Na-K-ATPase inhibitor dissociated from hypertension-associated plasma protein

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Na-K-ATPase Inhibitor Hypertension-Associated Elmav W.J. Weileu, Farhad Khalil-Manesh, and Dilip K. Senshavma

everal studies employing the sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique have described increased concentrations of a low molecular hypertension-associated protein (molecular

Received March 11, 1997. Accepted September 15, 1998. From the Division of Nephrology/Department of Medicine, The Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, and School of Medicine, University of California, Los Angeles, California (EWJW, FK-M, HCG); Department of Pharmacology, University of California, Irvine, California (BAP, REP); and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California (DKS). This investigation was supported by grants from Applied Medical Research, Inc., which are gratefully acknowledged. Address correspondence and reprint requests to Harvey C. Gonick, MD, Nephrology Section, Department of Medicine, CedarsSinai Medical Center, 8700 Beverly Boulevard, Becker Building, Room 227, Los Angeles, CA 90048; e-mail: [email protected]

0 1999 by the Americm ]ournnl of Hypertensio~z, Published b!y Elsevier Sckwce, Inc.


Dissociated From Plasma Protein

Harvey C. Gonick, Bruce A. Prim, Ralph E. Puvdy,

It has been demonstrated that human plasma contains a low molecular weight sodiumpotassium-stimulated adenosine triphosphatase (Na-K-ATPase) inhibitor, which can be dissociated from a circulating protein with a molecular weight of approximately 12,000 daltons. The dissociated factor was found to have a molecular weight ~500 daltons, and shared many characteristics with ouabain. Similar to ouabain, this factor was found to be a potent inhibitor of both the Na-K-ATPase and potassium-stimulated para-nitrophenyl phosphatase (K-pNPPase) enzyme systems, and to bind to both high- and low-affinity binding sites on Na-K-ATPase, but unlike ouabain did not crossreact with digoxin antibody. The factor was further separated by HPLC and electrochemical detection into two active compounds (p-NKAI-1 and p-

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NKAI-2). P-NKAI-1 was demonstrated on mass spectroscopy to have a molecular weight of 408 daltons. In a vasoconstrictor assay employing rabbit femoral artery segments, this compound was a direct vasoconstrictor and potentiated the vasoconstriction produced by norepinephrine. It behaved similarly to ouabain in counteracting the relaxing effect on rabbit femoral artery of increasing potassium concentrations in the tissue bath. Am J Hypertens 1999;12:364-373 0 1999 American Journal of Hypertension, Ltd.

WORDS: Na-K-ATPase, K-pNPPase, sodiumpotassium-activated adenosine-triphosphatase inhibitor, 3H-ouabain displacement, digoxin-like immunoreactivity.


weight ranging between 12 kD and 21 kD) in plasma samples derived from both hypertensive humans’” and rats with genetic hypertension6 In a previous publication7 we have characterized a 12-kD plasma protein as a so-called hypertensinogenic factor, markedly increased in plasma from a patient with primary aldosteronism, and increased in patients with essential hypertension. In the present paper we also describe a decrease in staining intensity in patients with congestive heart failure, attesting to its relationship to central volume. A semipurified preparation of this protein also exhibits many of the properties of the sodiumpotassium-activated adenosine triphosphatase (Na-KATPase, E.C. natriuretic hormone, namely, inducing natriuresis in rat bioassay,8 inhibiting purified hog cerebral cortex Na-K-ATPase,Y and displacing “H-ouabain from its Na-K-ATPase binding sites.Y 0895.7061/99/$20.00 PII SO895-7061(98)00242-S



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In addition, in the present study we have characterized a low molecular weight compound dissociated from the purified 12-kD circulating protein and compared its properties with those of ouabain in relationship to inhibition of the Na-K-ATPase and potassium-stimulated para-nitrophenyl phosphatase (K-pNPPase) enzyme system, binding to ouabain binding sites on purified Na-K-ATPase, cross-reactivity with digoxin antibodies, and in vitro vasoconstriction. MATERIALS



