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(Hypertension. 1995;26:781.)
© 1995 American Heart Association, Inc.

Articles

Endogenous Marinobufagenin-like Immunoreactive Factor and Na⁺,K⁺ ATPase Inhibition During Voluntary Hypoventilation

Presented in part at the XV European Section Meeting of the International Society for Heart Research, Copenhagen, Denmark, June 8-11, 1994.

Alexei Y. Bagrov; Olga V. Fedorova; Joy L. Austin-Lane; Renata I. Dmitrieva; David E. Anderson

From the Laboratory of Behavioral Sciences (A.Y.B., O.V.F., J.L.A.-L., D.E.A.), National Institute on Aging, 4940 Eastern Ave, Baltimore, Md, and the Laboratory of Pharmacology (A.Y.B., O.V.F., R.I.D.), Sechenov Institute of Evolutionary Physiology and Biochemistry, St Petersburg, Russia.

Correspondence to Dr Alexei Y. Bagrov, Laboratory of Behavioral Sciences, National Institute on Aging, 4940 Eastern Ave, Baltimore, MD 21224.

	Abstract

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Abstract In previous studies investigators found that conditioned hypoventilatory breathing potentiated a sodium-sensitive form of hypertension in dogs that was not mediated by sympathetic nervous system arousal. Our study investigated effects of 30 minutes of voluntary hypoventilation, maintained by a respiratory gas monitor and feedback procedure, in 16 normotensive humans of both sexes on (1) plasma concentrations of endogenous digitalis-like factors (ouabain-like and marinobufagenin-like immunoreactivity), (2) activity of erythrocyte Na⁺,K⁺-ATPase, (3) inhibitory activity of plasma Na⁺,K⁺-ATPase, and (4) blood pressure. Increased end tidal PCO₂ (41±0.78 mm Hg versus 37.6±1.03 mm Hg) was associated with (1) an increase in plasma marinobufagenin-like immunoreactivity (1.23±0.47 versus 4.96±1.19 nmol/L), (2) an inhibition of Na⁺,K⁺-ATPase in red blood cells (3.68±0.22 versus 2.15±0.25 mmol P_i · mL^-1 · h^-1; P<.01), (3) increase in plasma Na⁺,K⁺-ATPase inhibitory activity (34.9±4.0% versus 48.8±2.1%, P<.02), and (4) increases in systolic (112.4±2.6 versus 107.6±1.8 mm Hg) and diastolic (73.5±2.1 versus 68.8±2.1 mm Hg) blood pressures. Plasma levels of ouabain-like immunoreactivity did not increase significantly. Incubation of erythrocytes obtained during hypoventilation with antidigoxin antibody restored the Na⁺,K⁺-ATPase activity (3.99±0.34 mmol P_i · mL^-1 · h^-1). Cessation of hypoventilation was associated with decreases in diastolic blood pressure (70.5±2.2 mm Hg) and restoration of Na⁺,K⁺-ATPase activity in erythrocytes (2.99±0.43 mmol P_i · mL^-1 · h^-1). On the basis of organic extraction and thin-layer chromatography followed by separation with the use of reverse-phase high-performance liquid chromatography, the material coeluting with marinobufagenin was separated from human urine. This material cross-reacted with anti-marinobufagenin antibody. These results demonstrate the presence of a bufadienolide-like Na⁺,K⁺-ATPase inhibitor in human plasma and support the view that breathing pattern may participate in blood pressure control via release of a rapidly acting circulating Na⁺,K⁺-ATPase inhibitor.

Key Words: hypoventilation • Na⁺,K⁺-exchanging ATPase • bufanolides • hypertension

	Introduction

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Experimental hypertension can be produced in dogs within 14 days by a combination of aversive conditioning procedures and high sodium intake.¹ This form of hypertension is characterized by a positive sodium balance, is not prevented by renal denervation, and occurs only in dogs who develop a hypoventilatory breathing pattern (ie, subnormal breathing frequency with increased PETCO₂)^{2 3} under these conditions. On the basis of these observations, we hypothesized that hypoventilatory breathing pattern could affect sodium balance via decreases in plasma pH and stimulation of Na-H exchange in renal tubuli, resulting in retention of Na⁺ ions. Na retention may stimulate the release of one or more endogenous digitalis-like factors (EDLFs), which in turn could increase vascular tone.

