Pulse Pressure Correlates in Humans With a Proscillaridin A Immunoreactive Compound
Abstract Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2×10−9 mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L]=0.013×mm Hg−2.17, r=.25, P<.05), systolic pressure (log ED1 [nmol/L]=0.010×mm Hg−2.23, r=.32, P<.01), and pulse pressure (log ED1 [nmol/L]=0.019×mm Hg−1.80, r=.38, P<.0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.
- proscillaridin A
- hypertension, essential
- blood pressure
- pulse pressure
- Na+-K+-exchanging ATPase
Endogenous compounds with digitalis-like action are considered to circulate in increased concentrations in blood in essential hypertension and various conditions of volume expansion.1 2 3 4 The search for such compounds has revealed that ouabain or a ouabain isomer5 is produced by the adrenal cortex6 and is elevated in hypertension.4 7 A ouabain isomer is also found in the hypothalamus.8 A substance indistinguishable from digoxin has been isolated from human urine.9 A substance not identical with digoxin but cross-reacting with digoxin antibodies has been shown to exist in adrenals.10 It is well documented that a material cross-reacting with digoxin antibodies is available in increased concentrations in umbilical cord blood.11 Recently, 19-norbufalin has been isolated from human cataract lenses.12 An endogenous marinobufagenin-like immunoreactive factor circulating in blood and inhibiting the sodium pump has been found to increase with pressure response as the result of hypoventilatory breathing.13 Since toads are vertebrates and synthesize several bufadienolides in their skin,4 it seemed possible that other vertebrates, such as humans, may also be able to synthesize several bufadienolide-like compounds. Therefore, we raised antibodies against the bufadienolide proscillaridin A in rabbits and assayed its concentration in human plasma. This report shows that a material cross-reacting with antibodies against a bufadienolide circulates in blood and correlates with systolic and mean arterial BP values as well as pulse pressure.
Gelatin was obtained from Bio-Rad, heptafluorobutyric acid from Pierce Chemical Co, and the silicon oils AR 200 and AR 20 from Wacker Chemie. N-Acetylglucosaminyl-(β1-4)-N-acetylmuramyl-l-alanyl-d-isoglutamin (GMDP) was from Gerbu Biotechnik, the protein A column MemSep 1010 from Millipore, 86RbCl from Amersham/Buchler, and biotinylated goat anti-rabbit IgG as well as streptavidin conjugated to alkaline phosphatase from Jackson ImmunoResearch Dianova. All other chemicals were of the highest purity available and were obtained from Merck, Sigma Chemical Co, Roth, Boehringer, and Serva. Marinobufagenin was a kind gift from Dr A.Y. Bagrov, Laboratory of Pharmacology, Sechenov Institute of Evolutionary Physiology and Biochemistry, St Petersburg, Russia.
Plasma for use in this study was obtained from 22 essential hypertensive patients, 60 normotensive control subjects, and 19 patients with chronic renal failure. The characteristics of the patients are given in Table 1⇓. Patients were randomly recruited from among outpatients. All were free of major medical illness and did not have significant pain, elevated temperature, or any acute condition. Individuals with major medical illness and those who had taken prescription medications (including antihypertensive medication, nonsteroidal anti-inflammatory drugs, aspirin, or cardiac glycosides) were excluded. After subjects had rested for 10 minutes, seated BP measurements were obtained with conventional sphygmomanometric methods by the same individual. Subjects were considered normotensive if their BP was less than 140/90 mm Hg. A diagnosis of hypertension was based on a BP greater than or equal to 160/90 mm Hg or a diastolic BP of 95 mm Hg or higher. Diagnosis of chronic renal failure was based on serum creatinine concentration greater than or equal to 106 μmol/L. Chronic renal failure was due to chronic glomerulonephritis (n=11), interstitial nephritis (n=6), and polycystic kidney disease (n=2). None of the patients was undergoing hemodialysis.
[Nai]P was determined fluorimetrically by use of the sodium-sensitive fluorescent indicator sodium-binding benzofuran isophthalate.14 All other serum values—sodium (Na+), potassium (K+), calcium (Ca2+), glucose, creatinine, uric acid, and urea—were obtained by routine clinical chemical methods.