Collection of Blood and Urine Samples Pooled blood samples from 10 patients with well-documented essential hypertension, not taking any medications for at least 3 weeks, were collected into chilled vacutainers containing sodium ethylenediamine tetraacetic acid (EDTA) and Trasylol. Individual samples were also collected from patients with primary aldosteronism,3 congestive heart failure, before and after treatment,4 and normal controls.” The treatment of congestive heart failure used diuretics and vasorelaxants but avoided digitalis glycosides. Blood was centrifuged at 3000 rpm/$“C for 10 min. Plasma was removed with a Pasteur pipette, placed into clean glass containers, and stored at -80°C until further use. Pooled plasma samples were subsequently employed for purification of the low molecular weight plasma Na-K-ATPase inhibitor (p-NKAI). Five 24-h urine samples were collected from hypertensive patients and saved at -20°C until further processed. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was performed according the procedure described by Laemm1i.i’ In brief, plasma samples (100 pL) were mixed with 300 PL of sample buffer, containing 10% SDS, 0.5 mol/L Tris[hydroxymethyl]aminomethane-hydrochloric acidbuffer, and 4% mercaptoethanol, and incubated for 15 min at 50°C. The mixture (7 pL) was applied to a SDS gradient gel (4% to 27%). Protein bands were visually assessed by silver nitrate staining. Ultrafiltration Plasma samples were passed through a series of Amicon membranes (Millipore, Danvers, MA). The initial ultrafiltration step employed a 1-kD (YM-2) membrane. The filtrate was discarded. The retentate was reconstituted in water and heated for 10 min at 70°C in the presence of 4% /3-mercaptoethanol (Sigma, St. Louis, MO) and 1 mol/L formic acid.12 The solution was cooled down and subsequently placed on a 30-kD (YM-30) membrane. The resulting filtrate, containing the dissociated protein, was lyophilized and subjected to further purification.




Separation of a High Molecular Weight Plasma NaK-ATPase Inhibitor by SEP PAK C-18 Cartridges and Sephadex G-75 Chromatography The dissociated protein was adsorbed onto a SEP PAK C-18 cartridge (Millipore Corp., Milford, MA). Interfering compounds, eg, small peptides, hydrophobic substances, and so on, were retained on the SEP PAK C-18 cartridge. The protein of interest was eluted off the SEP PAK C-18 cartridge with distilled water. This fraction was lyophilized, reconstituted in 1 mL of distilled water, and subsequently separated on a Sephadex G-75 column (40 cm X 2.4 cm). Before any samples were loaded onto the column, at least three column volumes of ammonium acetate buffer were passed through to ensure complete equilibration. The plasma preparation was eluted off the column with 10 mmol/L ammonium acetate, pH 6.5; 4-mL fractions were collected. Each fraction ~was tested for Na-KATPase inhibitory activity. Fractions containing the Na-K-ATPase inhibitory material (12-kD protein), which eluted after the albumin peak, were pooled, lyophilized, and subjected to a series of assays. High Pressure Liquid Chromatography (HPLC) Acetonitrile (CHsCN) HPLC grade was purchased from Pierce, Rockford, IL. Water was purified through the Milli-Q system (Millipore Corp). All solvents were passed through 0.45-pm filters (Pierce, Rockford, IL) and degassed with helium. The reversed-phase HPLC analytical column, an Econosphere C,, (250 mm X 4.6 mm, Alltech, Deerfield, IL) was packed with Econosphere C,, silica, 5-p particle size. All purification procedures were carried out at room temperature. The reversed phase C,, column was equilibrated with triple-distilled water. The p-NKAI preparation, reconstituted in distilled water with the pH adjusted to pH 10, was applied onto the C,, column by a Rheodyne injection valve. The low molecular weight plasma Na-K-ATPase inhibitor (p-NKAI) was eluted off the column with a linear CH,CN (0% to 100% over a period of 30 min) gradient at a flow rate of 1 mL/min, which was initiated 2 min postinjection. The eluate was continuously monitored at 210 nm. One-minute fractions were collected, lyophilized, and subsequently tested for the presence of Na-K-ATPase inhibitory activity and digoxin reactivity. Urine samples were similarly processed after preliminary separation on Sephadex G-15.l’ Electrochemical Analysis P-NKAI was further purified by HPLC separation combined with electrochemical detection. Model 5100A Coulochem Detection System (ESA, Inc. Bedford, MA) was employed. The potential of the detector was set at 750 mV versus an Ag/AgCl reference electrode. A reversed-phase C,, column, 3 Frn) column (8 cm, 4.6-mm inner diameter)