In support of this hypothesis, we recently demonstrated that voluntary hypoventilatory breathing by healthy humans decreases plasma pH,⁴ evokes sodium and fluid retention, and stimulates urinary release of a digoxin-like immunoreactive material.⁵

Endogenous substances that inhibit Na⁺,K⁺-ATPase and cross-react with various anti-digitalis antibodies (ie, EDLF) are known to be stimulated by plasma volume expansion^{6 7 8} and have been implicated in the pathogenesis of volume-dependent forms of clinical and experimental hypertension.^{9 10 11} Recent studies showed that human plasma contains several substances with the ability to inhibit Na⁺,K⁺-ATPase and to cross-react with anti-digitalis antibodies.^{12 13} To date, the only EDLF to be purified from human plasma has been identified as ouabain.¹⁴ Amphibia have been shown to contain Na⁺,K⁺-ATPase inhibitors that have a bufadienolide (rather than cardenolide) structure.^{15 16} More recent studies have suggested that mammalian tissues may also contain bufadienolides¹⁷ and material that cross-reacts with antibodies raised against bufadienolides.^{18 19} Recently, we have shown that mammalian plasma cross-reacts with antibody raised against marinobufagenin, one of the bufadienolides from Bufo marinus toad.²⁰ In vitro, marinobufagenin has been shown to inhibit the Na⁺,K⁺-ATPase/Na⁺-K⁺ pump, cross-react with digoxin antibody, and display vasoconstrictor effects.^{21 22}

The goals of the present study were to investigate whether hypoventilatory breathing is associated with changes in (1) plasma concentrations of ouabain-like and marinobufagenin-like immunoreactive materials, (2) plasma Na⁺,K⁺-ATPase inhibitory activity, and (3) blood pressure. The possible role of EDLF in erythrocyte Na⁺,K⁺-ATPase inhibition was analyzed in vitro with an antidigoxin antibody that cross-reacts with bufadienolide EDLF.²¹ In addition, normal human urine was fractionated by high-performance liquid chromatography (HPLC). HPLC fractions were analyzed for the presence of marinobufagenin-like immunoreactive material.

	Methods

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Protocol of Experiment
Sixteen healthy volunteer white subjects who were not taking any prescription medication (9 men and 7 women; mean age, 31.4±2 years) were studied (May 1993 through August 1993). The study was approved by the Francis Scott Key Medical Center Institutional Review Board.

The protocol included a 1-hour training session and a 1-hour experimental session, occurring on separate days. During the training session, each subject practiced breathing into the mouthpiece of a respiratory gas monitor (RGM) (Ohmeda 5250), which displayed end tidal CO₂ (PETCO₂), and learned to maintain PETCO₂ above 40 mm Hg by maintaining decreased breathing frequency at a normal tidal volume.

During the experimental session, each subject was seated in front of the RGM display panel with his/her left arm placed on a table so that it extended behind a partition that screened a phlebotomist from the subject’s view. A polyethylene catheter filled with heparinized saline was then inserted into the left plantar vein.

After 10 minutes of quiet rest, subjects breathed into the RGM mouthpiece continuously during a 15-minute baseline period at a normal frequency and tidal volume without feedback from the RGM display panel. For the next 30 minutes, the RGM feedback of PETCO₂ was presented, and the subject attempted to maintain CO₂ as high above 40 mm Hg as could be comfortably maintained. At the conclusion of this interval, feedback was terminated and subjects resumed normal breathing into the RGM mouthpiece for another 20 minutes.

