Erythrocyte sodium/potassium ATPase activity in severe preeclampsia.

OBJECTIVE: Elevated blood levels of endogenous digitalis-like factors (EDLF) may decrease erythrocyte sodium pump activity in preeclampsia. As the highest EDLF levels might be expected in severe preeclampsia, we investigated sodium pump activity in that group of patients. STUDY DESIGN: Erythrocyte sodium pump activity was determined by (86)Rubidium uptake (in nM per hour per 10(6) cells) in women with severe preeclampsia and those with normal pregnancies, matched for gestational age, and in healthy nonpregnant women (n=12 in each group). Differences between groups were analyzed by a two-sided Student t-test. RESULT: Sodium pump activity was significantly increased in normotensive pregnancies as compared with normotensive non-pregnant women (81.4+/-8.4 vs 61.1+/-7.4, mean+/-s.d., p<0.05), and was decreased 43% in severe preeclamptic pregnancies as compared with normotensive pregnancies (46.4+/-14.1 vs 81.4+/-8.4, p<0.05). CONCLUSION: Severe preeclampsia is associated with significantly lower erythrocyte sodium pump activity than normotensive pregnancy. These data suggest that plasma levels of a biologically active EDLF are elevated in patients with severe preeclampsia.

Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

On the structure of endogenous ouabain.

The ouabain-like sodium pump inhibitor in mammals (so-called "endogenous ouabain") has been considered a subtle structural isomer of ouabain. Its structural investigation, however, has long been hindered by the paucity of sample material. Our recent purification of endogenous ouabain (3 micrograms) from bovine hypothalamus allowed the measurement of its 1H-NMR. The obtained spectrum as well as reexamination of past microscale structural studies on endogenous ouabain led us to identify the purified material as ouabain in an unusual manner. It turned out that the structural analysis had been complicated by a facile ouabain-borate complexation in borosilicate glassware. In retrospect, it is not surprising that the polyhydroxylated ouabain molecule serves as a polydentate ligand to inorganic species. In its physiological environment, ouabain may exist as some unknown complex. The chemical species giving rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified.

Hypoxia triggers release of an endogenous inhibitor of Na(+)-K(+)-ATPase from midbrain and adrenal.

An endogenous inhibitor of Na(+)-K(+)-ATPase has been isolated from bovine hypothalamus and human plasma and structurally characterized as an isomer of the plant cardiac glycoside, ouabain (A. A. Tymiak, J. A. Norman, M. Bolgar, G. C. DiDonato, H. Lee, W. L. Parker, L.-C. Lo, N. Berova, K. Nakanishi, E. Haber, and G. T. Haupert, Jr. Proc.Natl.Acad.Sci. USA 90: 8189-8193, 1993; N. Zhao, L.-C. Lo, N. Berova, K. Nakanishi, J. H. Ludens, and G. T. Haupert, Jr. Biochemistry 34: 9893-9896, 1995). This hypothalamic inhibitory factor (HIF) acts on cardiovascular and renal tissues consistent with physiological regulation in vivo. Stimuli for the release of HIF from tissue are unknown. Hypoxia may be a stimulus for the elaboration of digitalis-like activity in humans, and high NaCl concentration in central nervous system stimulates ouabain-like activity in animals. We examined the ability of low O2 tension in vivo and in vitro to stimulate HIF release from midbrain and adrenal tissues in Wistar rats. In both tissues, hypoxia stimulated a remarkable release of an inhibitor cochromatographing with HIF, and this release was enhanced by 300 mM NaCl. Plasma from hypoxic rats also showed increased levels of the purified inhibitory activity. We conclude that hypoxia is a potent stimulus for the release of HIF or HIF-like activity and discuss the possibility that an Na(+)-K(+)-ATPase inhibitor could be involved in energy-conserving cellular adaptive responses to hypoxic or ischemic insult through ATP conservation.

Na,K-ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis.

The specific, high affinity binding of plant-derived digitalis glycosides by the mammalian sodium and potassium transporting adenosine triphosphatase (Na,K-ATPase, or sodium pump), a plasma membrane enzyme with critical physiological importance in mammalian tissues, has raised the possibility that a mammalian analog of digitalis might exist. We previously isolated and structurally characterized from bovine hypothalamus a novel isomer of the plant glycoside, ouabain, which differs structurally only in the attachment site and/or the stereochemistry of the steroid moiety [Tymiak et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8189-8193]. Hamlyn and co-workers reported a molecule purified from human plasma which by mass spectrometry could not be distinguished from plant ouabain [Hamlyn et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6259-6263]. Since rhamnoside cardiotonic steroids are not known as natural products from mammalian sources, it became important to compare these two pure isolates to determine if the same or structurally distinct compounds has been found. Our results indicate that the human and bovine Na,K-ATPase-inhibitors are identical, but different from plant ouabain. This supports the notion that the human sodium pump may be under specific physiological regulation by a mammalian analog of the digitalis glycosides.