with a high-pressure injection valve was used. The mobile phase (Cat-A-Phase, containing methanol, phosphate buffer, and an ion-pairing agent) was provided by ESA, Inc. The flow rate was a constant 1 mL/min. The HPLC separation was carried out at room temperature. Mass Spectroscopy Mass spectrometric analysis of the samples was performed using a sector-field highresolution, double-focusing, and reverse-geometry mass spectrometer model ZAB-SE made by VG Analytical Ltd., England. The technique of fast atom bombardment (FAB) was used to ionize the sample molecules. The sample was first dissolved in m-nitrobenzyl alcohol and 1 PL of this solution was deposited on a target surface, which was then bombarded with a beam of xenon atoms at a kinetic energy of about 6 kV. The FAB gun voltage was set at 6 kV to give a discharge current of about 1 PA with xenon gas flowing through the gun. Several mass spectra were recorded for each sample using a computerized data system 11-250, supplied by VG Analytical Ltd. An average mass spectrum was generated from a set of spectra recorded for each sample to compensate for the fluctuations in peak intensities in each individual spectrum in a set. To control for the spectrum generated by the solvent system, this spectrum was subtracted from the results of the samples. Na-K-ATPase Inhibition Assay In these experiments a highly purified hog cerebral Na-K-ATPase preparation (Sigma) was employed. The incubation tubes contained a substrate solution providing final concentrations of 1 mmol/L adenosine 5’-triphosphate (ATP), 1 mmol/L MgCl,, 100 mmol/L NaCl, 20 mmol/L KCl, 0.1 mmol/L ethylene glycol-bis (p-amino-ethyl ether)-N,N,N,,N,-tetraacetic acid (EGTA), and 10 mmol/L imidazole-HCl buffer, pH 7.2. The tubes were preincubated with either the 12-kD protein or the purified plasma factor for 5 min at 37°C. The enzymatic reaction was initiated by adding 0.025 mL enzyme preparation (25 mg/mL), yielding a final incubation volume of 250 pL. The reaction was stopped by adding 1.0 mL ice-cold 10% trichloroacetic acid after an incubation time of 15 min. After centrifugation (1700 8 at 4°C for 10 min), 0.5 mL of supernatant was assayed for inorganic phosphate according to the procedure described by Fiske and Subbarow.13 Assays were carried out in duplicate; intraassay variations were less than 5%. K-pNPPase Inhibition Assay The enzyme assay was modified from the procedure described by Phillips et a1,14 utilizing purified hog cerebral cortex NaK-ATPase as the enzyme source. The incubation tube contained a substrate solution providing a final concentration of 5 mmol/L MgCl,, 10 mmol/L KCl, 100



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mmol/L Tris-HCl buffer (pH 7.4), p-NKAI, and 0.025 mL purified Na-K-ATPase. The tubes were preincubated for 15 min at 37°C. The enzymatic reaction was started by adding p-nitrophenylphosphate (pNPP), yielding a total volume of 250 PL with a final concentration of 5 mmol/L pNPP. The reaction was stopped after 30 min by adding 1 mol/L ice-cold sodium hydroxide. After centrifugation (1700 g for 10 min at 4°C) the supernatant was read at 410 nm to assess the concentration of p-nitrophenol (pNP) and results were expressed as percent inhibition of K-pNPPase. Assays were carried out in duplicate; intraassay variations were less than 5%. 3H-Ouabain Displacement Assay The ouabain displacement activity was determined by measuring the binding of 3H-ouabain to Na-K-ATPase in competition with ouabain or p-NKAI as the inhibitors. The assay was performed as follows: 100 PL Na-K-ATPase enzyme from hog cerebral cortex was incubated with 1 X 10e8 mol/L 3H-ouabain (approximately 120,000 counts; specific activity 0.25 mCi, purchased from New England Nuclear, Boston, MA) in a buffer solution with final concentrations of 2 mmol/L ATP, 2 mmol/L MgCl,, 100 mmol/L NaCl, and 100 mmol/L imidazole-HCl (pH 7.4). After an incubation period of 60 min at 37”C, at which time equilibrium was achieved, unlabeled ouabain at concentrations ranging from lo-” to lOPlo mol/L of p-NKAI were added. The final volume of the incubate was 1.0 mL. The reaction was stopped after a second incubation period of 60 min by addition of ice-cold imidazole buffer. Unbound 3H-ouabain was separated from the bound 3H-ouabain by a filtration technique using Millipore GSTF filters with a pore size of 0.22 pm. Complete removal of the free 3H-ouabain was achieved with three washings each with 5 mL buffer. 3H-ouabain bound to protein and retained on the filter was counted in an automatic liquid scintillation counter (Beckman LS 100, Fullerton, CA), at a counting efficiency of 44%. Digoxin Radioimmunoassay All HPLC fractions were assayed for the presence of digoxin-like immunoreactivity. The digoxin radioimmunoassay kit was purchased from Baxter Health-Care (Cambridge, MA) and the assay was performed according to the procedure described by Weiler et al.‘” The digoxin-like immunoreactivity was calculated from a digoxin standard curve with digoxin concentrations ranging between 0.5 ng/mL and 4.0 ng/mL. Ouabain crossreacts with this digoxin antibody, with detection from lop8 mol/L to 5 X 10P4 mol/L (personal observations). False positives have been noted clinically with this assay.