During this 65-minute experiment, 5 mL of blood was withdrawn from the catheter into heparinized tubes at minutes 5 (baseline, 10 minutes before hypoventilation), 45 (after 30 minutes of hypoventilatory breathing), and 65 (recovery, 20 minutes after termination of task breathing). Blood pressure and heart rate were measured oscillometrically (Physiocontrol 200, Lifestat) every 3 minutes via an inflatable cuff wrapped around the subject’s right arm.

Na⁺,K⁺-ATPase Activity
Activity of Na⁺,K⁺-ATPase in intact erythrocytes was determined, as reported previously²³ with minor modifications.²⁴ Erythrocytes and plasma were separated by centrifugation at 300g for 10 minutes. The plasma was decanted and the buffy coat removed. Packed erythrocytes were washed three times in an isotonic medium (0.145 mol/L NaCl in 0.02 Tris-HCl buffer; pH=7.6 at 4°C). The erythrocytes were incubated for 30 minutes in the medium (mmol/L): Na 100, K 10, MgCl₂ 3, EDTA 0.5, Tris 50, and ATP 2 (pH=7.1, T=37°C). The reaction was stopped by the addition of 0.2 mL 20% trichloroacetic acid. Total ATPase activity was measured by the production of P_i, and Na⁺,K⁺-ATPase activity was estimated as the difference between total ATPase activity in the presence and in the absence of 0.1 mmol/L ouabain. The results were expressed as mmol of P_i produced per hour per 1 mL of erythrocytes. The amount of P_i in the sample was determined spectrophotometrically (Beckman DU 65). Before the measurement of Na⁺,K⁺-ATPase activity, the whole blood was divided into two equal parts. Rabbit polyclonal antidigoxin antiserum (Sigma Chemical Co, 55 µg/1 mL of whole blood) was added to one part whole blood. Both samples were preincubated at room temperature for 30 minutes before the measurement of Na⁺,K⁺-ATPase activity.

Deproteinized plasma samples and HPLC fractions from urine were tested to determine their ability to inhibit purified Na⁺,K⁺-ATPase. Na⁺,K⁺-ATPase assay was performed using purified canine kidney Na⁺,K⁺-ATPase (Sigma Chemical Co) as reported previously,¹¹ with some modifications. ATP hydrolysis was assessed spectrophotometrically by measuring NADH oxidation at 340 nm with the use of the linked enzyme system, pyruvate kinase (PK) lactate dehydrogenase (LDH). The system contained 3 mmol/L MgCl₂, 0.2 mmol/L ATP, 100 mmol/L NaCl, 10 mmol/L KCl, 1 mmol/L phosphoenolpyruvate, and a suspension of PK (2 units/µL) and LDH (95.5 units/µL). Na⁺,K⁺-ATPase was determined as the difference between oxidation of NADH with and without 0.1 mmol/L ouabain. Protein was maintained between 4 and 5 µg/cuvette in a volume of 1.0 mL.

Plasma Electrolytes
Plasma concentrations of Na⁺ and K⁺ were determined with a Beckman Na/K analyzer (Beckman E2A, Beckman Instruments).

Immunoassays
Plasma concentrations of EDLF were measured in duplicate in protein-free plasma using two immunoassays: (1) a modified commercial ouabain kit (New England Nuclear) and (2) a marinobufagenin immunoassay. Plasma proteins were precipitated by pretreatment of the whole plasma with trichloroacetic acid as described previously in detail.²⁵

Ouabain-like Immunoreactivity
Plasma ouabain-like immunoreactivity was measured using a modified ouabain enzyme-linked immunosorbent assay kit (New England Nuclear), based on the competition between immobilized ouabain conjugate and sample ouabain for rabbit antiouabain antibody. Then, the bound rabbit antibody was detected using europium-labeled goat antirabbit antibody (Wallac Oy). Interactions of ouabain antibody with digoxin and marinobufagenin are presented in Fig 1A.

