Demonstration of a Ouabainlike Plasma Compound in
Hypertension Prone and Hypertension Resistant Rats
DAVID LICHTSTEIN, DAHLIA M I N C , YAKHIN SHIMONI, JOSEPH DEUTSCH,
JUDITH MEKLER, AND DRORI BEN-ISHAY
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SUMMARY Material extracted and partially purified from plasma of the Sabra hypertension prone
rats was found to be capable of 1) inhibiting the binding of 3H-ouabain to rat brain synaptosomes, 2)
inhibiting the activity of rat brain microsomal Na, K activated adenosine triphosphatase, and 3)
increasing the contractile force of rat heart muscle. The results demonstrate the presence of a
ouabainlike compound in the plasma of these rats. The plasma concentration of this compound in
Sabra hypertension prone rats was 698 ± 199 nmol/ml in ouabain equivalents (SKM; n = 11) versus
2543 ± 1140 nmol/ml (n = 9) in the Sabra normotensive strain. The presence of ouabainlike
compound in the plasma is consistent with the hypothesis that this compound functions as a hormone
that regulates Na, K activated adenosine triphosphatase activity and the physiological processes in
which this enzyme is involved. (Hypertension 7: 729-733, 1985)
KKY WORDS • digitalis • ouabainlike compounds • Na, K activated adenosine
triphosphatase * genetic hypertensive rats • heart contractility
O
VER the past 20 years numerous studies have
demonstrated the presence of a natriuretic
factor in mammalian blood and urine that is
activated or released by volume expansion.1-2 Recent evidence suggests that this factor is an inhibitor of Na, K activated adenosine triphosphatase
(Na, K-ATPase),-^ which probably interacts with the
ouabain binding site on the enzyme. 6 - 7
Theoretical considerations and experimental evidence suggest that inhibition of Na, K-ATPase can
increase the contractile force of vascular smooth muscle.9"1-1 Since this action may elevate peripheral vascular resistance and induce a rise in blood pressure," l3 a
link between the natriuretic factor and the etiological
process of some forms of hypertension was postulated.14"17 This hypothesis is supported by the identification of a circulating endogenous inhibitor of the Na, KATPase in hypertensive animals14"16 and humans. 171 *
Moreover, the concentration of the Na, K-ATPase inhibitor has been reported to be linearly related to the
level of blood pressure in normal and hypertensive
individuals.17
The presence of a digitalislike or ouabainlike compound (OLC) also has been reported in animal tissue.19"25 These studies were prompted by the demonstration of specific receptors for cardiac glycosides on
the Na, K-ATPase, which suggested the existence of
an endogenous ligand. Since compounds that can inhibit Na, K-ATPase activity by interacting with the
digitalis binding site on the enzyme were indeed extracted from mammalian brain19"23 and from toad skin
and blood,24 " we thought it reasonable to assume that
the natriuretic substance and the ouabainlike compound are identical.
The availability of the Sabra hypertension prone rats
(SBH) and Sabra normotensive rats (SBN) prompted a
search for a circulating ouabainlike inhibitor of the Na,
K-ATPase in this model. The two strains initially were
selected from the Hebrew University Sabra rat for their
respective sensitivity or resistance to deoxycorticosterone acetate salt hypertension.26 (For a detailed description of the main characteristics of these animals,
see refs. 27 and 28.) We also sought to determine
whether the disparate susceptibility to hypertension of
these rats could be related to the presence of a circulating Na, K-ATPase inhibitor in the plasma.
From the Department of Physiology (D. Lichtstein. D Minc. Y.
Shimoni) and the Department of Pharmaceutical Chemistry (J.
Deutsch). Hebrew University Hadassah Medical School. Jerusalem, and the Hypertension Unit, Hadassah University HospitalMount Scopus, Jerusalem. Israel (J. Mekler, D Ben-lshay).
Supported in part by the Israeli Academy of Sciences, the Israeli
Ministry of Health, and the Elcfant Research Fund.
Address for reprints: Dr. David Lichtstein. Department of Physiology, Hebrew University Hadassah Medical School, Jerusalem,
Israel.