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Bioassay The natriuretic property of the 12-kD plasma factor was assessed in an animal model. Duplicate bioassay procedures were performed according to the method described by Purdy et al? The sequence of injections after obtaining a stable baseline was control solution, consisting of injection of 0.3 mL isotonic saline delivered into the jugular vein as a single bolus injection given over 1 min, followed by four 15-min urine collections; then injection of test sample derived from 2.5 mL plasma dissolved in 0.3 mL saline and injected as described previously, followed by eight 15-min urine collections. The results were averaged for presentation. Vasoconstrictor Assay The purified Na-K-ATPase inhibitor (p-NKAI-1, vide infra) was tested for its vasoactive properties according to the procedure described by Purdy and Weber.17 Male New Zealand white rabbits were decapitated, femoral arteries were removed, and the tissues were cleaned and sectioned into 3-mm segments. The tissue segments were then mounted in a 30-mL tissue bath containing Krebsbicarbonate solution, and were continuously aerated with 95% 0,/5% CO, at 37°C. The tissue was connected to a force transducer (Grass, FT03C, Quincy, MA) and the contractile response was recorded with a Polygraph (Grass, Model 7, Quincy, MA.). The tissues were equilibrated for 60 min under a resting tension of 1.5 g. They were then exposed to 68 mmol/L potassium and allowed to contract to a steady-state response, after which the baths were drained and refilled twice with fresh Krebs solution. Subsequently, p-NKAI-1 was assayed for its vasoactive behavior in the presence and absence of norepinephrine. A dose-response curve was established for p-NKAI-1; the concentration of p-NKAI-1 yielding 1% contractile response was selected for the studies of synergy with norepinephrine. Functional Na-K-ATPase Assay Inhibition of Na-KATPase was measured functionally in the rabbit femoral artery according to the method of Webb and Bohr.” Artery rings were prepared as described previously. After exposure to, and recovery from, the contractile effect of 68 mmol/L K+, the Krebs solution was drained and replaced with K+-free Krebs solution. Artery rings were contracted with 10 nmol/L norepinephrine. When this contraction was at steady state, Kt was added cumulatively in 1-mmol/L increments and the magnitude of relaxation after each addition was measured. The experiment was repeated with p-NKAI-1 in the medium at a concentration of 6.0 X lop7 mol/L ouabain equivalents. In this procedure the artery ring is contracted to a steady state with norepinephrine, and the addition of Kt activates NaK-ATPase, resulting in increased electrogenic transport of sodium and potassium and, consequently, hy-