View larger version (24K): [in this window] [in a new window]   Figure 1. Left, single peak elution of ouabain (A), marinobufagenin (B), and digoxin (C) from Deltapak C18 reverse-phase columns (linear gradient of acetonitrile 10% to 80%, 0.1% trifluoroacetic acid). Solid line is absorbance at 300 nm, dotted line at 230 nm. Abscissa, time (in minutes); ordinate, concentration of acetonitrile. Right, cross-reactivity of ouabain (), marinobufagenin (), and digoxin () with antiouabain (A), antimarinobufagenin (B), and antidigoxin (C) antibodies. Fig 1B also demonstrates marinobufagenin-like immunoreactivity of three serially diluted deproteinized plasma samples (dotted lines).

Marinobufagenin-like Immunoreactivity
Marinobufagenin-like immunoreactivity was analyzed in protein-free plasma and in HPLC fractions of extracted urine using a solid-phase fluoroimmunoassay. The method is based on a competition between the immobilized conjugate (marinobufagenin-3-glycoside-RNAase) and a sample of EDLF for rabbit polyclonal antimarinobufagenin antibody. Marinobufagenin (3ß5ß-dihydroxy-14,15-epoxy bufadienolide) was separated from the venom derived from the parotid glands of Bufo marinus toads via thin-layer chromatography [Silica-gel 60 F_254+366, Merck, elution with ethyl acetate, ratio of fronts (R_f)=0.42, =300 nM, coefficient of molecular extinction (E)=18 600] as reported previously.^{26 27} Fig 1B demonstrates the elution profile of marinobufagenin and three other Na⁺,K⁺-ATPase inhibitors by reverse-phase HPLC (see description of the method below).

The marinobufagenin-3-glycoside was synthesized, as described by Koenigs and Knorr,²⁸ with some modifications.²⁹ Fifty milligrams of marinobufagenin was dissolved in 10 mL of absolute benzene; 80 mg of Ag₂CO₃ was added; and the solution was heated to the temperature of boiling. Then a solution of acetobromo-D-glucose in dry benzene (12 mg/mL, Fluka Chemicals) was added. The reaction was controlled by thin-layer chromatography on silica gel (60 F_254+366, Merck, elution with ethyl acetate); disappearance of the spot corresponding to marinobufagenin showed that conjugation of marinobufagenin with glucose had been accomplished. Marinobufagenin-3-glycoside bovine serum albumin (BSA) conjugate (for immunization) and marinobufagenin-3-glycoside-RNAase conjugate (19 molecules of marinobufagenin-3-glycoside per 1 molecule of RNAase, for coating of the solid phase) were prepared, and rabbits were immunized, as previously reported by Curd et al³⁰ for digoxin. The fraction of IgG from the serum (62 mg/mL) was purified as reported previously.³¹ Albumin and non-IgG proteins were precipitated with caprylic (octanoic) acid, and the IgG fraction was precipitated with ammonium sulfate.

Marinobufagenin-3-glycoside-RNAase conjugate (1 µg of conjugate in 100 µL of phosphate buffered saline [PBS] per well) was immobilized on the bottom of Nunc microtitration strip, as reported previously in detail.³² We added 40 µL of marinobufagenin standards and unknown samples to the coated wells, followed by 100 µL of antimarinobufagenin antibody (2.5 µg/mL protein). After 1 hour of incubation, the strips were washed twice (Dissociation Enhanced Lanthanide Fluoro Immune Assay [DELFIA], wash solution, Wallac OY), then 100 µL of secondary antibody (europium-labeled goat antirabbit antibody, Wallac OY) was added. After 1 hour of incubation, the wells were washed six times with the wash solution. Then, 200 µL of enhancement solution, which releases the europium conjugated with the secondary antibody, (Wallac OY) was added to each well, the strips were shaken for 5 minutes, and after 10 minutes more the fluorescence of free europium was measured (DELFIA 1234 Arcus Fluorometer, Wallac OY).