Metabolism of uridine in expanded extracellular volume states.

1. Uridine and uridine monophosphate (UMP) are natriuretic and a vasopressor in intact rats. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats metabolic clearance rate (MCR) of uridine is raised and basal plasma uridine diminished, suggesting that metabolism of uridine is linked to changes in extracellular space. 2. Plasma uridine concentration was raised in 38 patients with chronic renal failure compared with age- and sex-matched healthy controls (8.49 mumol/L, 4.37-13.74 mumol/L median, interquartile range, and 2.64 mumol/L 2.51-2.74 mumol/L, respectively, P < 0.001). Plasma uridine was significantly diminished after isotonic fluid removal by ultrafiltration (UF) from 7.25 mumol/L (3.7-11.08) to 5.07 mumol/L (3.3-8.3), P < 0.001, whereas concentration of marker solutes urea and creatinine remained unchanged. During haemodialysis (HD), plasma uridine fell significantly from its pre-HD level. 3. In an animal model of expanded extracellular space the one-kidney, one-clip rat, plasma uridine was significantly higher (20.56 +/- 1.19 mumol/L, P < 0.01) and MCR diminished (34.93 +/- 3.44 mL/kg per min, P < 0.01) compared with sham-operated animals (plasma uridine 12.14 +/- 1.07 and MCR 53.59 +/- 4.11 mL/kg per min). Uridine or UMP did not inhibit Na+, K(+)-ATPase in either of the two assay systems. 4. It was concluded that catabolism of uridine is reduced by extracellular expansion and probably increased by volume reduction by UF.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic Na+,K(+)-ATPase inhibitor constricts pulmonary arteries of spontaneously hypertensive rats.

Hypothalamic inhibitory factor (HIF) is an endogenous high-affinity inhibitor of Na+,K(+)-ATPase with ouabain-like properties and has been implicated in the pathogenesis of genetic systemic hypertension. We wondered whether HIF might also be associated with the recently demonstrated pulmonary hypertension of spontaneously hypertensive rats (SHRs). We compared HIF effects on the contractility of isolated 2- to 3-mm pulmonary artery (PA) rings from SHRs and age-matched normotensive Sprague-Dawley (SD) rats. HIF caused a reversible, concentration-dependent increase in tension in PA rings of SHR and SD rats, whereas ouabain did not. PA tension development with HIF (4 nM final concentration) was significantly higher in SHRs than in SD rats: 308 +/- 56 mg (mean +/- SE) vs. 137 +/- 26, respectively, p < 0.05. Abdominal aortic contractions induced by HIF did not differ between SHRs and SD rats. In SHRs, but not SD rats, the effect on PA rings was significantly greater than on aortic rings. In all cases, contraction was abolished by phentolamine but was unaffected by calcium-channel blockade using verapamil. HIF-induced tension development required external Ca2+. We conclude that PA rings from SHRs are more sensitive to Na+,K(+)-ATPase inhibitory effects of HIF than PA rings from SD rats, which may contribute to the observed pulmonary hypertension in SHR. Local modulation of the Na+,K(+)-ATPase-adrenergic neuroeffector interaction may be the vasoconstrictive mechanism of action of HIF in these vessels.

Use of two-sided bifunctional liposomes in the study of a hypothalamic Na,K-ATPase inhibitor.

Two-sided bifunctional (ATP-filled) Na,K-ATPase liposomes have been developed as a result of knowledge about the average liposome diameter and volume, the liposome size distribution, the average number of Na,K-ATPase molecules reconstituted per liposome, and the orientation of the reconstituted Na,K-ATPase molecules. The addition of 5-10 microM external 86Rb to the liposomes containing 50 mM encapsulated ATP provoked an impressive 86Rb accumulation by the cell-like-oriented pumps. The successive addition of external ATP activated the pumps in the reversed orientation of the same liposome, leading to total extrusion of the previously accumulated 86Rb. An inhibitor extracted from bovine hypothalamus (hypothalamic inhibitory factor) inhibited the cell-like-oriented population, i.e., acted like an extracellular inhibitor at 30 nM. Conversely, at 75 nM, the reversed pump population was also blocked, indicating that the inhibitor either transversed the membrane or was able to act also at the intracellular enzyme side at a higher concentration. Thus, the side of action as well as the membrane permeability of structurally unknown endogenous Na,K-ATPase inhibitors can be determined simultaneously in a single suspension of two-sided bifunctional Na,K-ATPase liposomes.