Received May 10, 1984; accepted February 13, 1985
729
730
HYPERTENSION
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Materials and Methods
Partial Purification of Ouabainlike Compound
Studies were performed on male SBH and SBN,
aged 2 to 5 months, maintained since weaning on regular laboratory chow (Ambar Food Mills, Hadera, Israel) containing 0.6 to 0.8% sodium and tap water. The
blood pressure (mean ± SE) measured in prewarmed,
unanesthetized animals was 151 ± 2.4 and 127 ± 1.3
mm Hg respectively (p < 0.01). The animals were
bled from the abdominal aorta under ether anesthesia,
the plasma of 3 to 10 rats was pooled, and methanol
was added (v/v). Proteins were precipitated by high
speed centrifugation (100,000 g), and the supernatant
decanted. The methanol was evaporated, and the samples were centrifuged again at 100,000 g. The aqueous
supernatant was separated and lyophilized. The residue was dissolved in 1.2 ml of methanol, and aliquots
of 20 to 200 ixl were fractionated by high-performance
liquid chromatography (model 985, Tracor Instruments, Austin, TX, USA) using an Alltech amino column. The column had been equilibrated with 90%
acetonitrile and eluted with a linear 90% to 20% acetonitrile gradient in 40 minutes at a flow rate of 1 ml/min.
The elution of the compounds was monitored at 300
nm with a Tracor 970A detector. Fractions of 2 ml
were collected and evaporated, and the residue was
dissolved in 1 ml of distilled water. Aliquots from this
solution were used for measurements of biological
activity.
The method used for the extraction and partial purification of the OLC is highly reproducible. When aliquots of pooled plasma extract were processed by this
method values of OLC obtained did not vary by more
than 5%.
Measurements of 3H-Ouabain Binding to Rat Brain
Synaptosomes
Rat brain synaptosomes were prepared as previously
described.21 Binding of-'H-ouabain was assayed by a
conventional filtration technique. The reaction mixture
(500 /AI final volume) contained the following constituents (in final concentrations): 50 mM tris(hydroxymethyl) aminomethane hydrochloride buffer (pH
7.4), 0.5 mM ethylenediaminetetraacetic acid, 80 mM
NaCl, 4 mM MgSO4, 2 mM adenosine triphosphate
(Tris, vanadium-free; Sigma Chemical Co., St. Louis,
MO, USA), 32 nM ['HJouabain (19.5 Ci/ mmol; New
England Nuclear Corp., Boston, MA, USA), and other additions as indicated. Reactions were initiated by
the addition of 200 /xl of a crude synaptosomal fraction, which resulted in a final protein concentration of
200 /xg per reaction. Binding equilibrium was
achieved after incubation for 1 hour at 37 °C. The
reactions were terminated by adding 3 ml of ice-cold
50 mM Tris-hydrochloride (pH 7.4), and the suspension was filtrated over Whatman GF/B filters (Whatman, Inc., Clifton, NJ, USA). Filters were washed
three times with 3 ml of the same Tris buffer, dried,
and assayed for radioactivity in a Packard liquid scintillation counter (Packard Instrument Co., Inc., Rockville, MD, USA) at a counting efficiency of approxi-
VOL 7, No
5, SEPTEMBER-OCTOBER
1985
mately 35%. Specific binding was calculated by
subtracting the binding observed in the presence of 100
/u,M unlabeled ouabain from that observed in the absence of unlabeled ouabain. Specific binding represented 93% of the total binding under control conditions. Binding of [3H]ouabain was a linear function
of membrane protein concentration in the range of
20 to 1000 fJLg per assay. Binding to the filters was
negligible when synaptosomes were omitted from the
incubation.
Measurements of Na, K Activated Adenosine
Triphosphatase Activity
Enzyme activity in rat brain microsomal fraction
was measured by the colorimetric determination of
inorganic phosphate29 after the incubation of synaptosomes (200 fig of protein per reaction) at 37 °C in a
solution containing (in final concentrations): 54 mM
Tris-hydrochloride (pH 7.4), 100 mM NaCl, 20 mM
KG, 4 mM MgCl2, and 4 mM adenosine triphosphate
(Tris, vanadium-free). The adenosine triphosphate
was added after a 10-minute preincubation to initiate
the reaction. Reactions were terminated by adding 5%
tricarboxylic acid, and the precipitate was removed by
centrifugation. The Na, K-independent ATPase activity was determined by omitting Na + and K + from the
reaction mixture or by adding I mM ouabain to the
complete assay mixture.