Production and Purification of Antibodies Against Proscillaridin A
Specific polyclonal antibodies against the bufadienolide proscillaridin A were raised in four New Zealand White rabbits. Proscillaridin A was coupled to bovine serum albumin according to a procedure similar to the one of Terano et al15 and Harris et al.16 Proscillaridin A (100 μg) coupled to bovine serum albumin in 0.5 mL of 0.9% NaCl solution was emulsified with 0.5 mL Freund’s complete adjuvant. This mixture was injected intradermally at five to six sites along the spinal column. Booster injections of 100 μg antigen dissolved in 0.5 mL of 0.9% NaCl solution and also in either 0.5 mL incomplete Freund’s adjuvant or 10 μg GMDP were administered at 4-week intervals as described above. Rabbits were bled 40 weeks after the start of the immunization procedure. The globulin fraction of antiserum in 1 mol/L glycine and 0.5 mol/L NaCl (pH 8.9) was applied to a protein A MemSep 1010 column. IgG was eluted with 0.1 mol/L citric acid (pH 3.0); the pH of the eluate was adjusted to 7 with Tris base and concentrated by ultrafiltration. The concentrated IgG fraction was stored in Tris-NaCl buffer (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.8) containing 0.02% NaN3 and 50% glycerol at −20°C.
Preparation of Plasma for Measurement of Proscillaridin A Immunoreactivity by Indirect ELISA and for Detection of Sodium Pump Inhibition With 86Rb+ Uptake Into Human Red Blood Cells
The extraction was performed as described previously.17 The water-soluble extract of 2 mL plasma after ethanol precipitation was applied to a LiChrospher 250-5 RP18 HPLC column and separated by a propanol/isopropanol gradient in 0.1% heptafluorobutyric acid according to Hamlyn et al.18 Three fractions, ED1, ED2, and ED3, with increasing hydrophobicity were collected and measured by an indirect competitive ELISA. Each of the ED1, ED2, and ED3 fractions was dissolved in 500 μL PBS, and volumes between 10 and 100 μL were included in the ELISA test system. All measurement was done in duplicate. Only those values showing a concentration dependency were included in this study.
For testing of the ability of the ED1, ED2, and ED3 fractions to inhibit the sodium pump, the residue was taken up in 250 μL distilled water after lyophilization.
Indirect ELISA for Proscillaridin A Immunoreactivity
Microtiter plates were coated with 100 μL antigen per well (proscillaridin A coupled to gelatin with a variation of the procedures of References 15 and 1615 16 ) in coating buffer (15 mmol/L Na2CO3, 35 mmol/L NaHCO3, pH 9.6) with a concentration of 100 μg/mL for 1 hour. Free binding sites were blocked with 1% gelatin in PBS for 30 minutes. Thereafter, 10 to 100 μL of the HPLC-purified plasma sample (0.04 to 0.4 mL plasma equivalents) was added and incubated with 10 μL proscillaridin A–IgG (1:1000 diluted in PBS) for 1 hour. Bound proscillaridin A–IgG was detected with 100 μL biotinylated goat anti-rabbit IgG (1:5000 in PBS for 1 hour) and 100 μL alkaline phosphatase coupled to streptavidin (1:500 in PBS for 30 minutes). Phosphatase activity was measured by addition of 100 μL p-nitrophenylphosphate (1 mg/mL) as a substrate in 0.5 mmol/L MgCl2 and 10 mmol/L diethanolamine (pH 9.5). The phosphatase reaction proceeded for 30 minutes and was stopped by addition of 50 μL of 3 mol/L NaOH. The yellow p-nitrophenolate formed was measured by a microtiter photometer (Multiscan MCC/340) at 405 nm. Between the incubation steps, washing was performed three times for 3 minutes with 200 μL of 150 mmol/L PBS, pH 7.4 (140 mmol/L NaCl, 3 mmol/L KCl, 1.5 mmol/L KH2PO4, and 5.5 mmol/L Na2HPO4).
This assay system was also used for measurement of the serum titer for proscillaridin A antibodies and testing of cross-reactivity of the purified anti–proscillaridin A–IgG with other cardiac glycosides and steroid hormones.