The sensitivity of the immunoassay was 0.001 nmol/L. Cross immunoreactivity of the assay was expressed as the ratio of the amount of marinobufagenin required to displace 50% of antimarinobufagin antibody from immobilized conjugate to the amount of the cross-reactant to give the same 50% displacement. Cross immunoreactivity was 0.1% (digoxin and cinobufagin), <0.01% (ouabain), 3% (digitoxin), 1% (bufalin), <5% (mixture of bufosteroids from Bufo marinus toad venom except marinobufagenin), <1% (proscillaridin), and <0.1% (prednisone, progesterone, and spironolactone). Interactions of marinobufagenin antibody with digoxin and ouabain are presented in Fig 1B.

Digoxin-like Immunoreactivity
Cross-immunoreactivity of digoxin antibody (Sigma Chemical Co) used for the treatment of erythrocytes with different substances was characterized using solid-phase fluoroimmunoassay. The procedure was the same as for the ouabain immunoassay. Digoxin-BSA conjugate was prepared, as previously reported by Curd et al,³⁰ and immobilized on the bottom of NUNC microtitration strip wells (0.025 µg in 100 µL of PBS per well). Forty microliters of digoxin standards and unknown samples was added to the coated wells, followed by 100 µL of digoxin (1:4000) antibody. After 1 hour of incubation, the plates were washed and incubated for an hour with a second (ie, europium-labeled mouse antirabbit) antibody. Sensitivity of digoxin immunoassay was 0.001 nmol/L. Interactions of digoxin antibody with marinobufagenin and ouabain are presented in Fig 1C.

HPLC Analysis
Twenty-four–hour urine samples were obtained from 5 healthy men and stored at -20°C. One-milliliter samples of urine were extracted using Sep-Pak C18 reverse-phase cartridges (Waters, 70%). Cartridges were prewashed with 5 mL distilled water, then with 5 mL of acetonitrile, and again with 5 mL of acetonitrile. The urinary sample (1 mL) was added to the cartridge. Each cartridge was washed twice with 5 mL of distilled water and then eluted with 5 mL of acetonitrile. The eluate was dried under vacuum and stored at -70°C. Concentrations of marinobufagenin-like immunoreactivity in extracted urinary samples were measured as described above for plasma, and 24-hour output of marinobufagenin-like immunoreactive material was determined.

Five liters of urine was extracted with 7.5 liters of chloroform; then chloroform was removed under vacuum, and the dry residue was dissolved in acetonitrile. The fraction having R_f similar to marinobufagenin was isolated by thin-layer chromatography as reported previously²² and as described above ("Marinobufagenin-like Immunoreactivity"). The partially purified material was further fractionated by HPLC on Deltapak C18 columns (3.9x150 cm, 300 A) using Gilson HPLC pump (model 303, detector model 116).

The columns were equilibrated with 0.1% trifluoroacetic acid and developed with a linear gradient of acetonitrile over 10 to 80% acetonitrile containing 0.1% trifluoroacetic acid for 1 hour at a flow rate of 1 mL/min.

The elution of the standards (marinobufagenin, ouabain, digoxin, bufalin, and spironolactone, Sigma Chemical Co) was monitored at wavelengths 230 and 300 nm (Fig 1).

Partially purified chloroform urinary extract was fractionated in 32% acetonitrile containing 0.1% trifluoroacetic acid (isocratic elution). One-minute fractions were tested for their ability to react with antimarinobufagenin antibody ("Immunoassays").

Statistics
Two-tailed Student’s t tests and two-tailed Wilcoxon-Mann-Whitney tests were used to analyze the significance of differences between individual means.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Breathing Pattern and Blood Pressure
As shown in Fig 2A, PETCO₂ at minute 30 of hypoventilation was significantly increased above baseline levels (37.6±1.0 versus 41.0±0.9 mm Hg; P<.01) and returned to baseline levels after cessation of the task (36.1±0.7 mm Hg). Fig 2B shows that increased PETCO₂ was accompanied by substantial decreases in frequency of breathing (12.5±1.0 versus 7.3±1.0; P<.01).