Measurements of Cardiac Muscle Contraction
Tension recordings were done from rat atrial strips
placed in a 2-ml chamber and perfused with control
solution containing 140 mM NaCl, 5.4 mM K G , 12
mM NaHCO,, 2 mM CaCl,, 1 mM MgCl2, 0.4 mM
Na,HPO4, and 5 mM glucose, continuously bubbled
with 95% O 2 , 5% CO2 with a pH of 7.4 at 36 °C. One
end was attached to an AKERS force transducer (Aksjeselskapet mikro-elektronikk, Horten, Norway) for
measuring the strength of contraction. Constant stimulation was applied at a rate of 0.5 Hz, and twitch
magnitude was monitored on a pen recorder and oscilloscope and stored on magnetic tape.
Results
An example of a chromatographic pattern is shown
in Figure I. The biological activity (i.e., inhibition of
'H-ouabain binding) was found in the peaks at a retention time of 12 to 20 minutes with a maximum in the
fraction at 15 minutes.
In the first set of experiments approximately 300 ml
of pooled plasma from SBH was used. The OLC was
extracted and partially purified as described in Methods. The material present in the fractions with a retention time of 12 to 20 minutes was collected, and aliquots were tested. As can be seen in Figure 2, the
endogenous material was capable of inhibiting both
ouabain binding and Na, K-ATPase activity. Both effects appeared to depend on the concentration of the
material in the assay. Like ouabain, the endogenous
compound inhibited Na, K-ATPase activity at concentrations almost 100 times higher than those need-
OUABAINLIKE COMPOUND IN HYPERTENSION/L/c/ime/n et al.
731
8mg
0
5
10
15
20
Rtttntion Tkn« t min )
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FIGURE I. High-performance liquid chromatography of
methanol extract of plasma obtained from Sabra hypertensive
rats. Methanol extract of 20 ml of plasma obtained from these
rats was processed as described in Methods. The optical density
is plotted (left ordinate) against retention time (abscissa). On
the right ordinate, the relative biological activity is represented
b\ hatched columns. This activity was measured by testing the
capability of each fraction to inhibit JH'-ouabain binding to rat
brain synaptosomes (see Figure 2).
9
"o
20 I
? 100
C
o
i? so
o
Z
60
•2
to
I
20
m
15 "a
•o-
>%
10 -f
5
10
20
30
40
SO
Initial Plasma Volumt I ml )
60
70
FIGURE 2. Inhibition of JH-ouabain binding and Na, K acti\xtted adenosine triphosphatase (Na, K-ATPase) activity by
plasma extracts separated from Sabra hypertensive rats. The
methods for synaptosome and microsome preparations from rat
brain and measurements of JH-ouabain binding and Na, KATPase have been described previously.21 22 The control binding in the absence of extract was 12.5 pmol JH-ouabain per
milligram of protein and was taken as 100%. Closed circles
represent the inhibition of binding by partially purified ouabainlike compound. The ouabainlike compound obtained from
pooled plasma was diluted, and the equivalent initial plasma
\xtlue was calculated. Control ATPase activities were 45.0,
19.3, and25.7 p.mol PJmg proteinlhrfor total, Na, K-dependent, and Mg-dependent enzymes respectively. Open circles
show the inhibition ofNa, K-ATPase at different concentrations
of plasma.
FIGURE 3. The positive inotropic effect of the partially purified material on rat atrium. The ouabainlike compound was
extracted from pooled rat plasma and partially purified as described in Methods. Top tracing: Tension traces are shown
from left to right in a control solution and after exposure to 0.5
\iM of the compound for 9 minutes and 13 minutes, and after 20
minutes of washout with control solution. Bottom tracing:
Superposition of tension traces in control and after 13 minutes
of exposure to the ouabainlike compound.
ed to inhibit 3H-ouabain binding.8 Furthermore, at
concentrations that caused 50% inhibition of Na,
K-ATPase activity, no change in Mg-ATPase activity
was detected.