perpolarization. In turn, the hyperpolarization causes relaxation. When an agent that inhibits the Na-KATPase is present in the medium, the ability of the addition of potassium to cause relaxation is impaired. Thus, in this functional assay, reduction of relaxation may be taken as an indicator of Na-K-ATPase inhibition in vascular smooth muscle. We have previously demonstrated that this functional assay is more sensitive than inhibition of *‘Rb uptake.16 RESULTS SDS-PAGE As demonstrated in Figure 1, a 12-kD protein band appeared to correlate with the status of effective arterial volume. The staining intensity of this band with silver nitrate was increased in all patients with primary hyperaldosteronism and decreased in decompensated congestive heart failure, returning towards normal after recovery. Isolation of the Low Molecular Weight Plasma Protein Pooled plasma samples from hypertensive subjects were initially passed through an Amicon membrane (YM-2) with a molecular weight cutoff of 1 kD. The retentate was chromatographed on a Sephadex G-75 column using 10 mmol/L ammonium acetate as eluent. An Na-K-ATPase inhibitor, which yielded a 12-kD protein band on subsequent SDS-PAGE separation, was found in fractions eluting in Ve/Vo 2.2 to Ve/Vo 3.1. The lyophilized active fraction was further purified on a SEP-PAK Cl8 cartridge with water as eluant, then rechromatographed on Sephadex G-75. The fraction with maximum Na-K-ATPase inhibitory activity contained a single ultraviolet peak at 280 nm, and yielded three protein bands on SDS-PAGE, with estimated molecular weights of 10 kD, 12 kD, and 14 kD (predominantly 12 kD). In the following sections, the low molecular weight protein will be referred to as a 12-kD protein. In Vivo Characterization of the 12-kD Plasma Protein The equivalents of 2.5 mL of plasma were tested for their natriuretic properties. The material was injected slowly into the bioassay rat via jugular vein after obtaining stable baseline values for sodium excretion for four consecutive 15-min collection periods. The results shown in Figure 2 illustrate that both an early and sustained increase in sodium excretion is observed in response to administration of the semipurified 12-kD Na-K-ATPase inhibitor. In Vitro Characterization of the 12-kD Plasma Protein Na-K-ATPase Inhibition Assay The dose-response curve for Na-K-ATPase inhibition of the semipurified 12-kD protein parallelled the dose-response curve for ouabain. Fifty percent inhibition of Na-K-ATPase





FIGURE 1. SDS-PAGE separation protein bands from the plasma of patients zuith various volume-related disease states. Bands were stained with silver nitrate. Myoglobin protein markers of 17.2 kD, 14.6 kD, and 8.2 kD molecular weight are shown in lane 1. Lane 2: Normal control. Lane 3: Hyperaldosteronism. Lane 4: Congestive heart failure, pretreatment. Lane 5: Congestive heart failure, posttreatmerit. These gel separations are from the plasma of individual patients. Plasma from other patients within the same groups showed similar results.


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17.2 14.6 12 kD 8.2


(I&), corresponding to 5 X 10Ph mol/L ouabain, was produced by the 12-kD inhibitor in a fraction containing 2.7 mg/mL Lowry protein, representing 120 PL of original plasma. The ouabain displacement 3H-O~abain Receptor Assay assay revealed that the 12-kD protein factor displaces 3H-ouabain from its receptor in a dose related manner, similar to ouabain. Determination of Digoxin-like Immunoreactivity of 12-kD Plasmn Protein The low molecular weight Na-K-

ATPase inhibitory material did not show crossreactivity with digoxin antibody.


FIGURE 2. Rat natriuretic bioassay of semipurified pooled 12-kD protein representing 2.5 mL of original plasma. First arrozu designates time of injection of a control solution. The second arrow designates the time of injection of the 12-kD protein.



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Electrochemical Analysis 011 rechromatography, p-NKAI appeared versed-phase C,, at 4% CH,CN. No ultraviolet absorption was observed at 210 nm. This purified plasma Na-K-ATPase inhibitor was shown not to crossreact with digoxin antibodies and to coelute with a low molecular weight Na-K-ATPase inhibitor derived from pooled human urine of hypertensive patients.‘” (Figure 3 ). p-NKAI was ultrafilterable through an Amicon YM-05 membrane that has a 500 molecular weight exclusion limit. Rechromatography of active fractions on a 3-pm C,, column monitored electrochemically yielded two active compounds, p-NKAI-1 and p-NKAI-2 (Figure 4 ), HPLC




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FIGURE 3. Comparison of elution patterns of urinary and plasma-derived Na-K-ATPase inhibitors and digoxin-l&e immunoreactivity (DLI) on a reversed-phase C,, HPLC column. Na-KATPase inhibitor is indicated by hatched bars and DLI by stippled bars. The dotted line represents acetonitrile gradient.

both of which were inhibitors of the Na-K-ATPAse enzyme system. P-NKAI-1 caused 50% inhibition and P-NKAI-2 caused 8% inhibition, in a volume of inhibitor corresponding to 187 FL original plasma. The remaining fractions were without inhibitory activity. The purified p-NKAI-1 was used for the vasocon-