Quantification of Sodium Pump Inhibitor by Measurement of Ouabain-Sensitive 86Rb+ Uptake Into Human Red Blood Cells
To examine whether HPLC fractions cross-reacting with proscillaridin A antibodies inhibit the sodium pump, we tested their potency to inhibit ouabain-sensitive 86Rb+ uptake into human red blood cells by a procedure described previously.19 For the quantification of the inhibitor concentration, it was assumed that the endogenous inhibitor binds with the same affinity as ouabain binds to the sodium pump of human red blood cells. Half-maximal inhibition of the sodium pump of human red blood cells was seen at 4×10−8 mol/L ouabain. Concentrations of the HPLC-purified plasma extract giving half-maximal inhibition were assumed to contain 4×10−8 mol/L inhibitor units. Half-maximal inhibition was extrapolated after subtraction of the ouabain-independent 86Rb+ uptake from all values from a double logarithmic plot (% inhibition/100−% inhibition) against the concentration of the inhibitor.19
Statistical analysis was done by the BMDP computer programs20 and GraphPad Prism (GraphPad Software). Because the statistical distribution of the ED1, ED2, and ED3 concentrations were nearly log normal, their values are represented as geometric mean with dispersion factor. All other data are represented as mean value and SE. The geometric means of the proscillaridin A immunoreactivities ED1, ED2, and ED3 of the three groups of patients (normotensive, hypertensive, and patients with chronic renal failure) were compared by the nonparametric Kruskal-Wallis test as well as multiple comparison (Nemenyi test) using the BMDP-3S program. Regression analysis of log ED1, log ED2, and log ED3 was tested as a function of systolic, diastolic, and mean arterial BP values as well as of pulse pressure, [Nai]P, age, body mass index, and serum concentrations of Na+, K+, Ca2+, glucose, creatinine, uric acid, and urea with GraphPad Prism. Mean values of parameters other than ED1, ED2, and ED3 were compared in a two-sided Student’s t test or Mann-Whitney test with comparison related significance level. The two-tailed level of significance was set at a value of P<.05.
Development of an Indirect ELISA for Proscillaridin A and Bufadienolide-Like Substances
To develop an immunological assay for the bufadienolide proscillaridin A and bufadienolide-like immunoreactive substances, we immunized rabbits with proscillaridin A coupled to bovine serum albumin. Rabbits produced antibodies with a titer of 1:103 after 40 weeks and a Kd value of 2×10−9 mol/L. This titer and affinity remained constant from the 25th to the 120th week after immunization. When the specificity of the antibodies was tested, only bufadienolides such as proscillaridin A and bufalin were recognized (marinobufagenin was almost not recognized); cardenolides such as ouabain and digoxin or other steroid hormones were not recognized (Table 2⇓). With these antibodies, concentrations as low as 100 fmol per well or 10−9 mol/L could be detected. The range for determinations of proscillaridin A was 10−7 to 10−10 mol/L. The interassay coefficient of variation of the method was 18.6%, and the intra-assay coefficient was 8.8%. The curve of proscillaridin A immunoreactivity as a function of plasma volume was superimposed over the proscillaridin A standard curve (Fig 1⇓).
Characterization of Patients
All three groups of patients—normotensive subjects, patients with hypertension, and patients with chronic renal failure—had normal and equal serum levels of Na+, K+, Ca2+, and glucose. Patients with chronic renal failure had significantly elevated levels of creatinine (P<.001), uric acid (P<.05), and urea (P<.001) compared with normotensive subjects and hypertensive patients (Table 1⇑). Body mass index of all groups did not differ. The hypertensive patients had a significantly higher (P<.05) diastolic BP compared with normotensive subjects and patients with chronic renal failure (Table 3⇓). Patients with chronic renal failure had a significantly (P<.05) higher [Nai]P compared with normotensive subjects and hypertensive patients (Table 3⇓).
