spectrometric, immunoreactive, and binding characteristics, suggested that the EDLF purified in the present study was either ouabain or an isomer of ouabain. This material may represent a plasma-borne, naturally occurring, selective, high-affinity ligand for the digitalis binding site that may play a significant role in the modulation of the sodium pump and thereby cellular electrolyte homeostasis in humans.

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

A sodium pump inhibitor has been isolated from human plasma and extensively purified. This material, endogenous digitalislike factor, was examined by a variety of mass spectrometric techniques. A low-resolution fast atom bombardment mass spectrometric analysis of a sample of purified endogenous digitalislike factor revealed a single unique molecular ion in the mass range 100-2,500. The accurate mass was determined to be 585.295 Da in a second high-resolution fast atom bombardment mass spectrometric experiment. Based on this accurate mass, the elemental composition of endogenous digitalislike factor was determined and found to be identical to the elemental composition of the known cardenolide ouabain. Direct comparison of ouabain and endogenous digitalislike factor by linked scan tandem mass spectrometry, derivatization with acetic anhydride coupled with fast atom bombardment mass spectrometry, and analytical high-performance liquid chromatography failed to reveal any differences. We conclude that the endogenous digitalislike factor isolated from human plasma is ouabain or a closely related isomer.

Development of an immunoassay for endogenous digitalislike factor.

Recently, attempts to purify and identify a circulating inhibitor of the sodium pump have been successful. Based on the outcome of mass spectral analysis of purified inhibitor, we raised in rabbits antibodies to conjugates of the commercially available cardenolide ouabain and used them in the development of an indirect enzyme-linked immunosorbent assay (ELISA) for endogenous digitalislike factor (EDLF). Antisera obtained were of high antibody titer (1:2 x 10(6)) and showed full cross-reactivity with purified EDLF. The antisera were highly specific for ouabain and structurally related cardenolides but showed no cross-reactivity with numerous endogenous steroids and peptides. At each step in the purification of EDLF, inhibition of the sodium pump and immunologic cross-reactivity were inseparable. The ELISA as developed had a working range of 5-2,000 fmol, with an IC50 of 80 fmol/well. Using solid-phase extraction and the ELISA, we determined the circulating level of EDLF in plasma from normal human volunteers to be 138 +/- 43 fmol/ml, whereas patients on total parenteral nutrition for at least 1 week had a circulating level of 108 +/- 17 fmol/ml, suggesting that the circulating factor was of endogenous origin. The ELISA developed appears to measure a naturally occurring counterpart to the cardenolides that could play a role in modulating the sodium pump and thereby cellular electrolyte homeostasis.

Effects of an endogenous ouabainlike compound on heart and aorta.

An endogenous ouabainlike compound (OLC) has been purified from human plasma, and mass spectrometry has shown it to be indistinguishable from plant-derived ouabain. This human OLC was tested for its effects on evoked tension in guinea pig left atria and aortic rings. The tissues were incubated at 37 degrees C in bicarbonate-buffered physiological salt solution gassed with 95% O2-5% CO2. In atria stimulated electrically at 1 Hz, 85 and 170 nM human OLC increased peak active force to 177 +/- 15% and 313 +/- 32% of control, respectively (n = 3), with little effect on the duration of contraction. On washout of the OLC, peak systolic force returned to the control level with a half-time of 4.3 +/- 0.5 minutes. Similar results were obtained with 160 nM plant-derived ouabain: peak systolic force increased to 310 +/- 31% of control (n = 4) and returned to the control level with a half-time of 3.8 +/- 0.2 minutes during washout. In aortic rings, neither 170 nM human OLC nor 160 nM plant ouabain (30-minute treatments) affected resting (unstimulated) tension, but they increased the contractions evoked by histamine (0.2-1.0 microM) to 156 +/- 13% (n = 4) and 143 +/- 6% (n = 4) of control responses, respectively. The mean half-time for washout of the OLC and plant ouabain-induced augmentation of histamine-evoked tension exceeded 35 minutes. These data show that human OLC has cardiotonic and vasotonic actions qualitatively and quantitatively similar to those observed with plant ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Left ventricular mass, stroke volume, and ouabain-like factor in essential hypertension.

Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased (P<0.0001) in patients with EH (377+/-19 pmol/L) versus normotensive (253+/-53 pmol/L) subjects. The distribution of plasma OLF was unimodal in normotensives, whereas it was bimodal in EH. Twenty-four-hour diastolic ambulatory blood pressure was slighter higher in EH with high OLF compared with EH with normal OLF (93.2+/-1.14 versus 89.4+/-1.33 mm Hg, P=0.03). Left ventricular mass index and stroke volume in EH with high OLF were greater than in EH with normal OLF (101.9+/-3.3 versus 86.1+/-2.5 g/m(2), P=0.0003, and 57.10+/-1.48 versus 52.30+/-1.14 mL/m(2), P=0. 02, respectively), although heart rate was slower (74.2+/-1.3 versus 80.5+/-1.3 bpm, P=0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that approximately 50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events.

Long-term ouabain administration produces hypertension in rats.

Ouabain has recently been identified as an endogenous Na(+)-K+ pump inhibitor. We administered ouabain chronically to normotensive rats with varying degrees of reduced renal mass (RRM) and to normal two-kidney rats to see whether hypertension could be produced. Normal male Wistar rats and rats with 25%, 60%, and 70% RRM received ouabain (13.9 micrograms/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 micrograms/kg per day IP) for 3 to 4 more weeks. Respective control animals received vehicle only. Blood pressure was recorded weekly by tail plethysmography. Animals received tap water and standard rat chow, except for 70% RRM rats, which received distilled water and sodium-free chow. After 6 to 8 weeks of treatment, with rats under thiobutabarbital anesthesia, direct blood pressure was determined. Plasma, tissue, and urinary ouabain levels were measured with a specific radioimmunoassay. Animals receiving ouabain developed significant increases in mean blood pressure compared with control animals (70% RRM, 147 +/- 4 vs 116 +/- 4 mm Hg; 60% RRM, 140 +/- 4 vs 107 +/- 3 mm Hg; 25% RRM, 131 +/- 5 vs 100 +/- 2 mm Hg; no RRM, 116 +/- 4 vs 98 +/- 5 mm Hg). Plasma ouabain levels measured 24 hours after the last ouabain dose were not different in animals receiving ouabain vs those receiving vehicle. However, kidney tissue ouabain levels were significantly greater (6.39 +/- 1.17 vs 2.36 +/- 0.52 micrograms/kg, P < .05) in animals receiving ouabain. In conclusion, ouabain, given chronically, is associated with the development of hypertension in RRM rats as well as in normal rats. Blood pressure was greater in animals with greater degrees of RRM for a given ouabain dose.

Sodium transport inhibition, cell calcium, and hypertension. The natriuretic hormone/Na+-Ca2+ exchange/hypertension hypothesis.

Sodium plays a critical role in the etiology of essential hypertension, but the mechanism by which excess dietary sodium actually leads to the elevation of blood pressure is not understood. The hypothesis described shows how an excessive sodium load can lead to the development of hypertension. The underlying factor must be a genetic or acquired deficiency or limitation in renal sodium excretion that may be undetectable by standard renal function tests. The resultant tendency towards sodium, water, and extracellular fluid volume expansion is compensated by the secretion of a natriuretic hormone that promotes sodium excretion by inhibiting sodium pumps in the kidney tubule cells. The hormone also inhibits sodium pumps in other cells, including vascular smooth muscle cells, causing intracellular sodium to increase. Then, because the vascular smooth muscle cells contain a Na+-Ca2+ exchange transport system in their plasma membranes, more calcium than normal is delivered to these cells. This causes the increased contractility and reactivity that underlies the increased vascular tone and peripheral vascular resistance that elevates the blood pressure.

Endogenous ouabain, sodium balance and blood pressure: a review and a hypothesis.

OBJECTIVE: To assess possible relationships between endogenous ouabain, sodium balance and blood pressure. CONTENT: This review concerns the structure of endogenous ouabain, circulating levels of this steroid in various disorders of fluid and electrolyte balance, recent evidence for the association of endogenous ouabain with human hypertension, the influence of sodium and volume factors on ouabain-induced hypertension, and possible mechanisms for the hypertensinogenic activity of ouabain. CONCLUSIONS: The human circulation contains a closely related isomer of ouabain of putative adrenocortical origin. Elevated circulating levels of this 'endogenous ouabain' are common but not universal in physiologic and pathologic states associated with positive sodium balance or high blood pressure, or both. In the absence of adrenal hyperfunction, elevating circulating levels of endogenous ouabain appear to be secondary to impaired renal clearance. Prolonged elevation of circulating ouabain in the rat induces sustained hypertension. This model exhibits normal plasma renin activity, increased levels of ouabain in the hypothalamus, pituitary, and kidney, and responds to angiotensin converting enzyme inhibitor. In rats with normal kidney function, ouabain-induced hypertension is primarily sodium-insensitive although maneuvers that hinder renal sodium excretion augment the pressor effect of this steroid. Prolonged administration of ouabain into the brain ventricles augments sympathetic nervous system activity and induces sustained hypertension. These observations lead us to propose the following hypothesis. Among Caucasian patients with essential hypertension, a large fraction have elevated circulating levels of endogenous ouabain, possibly caused by an inherited or acquired renal defect in clearance of this steroid. In these patients, and in rats with ouabain-induced hypertension, increased local generation of, or increased target organ sensitivity to, angiotensin II, or both, may contribute critically to heightened vasoconstriction and a sustained increase in blood pressure. Investigations of the efferent pressor mechanisms and the renal handling of endogenous ouabain are novel approaches to the etiology and therapy of several common cardiovascular disorders.

Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure.

OBJECTIVES: To determine the steady-state dose-dependence of blood pressure, plasma and tissue ouabain during continuous infusion of ouabain in the rat, and to evaluate the adrenal dependence and effect of a high salt intake on this form of hypertension. DESIGN AND METHODS: Ouabain was administered, via subcutaneous osmotic pumps, to normal and adrenalectomized male Sprague-Dawley rats for 5 weeks. Blood pressure, plasma renin and aldosterone, and circulating and tissue levels of ouabain were determined. RESULTS: Following a latent period, blood pressures and circulating ouabain were significantly elevated dose-dependently in glycoside-infused rats at 5 weeks. Upon withdrawal of the ouabain infusion, blood pressure and plasma ouabain levels normalized within 1 week. In rats that received 30 micrograms ouabain/kg per day, the circulating, kidney, hypothalamic and anterior pituitary levels of ouabain were increased significantly (by 7-, 15-, 2.8- and 2.1-fold, respectively), whereas the content of other tissues tested was unchanged. Blood pressure and plasma levels of ouabain correlated with hypothalamic and kidney glycoside content in the infused rats. High-performance liquid chromatography of the adrenal, renal, hypothalamic and pituitary extracts showed one major peak of ouabain immunoreactivity, with a retention time equivalent to that of commercial ouabain. Plasma renin activity was normal, whereas aldosterone levels were increased significantly to 2.9- and sevenfold in rats that received 10 and 30 micrograms ouabain/kg per day, respectively. Dietary salt loading suppressed aldosterone and did not exacerbate hypertension. In bilaterally adrenalectomized rats the ambient circulating and kidney levels of ouabain were low and ouabain infusion raised glycoside levels and blood pressure significantly. CONCLUSIONS: Prolonged infusion of ouabain in the normal rat raises the circulating, kidney, hypothalamic and anterior pituitary levels and induces a reversible hypertension with normal plasma renin activity. Although characterized by raised aldosterone levels, the hypertension does not require the adrenal glands and is not salt-sensitive. This model may be useful for exploring novel mechanisms of long-term regulation of blood pressure.

Vasopressin gene transcripts in mineralocorticoid hypertension: an in situ study.

OBJECTIVE: To test the hypothesis that enhanced expression of the vasopressin gene accompanies the development of deoxycorticosterone acetate (DOCA)-salt hypertension in the rat and to compare the response with those observed during chronic hypernatremia. METHODS: Transcript levels were determined by measurement of vasopressin messenger RNA (mRNA) in the supraoptic nucleus and paraventricular nucleus by in situ hybridization, autoradiography and image analysis. Plasma, urinary and pituitary vasopressin were determined by radioimmunoassay. DESIGN: High-resolution localization and measurement of specific mRNA in the supraoptic and paraventricular nuclei before and during development of DOCA-salt hypertension were compared with corresponding results in both age-matched controls and normal rats that drank hypertonic saline. RESULTS: Vasopressin mRNA levels were increased in the paraventricular nucleus during the established and chronic stages of DOCA-salt hypertension, but were unchanged in the supraoptic nucleus. Urinary excretion of vasopressin was increased in the prehypertensive, established and chronic phases of DOCA-salt hypertension, whereas plasma vasopressin levels were increased only in the chronic phase. Pituitary vasopressin levels were unchanged. In comparative studies, vasopressin mRNA levels in both the supraoptic and paraventricular nuclei and plasma vasopressin were significantly increased in normal rats drinking 2% saline. CONCLUSION: Whereas hypernatremic rats showed markedly elevated vasopressin transcripts in the supraoptic and paraventricular nuclei, DOCA-salt hypertension is associated with increased vasopressin mRNA in the paraventricular but not the supraoptic nucleus. The response in the paraventricular nucleus may explain part of the increased peripheral vasopressin levels and suggests that this nucleus makes a critical contribution to the pathogenesis of DOCA-salt hypertension.