View larger version (19K): [in this window] [in a new window]   Figure 2. PETCO2 (A) and breathing frequency (B) at baseline (BL), during hypoventilation (HV), and after hypoventilation (recovery, RC). Values are mean±SEM from 16 experiments. **P<.005 vs baseline. ##P<.01 vs hypoventilation.

As presented in the Table, systolic and diastolic pressures both increased during hypoventilatory breathing. The decreases in diastolic pressure after cessation of task breathing were significant, but the changes in systolic pressure were not. No significant changes in heart rate were observed during the period of task performance or after termination of the task (see the Table).

View this table: [in this window] [in a new window]   Table 1. Effects of Sustained Mild Hypoventilation

Na⁺,K⁺-ATPase Inhibition
As shown in Fig 3A, a significant decrease in the activity of Na⁺,K⁺-ATPase in erythrocytes (3.66±0.17 versus 2.15±0.28 mmol P_i · mL^-1 · h^-1, P<.001) was observed after 30 minutes of hypoventilation. After cessation of the task, activity of Na⁺,K⁺-ATPase in red blood cells was restored (3.20±0.27 mmol P_i · mL^-1 · h^-1, P<.02). As shown in Fig 3B, treatment of erythrocytes obtained before hypoventilation with 55 µg/mL antidigoxin antibody did not change activity of Na⁺,K⁺-ATPase (3.66±0.23 mmol P_i · mL^-1 · h^-1). Treatment of erythrocytes obtained during hypoventilation with antidigoxin antibody reversed the inhibition of Na⁺,K⁺-ATPase (4.11±0.34 mmol P_i · mL^-1 · h^-1) (Fig 3B).

View larger version (24K): [in this window] [in a new window]   Figure 3. Activity of Na+,K+-ATPase in nontreated erythrocytes (A) and erythrocytes treated with digoxin antibody (B) at baseline (BL), during hypoventilation (HV), and after hypoventilation (recovery, RC). Values are mean±SEM. *P<.01 vs baseline; #P<.01 vs nontreated erythrocytes; @P<.05 vs hypoventilation.

As shown in Fig 4A, deproteinized plasma obtained during hypoventilation demonstrated greater ability to inhibit dog kidney Na⁺,K⁺-ATPase. Plasma Na⁺,K⁺-ATPase inhibitory potency correlated to activity of Na⁺,K⁺-ATPase in erythrocytes (r=.40, P<.02, Fig 4B).

View larger version (21K): [in this window] [in a new window]   Figure 4. A, Plasma Na+,K+-ATPase inhibitory activity at baseline (BL), during hypoventilation (HV), and after hypoventilation (recovery, RC). *P<.05 vs BL. B, Relationship between erythrocyte Na+,K+-ATPase activity and plasma Na+,K+-ATPase inhibitory activity in 16 subjects during the experiment (BL, HV, and RC). r=.40, P<.02.

Plasma Digitalis-like Factors
Mean plasma levels of marinobufagenin-like immunoreactive material before hypoventilation were 1.23±0.47 nmol/L. As shown in Fig 1B, serially diluted plasma samples demonstrated concentration dependency in their ability to inhibit the binding of antimarinobufagenin antibody to the marinobufagenin-glycoside-RNAase conjugate in DELFIA immunoassay.

Within 30 minutes of voluntary hypoventilation, plasma concentrations of marinobufagenin-like immunoreactive material were increased in 14 of 16 subjects (6.69±2.0 nmol/L; P<.03, Fig 5A). After cessation of the task, mean plasma levels of marinobufagenin-like immunoreactivity were decreased (2.70±0.68 nmol/L).

View larger version (23K): [in this window] [in a new window]   Figure 5. Plasma concentration of digitalis-like substances at baseline (BL), during hypoventilation (HV), and after hypoventilation (recovery, RC). A, Plasma marinobufagenin-like immunoreactivity. B, Plasma ouabain-like immunoreactivity. Values are mean±SEM. *P<.01 vs baseline; #P<.05 vs hypoventilation.