strictor assays described later. .I’-NKAI-2 will be described in a subsequent communication. When the starting material, p-NKAI, was applied to an alumina extraction column, both p-NKAI-1 and p-NKAI-2 were recovered in the eluate. This is in contrast to the catecholamines, norepinephrine, epinephrine, and dopamine, which typically adsorb to the column.” Thus p-NKAI-1 and p-NKAI-2 are not catecholamines. On the 3-pm C,, HPLC column pNKAI-1 elutes before the loci of norepinephrine and epinephrine, whereas p-NKAI-2 elutes at the same locus as dopamine. Mass Spectroscopy The mass spectrum of p-NKAI-1, a compound of molecular weight 408, showed a fairly intense protonated molecular ion at mass 409 (Figure 5). Two other related ions of interest were also detected at masses 431 and 447, which are sodium and potassium adduct ions. In the mass spectrum proton and sodium cation-held dimers of the compound were also observed at masses817 and 839, respectively. The remaining peaks reflect minor impurities in the material. Na-K-ATPase and K-pNPPase Inhibition Assay PNKAI was shown to inhibit both the Na-K-ATPase and K-pNPPase enzyme systems in a dose-related manner, analogous to ouabain”’ (Figure 6A,B). The IC,, for inhibition of Na-K-ATPase by p-NKAI corresponds to 8 x 10e7 mol/L ouabain equivalents. Ouabain Displacement Assay Figure 7 shows that p-NKAI binds to both the high- and the low-affinity binding sites of the Na-K-ATPase enzyme system, as seen for the glycoside ouabain.


LMW p-NKAI-2 FIGURE 4. and p-N&II-2.


of [email protected] low molecular weight p-NKAI,

monitored electrochemically. Arrows indicate the loci of p-NKAI-I



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FIGURE 5. Mass spectroscopyanalysis of p-NKAI-1. peaks at masses 409, 431, and 447.


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Vasoconstrictor Assay As illustrated in Figure 8A, p-NKAI-1 possessesa dose-related intrinsic vasoconstrictor activity and also potentiates the vasoconstrictor effects of norepinephrine (lo-’ mol/L)(Figure 8B).


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Although the focus of interest in the past has been primarily on low molecular weight forms (~1 kD) of NKAI in deproteinized plasma,35,41there have been previous reports of a circulating high molecular weight (5 kD to 70 kD) natriuretic compound in volume-expanded sheep and humans,42 as well as a 12-kD to 21-kD protein in hypertensive humans’-” and rats.’ We have previously demonstrated that a high molecular weight form of the Na-K-ATPase inhibitor predominates over low molecular weight forms (< 1000 daltons) in essential hypertension.7 In the present report, our laboratory has described a volume-responsive and hypertension-related 12-kD plasma protein, which has properties similar to NKAI, namely, induction of natriuresis and inhibition of both


Na-K-ATPase Functional Assay As indicated in Figure 9, p-NKAI-1 serves as a functional inhibitor of the Na-K-ATPase enzyme system in blood vessels. P-NKAI-1 at a concentration of 0.23 X 10P6 mol/L ouabain equivalent prevented the complete relaxation of the blood vessel induced by potassium, indicative of an inhibitor of Na-K-ATPase, which is competitive with potassium.

. p-NKAI 0 Ouabain

DISCUSSION Extracellular fluid volume expansion induces synthesis and release of at least two natriuretic hormones, an Na-K-ATPase-inhibiting compound (NKAI), and an atria1 natriuretic factor (ANF). The site of origin of NKAI is most likely the hypothalamus21-24 or the adrenal gland,2”,26 whereas the principal locus for ANF is the heart.27 Whereas NKAI acts as a vasoconstrictor, 28-30 ANF is a vasodilator,31,32 and although NKAI acts as an inhibitor of Na-K-ATPase, ANF does not.33 Low molecular weight forms of NKAI have been reported to increase renal sodium excretion, thus qualifying as a natriuretic compound8,9,34; to inhibit the transport enzyme, Na-K-ATPase9,35Z36;and to displace 3H-ouabain from its receptors.’ Although NKAI also has been reported to crossreact with antibodies to digoxin,s,37 this property remains controversial.38 Blaustein39 and dewardener and MacGregor4’ have hypothesized that circulating NKAI might play a role in the pathogenesis of hypertension through enhancement of vascular muscle contractility. It is assumed that reduction of the active transport of sodium results in an increase in intracellular sodium and calcium, thus making the cells more sensitive to the contractile effect of vasoactive agents such as norepinephrine and angiotensin II.