Determination of Proscillaridin A Immunoreactivity in Serum of Healthy Normotensive Subjects and Patients With Essential Hypertension and Chronic Renal Failure
Before measurement of immunoreactivity, we separated the ethanolic extracts by reverse-phase C18 HPLC using a propanol/isopropanol gradient as described by Hamlyn et al.18 Chromatograms of the ethanolic plasma extracts are shown in Fig 2⇓. It is evident that three peaks of proscillaridin A immunoreactive material with different polarities and retention times were eluted from the C18 reverse-phase column (Fig 2B⇓). These three peaks also inhibited the cardiac glycoside–sensitive 86Rb+ uptake into human red blood cells (Fig 2A⇓). The peak with a retention time of 20 minutes and almost the same polarity as ouabain was denoted as ED1 in all further investigations. In addition, more hydrophobic fractions with proscillaridin A immunoreactivity were seen. The peak with a retention time of approximately 40 minutes was called ED2, and the most lipophilic fraction with a retention time of 60 minutes and a polarity similar to that of proscillaridin A was named ED3 (Fig 2⇓).
An analysis of the plasma concentrations of proscillaridin A immunoreactivity of 60 normotensive subjects revealed a non-gaussian distribution of the individual values. However, it showed a logarithmic distribution curve like other hormones. Therefore, geometric means and dispersion factors were calculated for each fraction of ED1, ED2, and ED3 proscillaridin A immunoreactivity. When the concentrations of ED1, ED2, and ED3 were compared, for all groups of patients, ED1<ED2<ED3 (Table 3⇑). Although inspection of the ED1, ED2, and ED3 values of proscillaridin A immunoreactivity indicates a tendency for higher values in hypertensive patients compared with normotensive subjects for the geometric mean (Table 3⇑), statistical analysis by the Kruskal-Wallis test and Tukey comparison was unable to detect any significant differences between the patient groups. However, we cannot exclude the possibility that a higher number of investigations may reveal such differences.
No correlations were found between the ED2 and ED3 concentrations of proscillaridin A immunoreactivity and the individual systolic, diastolic, and mean BP values of their plasma donors; pulse pressure; body mass index; and age of the patients. There also was no correlation with [Nai]P and other plasma concentrations such as Na+, K+, Ca2+, glucose, creatinine, uric acid, and urea. The ED1 values of proscillaridin A immunoreactivity also did not correlate with these plasma concentrations, the [Nai]P of their donors, body mass index, or age. However, the ED1 concentration of proscillaridin A immunoreactivity showed a significantly positive correlation with systolic BP (P<.01, Fig 3A⇓), mean BP (P<.05, Fig 3B⇓), and pulse pressure (P<.0001, Fig 3C⇓), but it did not correlate with diastolic BP.
Increased circulating levels of sodium pump inhibitors have been implicated in the pathogenesis of hypertension.2 19 21 22 The search for sodium pump inhibitors or for substances with endogenous digitalis-like action in human serum has led to the detection of an isomer of the cardenolide ouabain5 6 7 that is twofold to threefold elevated under the conditions of hypertension. Elevated concentrations were also found in patients with adrenocortical tumors, aldosteronism, and heart failure (for a recent review see Reference 2323 ). Evidence is accumulating that the compound is produced in the adrenal cortex and that its release and synthesis are increased by corticotropin and angiotensin II.6 23 Endogenous ouabain, however, does not seem to be the sole compound with cardiac glycoside–like action in serum. Numerous reports show elevated levels of a compound or compounds cross-reacting with digoxin antibodies in hypertension.3 11 This assay system is not digoxin specific because compounds indistinguishable from ouabain have been isolated by affinity chromatography on digoxin antibodies as well.24 However, a compound resembling digoxin in its polarity has been detected in adrenals,10 and a substance that cannot be distinguished from digoxin was isolated from the urine of individuals not receiving any digoxin treatment.3 The body of evidence for the existence of a biosynthetic pathway for the cardenolides in adrenal glands is growing,6 10 and recent reports add the suggestion that bufadienolides may be formed in mammals as well. The bufadienolide 19-norbufalin has been isolated from cataracts in human lenses.12 Recently, Bagrov et al13 reported on the existence of a marinobufagenin-like compound in blood and urine of humans that seems to be related to the rise in BP. This compound is more hydrophobic than ouabain. Interestingly, the hydrophobic bufalin has been shown to raise BP when administered acutely25 and to be a more potent vasopressor agent than ouabain.26 Since toads are vertebrates and synthesize several bufadienolides, we speculated that mammals may be able to synthesize such compounds as well.