Baseline plasma concentration of ouabain-like immunoreactive material was 0.163±0.047 nmol/L. After 30 minutes of hypoventilation, plasma levels of ouabain-like immunoreactivity did not change significantly (0.193±0.058 nmol/L; Fig 5B).

Plasma Electrolytes
Plasma concentrations of Na⁺ and K⁺ did not change significantly from baseline levels during or after task performance (see the Table).

HPLC Analysis
Total 24-hour urinary output of marinobufagenin-like immunoreactive material by 5 healthy men was 8200±2907 nmol/day. Mean 24-hour urine output was 967±201 mL. As shown in Fig 1B, a linear gradient of acetonitrile marinobufagenin standard eluted from Deltapak column in 7 minutes (Fig 1B). Ouabain (Fig 1A), digoxin (Fig 1C), bufalin, and spironolactone standards were eluted at minutes 6.15, 12.86, 15.8, and 12.28, respectively. As presented in Fig 6A and 6B, during isocratic elution with 32% acetonitrile, >90% of marinobufagenin-like immunoreactivity eluted as a sharp single peak at 8 minutes. Fig 6C shows that the addition of marinobufagenin standard to extracted urine resulted in an increase of a peak corresponding to fraction number 8. There was no separation of the 8-minute peak from the urinary sample with the added marinobufagenin standard (Fig 6B and 6C).

View larger version (11K): [in this window] [in a new window]   Figure 6. A and B, Elution profile of marinobufagenin-like immunoreactive substance from urinary extract on HPLC (Deltapak C18, isocratic elution with 32% acetonitrile and 0.1% trifluoroacetic acid, absorbance at 300 nm). In B, Small arrow indicates the peak obtained at 8 minutes and corresponds to fraction 8 in A. C, Addition of a marinobufagenin standard (20 µL of 100 µmol/L solution) to the urinary extract results in an increase of peak 8 with no separation.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major observations of these experiments are that a period of 30 minutes of hypoventilation is associated with (1) a fourfold increase in plasma concentrations of marinobufagenin-like immunoreactive material; (2) a marked inhibition of the activity of Na⁺,K⁺-ATPase in intact erythrocytes, which correlated with plasma Na⁺,K⁺-ATPase inhibitory activity and was reversed by digoxin antibody; and (3) a small but consistent pressor response. The results of the present experiments provide further evidence that human plasma contains a bufadienolide (marinobufagenin-like immunoreactive) Na⁺,K⁺-ATPase inhibitor. HPLC fractionation of urinary extract shows that urinary marinobufagenin-like immunoreactive material is very similar to marinobufagenin purified from Bufo marinus toad.

In previous studies in humans, activity of red blood cell Na⁺,K⁺-ATPase was shown to reflect the plasma levels of EDLF in essential hypertension,^{33 34} in prehypertensives,³⁵ and in hypertension of the third trimester of pregnancy.³⁶ Balzan et al,³⁷ using in vitro pretreatment of the blood with antidigoxin antibody, reversed the inhibition of erythrocyte Na⁺-K⁺ pump accompanying increased plasma EDLF concentrations in newborns. In the present study, inhibition of Na⁺,K⁺-ATPase in erythrocytes observed during the period of hypoventilation was reversed by in vitro pretreatment of the whole blood with antidigoxin antibody. The fact that digoxin antibody did not affect the activity of Na⁺,K⁺-ATPase from erythrocytes obtained during the baseline period suggests that the inhibitory effect is likely to have occurred because of the action of EDLF. Further support for this conclusion is provided by observations of increases in (1) plasma Na⁺,K⁺-ATPase inhibitory activity and (2) plasma levels of marinobufagenin-like immunoreactivity during hypoventilation.

In the present study, marked inhibition of Na⁺,K⁺-ATPase in erythrocytes, a fourfold increase in plasma levels of marinobufagenin-like immunoreactivity, and a pressor response developed within 30 minutes of hypoventilatory breathing. Within 20 minutes after cessation of task performance, activity of Na⁺,K⁺-ATPase was restored, and diastolic blood pressure and plasma marinobufagenin-like immunoreactivity decreased.