Hypothalamic Na(+)-K(+)-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na(+)-K(+)-ATPase.

The functional characterization of putative endogenous inhibitors of the Na(+)-K(+)-ATPase has been greatly hindered by spare amounts extractable from biological sources. We therefore used a miniaturized, two-sided test system consisting of ATP-filled liposomes containing dispersed, randomly oriented renal Na(+)-K(+)-ATPase molecules to study effects of a low-molecular-weight, nonpeptidic Na(+)-K(+)-ATPase inhibitor extracted from bovine hypothalamus. With this test system, Na(+)-K(+)-ATPase inhibition produced by a single dose of 0.1 U (congruent to 75 fmol) of the hypothalamic inhibitory factor (HIF) as well as the membrane permeation of a single unit (approximately equal to 750 fmol) became measurable, and an estimation of the minimal number of HIF molecules per unit could be made. By a molecular mechanism involving positive cooperativity, HIF potently and completely blocked active 86Rb+ transport catalyzed by the right-side-out-oriented pump population, with an average 50% inhibitory concentration of 3.5 x 10(-8) M, indicating a roughly 30-fold higher apparent affinity than ouabain. By studying inhibition of the inside-out-oriented pump population, comparison of the membrane permeability of HIF to that of various cardiac glycosides of known hydrophobicity further indicated that HIF is not entirely ouabain-like as HIF penetrates the liposomal membrane, whereas ouabain does not. Besides the cardiac glycosides, HIF is the only compound tested thus far in the purified system that displays such striking transport inhibition. Other known or proposed endogenous Na(+)-K(+)-ATPase inhibitors, including unsaturated fatty acids, palytoxin, dehydroepiandrosterone, and vanadate, produce only partial transport inhibition even at high concentration.

Positive inotropic effects of the endogenous Na+/K(+)-transporting ATPase inhibitor from the hypothalamus.

Bovine hypothalamus contains a nonpeptidic substance that inhibits purified Na+/K(+)-transporting ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] reversibly with high affinity by a mechanism similar to, but not identical to, that of the cardiac glycosides. It possesses some of the characteristics ascribed to a putative endogenous "digitalis-like" compound that has been implicated in the control of renal sodium excretion and the pathogenesis of essential hypertension in man. To determine whether this hypothalamic Na+/K(+)-transporting ATPase inhibitor might have physiologic properties in cardiac tissues, its effects on Na+ pump inhibition, accumulation of cytosolic free calcium, and contractile response were studied in cultured, spontaneously contracting neonatal rat cardiocytes. The hypothalamic factor potently inhibited the Na+ pump in these cells, increased myoplasmic free calcium in a dose-dependent manner, and reversibly enhanced myocyte contractility by up to 40%, comparable in degree to maximal positive inotropic effects caused by the cardiac glycoside ouabain. Comparative studies further indicate that cardiotoxic effects of ouabain in the myocytes may be more complex than simple progressive elevation of intracellular free calcium concentration because at a free calcium concentration in excess of that produced by a toxic dose of ouabain, no toxicity with the hypothalamic Na+/K(+)-transporting ATPase inhibitor occurred.

Na+ pump in renal tubular cells is regulated by endogenous Na+-K+-ATPase inhibitor from hypothalamus.

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na+-K+-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK1) were studied using 86Rb+ uptake and [3H]ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na+-K+-ATPase in LLC-PK1 cells. In contrast to membrane studies, no prolonged incubation with LLC-PK1 was needed to observe inhibition of Na+-K+-ATPase. HF caused a 33% inhibition of ouabain-sensitive 86Rb+ influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK1 86Rb+ uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na+-K+-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.

Atrial natriuretic peptide regulates release of Na+-K+-ATPase inhibitor from rat brain.

Tissue culture media from incubations of fragments of rat brain were collected and partially purified. These supernatants were effective in inhibiting the Na+-K+ pump as indicated by a 77% reduction of ouabain-sensitive 86Rb+ uptake into human erythrocytes. Release of the Na+-K+-ATPase inhibitor depended on the amount of tissue, the temperature, and the length of incubation. Atrial natriuretic peptide (ANP) injected intravenously, or included (10(-8) M) in the in vitro incubation of brain tissue, decreased the release of the Na+-K+-ATPase inhibitor by 74 and 42%, respectively. Control experiments using the neuropeptide arginine vasopressin showed no effect on release of the inhibitor. These studies indicate that ANP is capable of regulating the release from brain of a Na+-K+-ATPase inhibitor with similar chromatographic characteristics to the one previously obtained from extraction of bovine hypothalamus and raise the possibility that the two factors are interrelated in the regulation of fluid and electrolyte balance.