The present report shows that in fact several substances cross-reacting with an antibody against the bufadienolide proscillaridin A and inhibiting the sodium pump of red blood cells circulate in human blood (Fig 2⇑). However, only the most polar substance in the serum with a retention time similar to that of ouabain correlated with the rise of mean and systolic BP values and pulse pressure (Fig 3⇑). Since the concentrations of the hydrophilic ED1 proscillaridin A immunoreactivity did not correlate with diastolic pressure, this substance does not seem to be responsible for the increase of peripheral resistance in hypertensive patients. Although stroke volume is only one determinant of pulse pressure, the correlation of ED1 levels with pulse pressure is compatible with the hypothesis that ED1 may have a positive inotropic effect and thereby affect pulse pressure (Fig 3C⇑). The anti–proscillaridin A–IgG used in the present study did not cross-react with cardenolides but did cross-react with a different bufadienolide, bufalin, and slightly with marinobufagenin (Table 2⇑). It is unlikely, therefore, that ouabain was measured in the present study despite the similarity in retention times with authentic ouabain in the HPLC system used to detect endogenous material cross-reacting with proscillaridin A antibodies. Although the antibody used in the present study cross-reacts slightly with marinobufagenin (Table 2⇑), we did not assay this compound. Marinobufagenin is more hydrophobic than ouabain13 and elutes with a higher retention time than ouabain in the HPLC system used for the clean up of proscillaridin A immunoreactivity. Additionally, the marinobufagenin antibody of Bagrov et al13 does not cross-react with proscillaridin A, and the endogenous marinobufagenin shows different physiological properties.13 Therefore, it is highly probable that two different inhibitors of the sodium pump are tested by antibodies against proscillaridin A and marinobufagenin.
Considering the fact that 19-norbufalin12 and an endogenous marinobufagenin13 exist in humans, it is tempting to speculate that several bufadienolides may circulate in blood. Since bufadienolides show a distinct UV maximal absorbance at 300 nm, it should not be difficult to substantiate this speculation. Preliminary studies with affinity purification of a material from bovine adrenals cross-reacting with proscillaridin A antibodies led to the isolation of a pure compound that failed to show UV absorbance at 300 nm.27 Consequently, it is likely that an inotropic substance other than a bufadienolide may circulate in blood and mimic the action of a bufadienolide by binding to the sodium pump and to antibodies against proscillaridin A.
The geometric mean of the ED1 proscillaridin A immunoreactive material in serum was 0.102 nmol/L (with a dispersion factor of 8.77) in 60 normotensive subjects. Endogenous ouabain in 11 normotensive human volunteers has been found by Harris et al16 to be 0.138±0.043 nmol/L. Evidently, both substances exist in humans in the same concentration range. It is unclear thus far whether the actions of both substances are additive. It is well known that multiple digitalis receptor sites differing in their affinities exist in mammals.28 The physiological meaning of this observation is unknown. It will be interesting to determine whether the various substances with endogenous digitalis-like actions affect specific isoforms of the sodium pump.
Selected Abbreviations and Acronyms
|ELISA||=||enzyme-linked immunosorbent assay|
|HPLC||=||high-performance liquid chromatography|
|[Nai]P||=||cytosolic free Na+ concentration in platelets|
This work was supported by grants from the Deutsche Forschungsgemeinschaft/Bonn through Scho 139/20-1; the Fonds der Chemischen Industrie, Frankfurt/M; and the Giessener Hochschulgesellschaft eV, Giessen. We are grateful to Dr Klaus Failing of the Biomathematic Group of the Institute of Veterinary Physiology for his advice in handling the statistics in this study and to Dr Jack Muth for correcting the English of the manuscript.
Reprint requests to Dr Wilhelm Schoner, Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität Giessen, Frankfurter Str 100, D-35392 Giessen, Germany. E-mail firstname.lastname@example.org.
- Received October 10, 1995.
- Revision received October 31, 1995.
- Accepted January 22, 1996.
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