Unlike marinobufagenin, ouabain-like immunoreactivity did not increase significantly over the course of the experiment. Recent evidence suggested that plasma levels of endogenous ouabain may be too low to produce a functionally significant inhibition of the Na pump and that ouabain-like immunoreactive material does not represent ouabain itself.^{38 39} However, endogenous ouabain-like immunoreactive substance may represent a stereoisomer of ouabain.⁴⁰

In our experiment, Na⁺,K⁺-ATPase inhibition in erythrocytes was reversed by digoxin antibody. As illustrated in Fig 1C, in the digoxin immunoassay marinobufagenin demonstrated greater digoxin-like immunoreactivity than ouabain. In the marinobufagenin immunoassay (Fig 1B), digoxin showed marinobufagenin-like immunoreactivity greater than did ouabain. Analysis of the interaction of three Na⁺,K⁺-ATPase inhibitors with anti-ouabain antibody (Fig 1A) shows that digoxin and marinobufagenin share similar immunoreactivity. These results suggest, therefore, that the so-called endogenous digoxin-like factor is more likely to represent a circulating endogenous bufadienolide than endogenous ouabain. Naomi et al,⁴¹ using several digoxin antisera and several substances considered to be potential EDLFs, established immunological profiles of plasma EDLF from different groups of patients and suggested that circulating EDLF may have a bufadienolide nature. Recently, Lichtstein et al¹⁷ demonstrated with mass spectrometry that digoxin-like immunoreactive and Na⁺,K⁺-ATPase inhibitory material from human eye lenses has a bufadienolide nature and represents bufalin derivatives.

Our present observation that marinobufagenin-like immunoreactivity from human urinary extract HPLC fractions coelutes with authentic marinobufagenin, supports the evidence that mammals have a bufadienolide Na⁺,K⁺-ATPase inhibitor. In our study, EDLF from HPLC fraction that cross-reacted with anti-marinobufagenin antibody, demonstrated absorbance at =300 nm, which is typical for bufadienolides.¹⁵ Earlier, Lichtstein et al⁴² separated EDLF from human cerebrospinal fluid that also showed absorbance at 300 nm. In marinobufagenin immunoassay, serially diluted plasma samples demonstrated concentration dependency in inhibition of anti-marinobufagenin antibody binding to the solid phase in DELFIA. Previously, a compound that was chromatographically indistinguishable from digoxin was separated from human urine.⁴³ As presented in Fig 1, digoxin and marinobufagenin eluted from the Deltapak reverse-phase column at different times, 12 and 7 minutes, respectively. Therefore, marinobufagenin-like immunoreactive substance is unlikely to represent digoxin.

Our data suggest that a marinobufagenin-like immunoreactive factor may be involved in short-term cardiovascular control. The possible role of endogenous marinobufagenin-like immunoreactive material in long-term blood pressure regulation remains to be investigated.

The focus on hypoventilatory breathing as a stimulus to EDLF was prompted by previous observations of a model of canine hypertension involving hypoventilatory breathing and high sodium intake.⁷ In this regard, it is noteworthy that one of the earliest studies indicating the existence of EDLF suggested that induction of hypoxia in cats elicited a "strophanthin-like" compound.⁴⁴ The results of the present study confirm the view that breathing pattern may participate in blood pressure control via release of circulating Na⁺,K⁺-ATPase inhibitors.

	Acknowledgments

 
Purification of marinobufagenin and production of antimarinobufagenin antibody were carried out in the Laboratory of Pharmacology, Sechenov Institute of Evolutionary Physiology and Biochemistry, St Petersburg, Russia, and were supported by Biomedical Sciences Research Laboratories Inc, Millersville, Md. The authors are grateful to Georgii P. Kazakov, Verta Peptides Inc, St Petersburg, Russia, for help in performing the HPLC analyses.

Received March 2, 1995; first decision April 8, 1995; accepted August 16, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

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