Circulating inhibitors of sodium transport at the prehypertensive stage of essential hypertension.

Alterations in cellular calcium metabolism are presumed to be the basis of the vasoconstrictive process that sustains elevations in arterial pressure. Altered membrane sodium transport can effect changes in intracellular free calcium concentration through changes in transmembrane sodium gradients and membrane depolarization. Thus, changes in membrane sodium transport could produce calcium accumulation in vascular smooth muscle, resulting in vasoconstriction and arterial hypertension. Multiple sodium transport abnormalities exist in tissues of genetically hypertensive rats, and blood cells of human essential hypertensives. Na+-K+ cotransport and Na+-Li+ countertransport abnormalities appear to be primary membrane defects, but a direct physiologic link of these to vasoconstriction remains to be established. Evidence for circulating Na,K-ATPase inhibitors in hypertension is now widely reported, and Na,K-ATPase inhibition provides a rationale for vasoconstriction through altered calcium and/or neurotransmitter metabolism. Na,K-ATPase inhibition in hypertensive disease appears to arise not as a primary abnormality in membrane transport, nor as a phenomenon secondary to hypertension per se, but as a physiological response to compensate for excess extracellular fluid volume accumulation.

Regulation of Na+, K+-ATPase by the endogenous sodium transport inhibitor from hypothalamus.

We characterized the effect of a small, nonpeptidic molecule isolated from bovine hypothalamus on mammalian Na+, K+-adenosine triphosphatase (ATPase). This hypothalamic factor has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity. This report reviews the mechanism of inhibition. Hypothalamic factor inhibits Na+, K+-ATPase only from the extracellular surface. It prevents the phosphorylation from magnesium and inorganic phosphate of the active site aspartate residue of Na+, K+-ATPase and stabilizes the enzyme in an E2 conformation, preventing a sodium-induced shift from E2 to E1. Binding and dissociation reactions of hypothalamic factor in cultured renal tubular epithelial cells show a time frame different from that in isolated membranes and consistent with physiological relevance. A possible mechanism for the physiological regulation of Na+, K+-ATPase, including a cycle of binding and rapid dissociation in intact renal tubular cells, is discussed.

The search for a hypothalamic Na+,K+-ATPase inhibitor.

Accumulating experimental evidence suggests that natriuresis in response to intravascular volume expansion is promoted by an endogenous regulator of Na+,K+-adenosine triphosphatase (ATPase). Efforts to purify this substance by a number of laboratories have as yet been unsuccessful. The properties of partially purified inhibitors from plasma, urine, and tissue often fail to possess the characteristics thought to be consistent with those of a physiological regulator. These include potency (Ki of approximately 1 nM), reversibility of inhibition, specificity for Na+,K+-ATPase, and responsiveness to relevant physiological stimuli. Two rather different candidate substances, extracted from urine and hypothalamus, have been purified to a high degree. Neither is a peptide, and both are of low molecular weight and resistant to acid hydrolysis. The substance from urine is rather nonpolar and interacts with digoxin-specific antibodies, while that from hypothalamus is polar and does not appear to share epitopes with the cardiac glycosides. On the serosal surface of the toad urinary bladder, the hypothalamic substance causes a reversible inhibition of Na+ transport, inhibits rubidium uptake in red blood cells by acting on the membrane's exterior surface, inhibits binding of ouabain to purified Na+,K+-ATPase, and reversibly inhibits hydrolysis of adenosine 5'-triphosphate by the enzyme with a Ki of 1.4 nM. The hypothalamic inhibitor may be differentiated from ouabain by their respective ionic requirements for optimal inhibition of enzymatic activity, and although both ouabain and the hypothalamic inhibitor fix Na+,K+-ATPase in its E2 conformation, the hypothalamic inhibitor does not promote phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. Ionic requirements for inhibition also differentiate the hypothalamic inhibitor from vanadate ion, as does the inhibitor's activity in the presence of norepinephrine. Further enzymological and physiological studies will be facilitated by structural characterizations of the inhibitory substances and by the availability of a method to measure their concentrations in physiological fluids.