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FIGURE 6. Comparison of inhibitory effect of the purified NKAI (@) and ouabain (+) on Na-K-ATPase (A) and pNPPase (B) activity. Concetztration of p-N&II is indicated the abscissa as PL plasma concentrate (corresponding to 25 original volume).

pKon x



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254 Ouabain . p-NKAI







60 Bc



80 100




FIGURE 7. Scatchard plot analysis of purified plasma Na-KATPase inhibitor compared with ouabain. 0 indicates p-N&II. + indicates ouabain. The ratio of free to bound ouabain counts is plotted against bound ouabain counts, yielding two intersecting lines, indicative of both high- and low-affinity binding.



potency of norepinephrine. Its vascular effect was shown to be mediated through inhibition of the NaK-ATPase enzyme system, as demonstrated by a functional Na-K-ATPase inhibitory assay in isolated vascular strips, which depends on the observations of the effect of increasing concentrations of potassium on reversing norepinephrine-induced contraction.i8 Similar to ouabain,i8 p-NKAI-1 exer ted ‘ti s maximum contractile effect in the presence of a low-potassium concentration in the incubation solution, with gradual diminution of its contractile effect as the potassium concentration increased. The primary significant functional difference between p-NKAI and ouabain was the failure of p-NKAI to crossreact with the digoxin antibody. In this respect, p-NKAI differs also from the endogenous ouabain purified from bovine plasma by Ludens et a1.46Although initial studies performed by Gruber et aP7

Na-K-ATPase activity and 3H-ouabain displacement in a dose-related manner. In the present study, we have also successfully dissociated from this 12-kD protein a low molecular weight compound; the molecular weight of this factor was determined by means of Amicon filtration technique to be less than 500 daltons. The dissociation technique used P-mercaptoethanol, which is known to cleave S-S bonds, as well as alteration in pH, a measure known to release carrier-bound compounds.43,44 In one of the classical studies of carrier-bound hormones, Lindner et a143demonstrated that the application of heat and formic acid to plasma permits the separation of oxytocin and vasopressin from their carrier-protein, neurophysin. We believe that the use of P-mercaptoethanol and alkalinization induced the dissociation of the low molecular weight p-NKAI from its circulating carrier.44 120-1 P-NKAI was found to inhibit both Na-K-ATPase I M and K-pNPPase, the E-2 form of Na-K-ATPase, in a dose-related manner, as would be expected from a ouabain-like compound.20 Receptor assay studies, employing purified Na-K-ATPase, also demonstrated that p-NKAI binds to both high- and low-affinity binding sites on the Na-K-ATPase enzyme system, in a manner similar to ouabain.45 The homogeneity of p-NKAI could not be assessed by ultraviolet absorption at 210 nm. Therefore, an electrochemical detection system was employed. This resulted in the separation of two more purified Na-KATPase inhibitors, p-NKAI-1 and p-NKAI-2. Mass spectroscopy of p-NKAI-1 revealed a molecular weight of 408 daltons. FIGURE 8. Efiect of p-NKAI-1 on contractile response of rabbit The more purified p-NKAI-1 was found to be a arteries in the absence (A) and presence (B) of lop* mol/L norepinephrine. On the abscissa is indicated microliters of p-NKAI-I vasoconstrictor and to potentiate the vasoconstrictive






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FIGURE 9. Functional assay of Na-K-ATPase inhibition by p-NKAI-1 as represented by modification of the vasoconstrictor effect in rabbit arteries in the presence of increasing potassium concentrations. Solid bars represent control observations. Hatched bars represent observations in the presence of p-N&II-l,






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for their


Gonick HC, Weiler EWJ, Prins BA, Purdy RA: Comparison of low molecular weight plasma and urine Na-KATPase inhibition/hypertensive factors. J Cardiovasc Pharmacol 1993;22(suppl 2):569-571.

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that plasma Na-K-ATPase inhibitors crossreact with the digoxin antibody, it has become apparent subsequently that the digoxin antibody-reacting substances and Na-K-ATPase-inhibiting substances can be dissociated in both plasma and urine by further purification.10,38 In summary, our results show that purified low molecular weight p-NKAI, which has been dissociated from its 12-kD plasma protein carrier, inhibits Na-KATPase and K-pNPPase enzyme systems in a doserelated manner, binds to both high- and low-affinity binding sites on purified Na-K-ATPase, and produces in vitro vasoconstriction, analogous to the cardiac glycoside ouabain. However, in contrast to ouabain, pNIL41 does not crossreact with the digoxin antibody.


ACKNOWLEDGMENTS We wish to thank Lydian Reitz and Ruby McCarty assistance in the preparation of this manuscript.


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