Since cardiac glycosides increase the contractile
force of heart muscle, demonstration of such an effect
by the endogenous compound might provide further
indication of its ouabainlike activity. As can be seen in
Figure 3, the addition of the partially purified material
to a preparation of rat atrial muscle resulted in a
marked increase (30%) in the contractile force that was
completely reversible on washout. In two other experiments, 20 ixM ouabain resulted in an increase in the
force of contraction by 70% and 100% (data not
shown). The results of this set of experiments are consistent with the contention that the endogenous compound is ouabainlike in nature.
In a second set of experiments, the compound partially purified from plasma of several groups of SBH
and SBN (with the use of the chromatographic system
described in Methods) was compared for its ability to
inhibit JH-ouabain binding. To avoid possible bias the
tests were carried out on coded samples. Each specimen was tested at least twice and was found to vary by
no more than 5%. The K level in all the tested fractions
was negligible and therefore could not have affected
ouabain binding.
As shown in Table 1, the plasma levels of the J Houabain displacing material were extremely variable,
averaging 2543 zh 1140 and 698 ± 199 pmol/ml of
plasma (mean ± SEM) for SBN and SBH respectively.
The ability of the compound to inhibit Na, K-ATPase
732
HYPERTENSION
TABLE I. JH-Ouabain Displacing Material in Plasma of Sabra
Normotensive (SBN) and Hypertensive (SBH) Rats
3
Experiment
1
2
3
4
5
6
7
8
9
10
11
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Mean±sEM
H-ouabain displacing material
(pmol/ml plasma)
SBN
SBH
287
6375
282
134
3590
1310
2151
1670
207
1700
20
565
30
533
250
175
9660
1122
—
—
439
70
2543±1140
698±199
The concentration of the compound in pooled plasma of 3 to 10
rats was estimated by testing the ability of extracted and separated
material to inhibit 3H-ouabain binding to rat brain synaptosomes.
Each experiment represents a different pool. The difference in the
concentration of the compound between SBH and SBN was not
statistically significant.
activity was not assessed in this study since, as previously stated, large volumes of plasma are needed for
this assay.
Discussion
This study provides evidence for the presence of an
endogenous inhibitor of Na, K-ATPase in the plasma
of hypertension prone Sabra rats. The compound also
appears to be capable of inhibiting 3H-ouabain binding
to its specific receptors on the plasma membrane.
These findings indicate that the inhibition of the enzyme activity by the endogenous compound is due to
its interaction with the digitalis binding sites of Na, KATPase.
In addition, our results demonstrate that the endogenous plasma compound, like digitalis, increases the
contractile force of heart muscle. The increase in the
positive inotropic effect amounted to 30% and was
observed at a concentration of 0.5 /xM ouabain equivalents. The concentration of the OLC was approximated from the binding experiments under the assumption
that the endogenous material and ouabain have the
same affinity for their common binding site. Evidently, this is a crude estimation, and the actual plasma
concentration of the material remains to be determined. Moreover, it is premature to compare the relative potencies of OLC to cardiac glycosides in their
ionotropic effect. Atrial contractility may be influenced by other factors including local release of endogenous catecholamines and alterations in calcium metabolism. However, the observation that the same
material that inhibited [3H]ouabain binding and Na, KATPase activity also increased the contractile force of
VOL 7, No 5, SEPTEMBER-OCTOBER
1985
the myocardium strongly supports the contention that
the endogenous compound is ouabainlike in nature.
The finding of an increase in the contractile force of
heart muscle by material isolated from rat blood is in
accordance with our recent report of a similar effect of
compounds partially purified from toad skin and sheep
brain.30 Due to the small amount of OLC obtained from
the rat plasma, we were unable to characterize the
inotropic effect with respect to its dose dependence,
sensitivity to a-adrenergic and /3-adrenergic blockers,
extracellular ion concentration, and rate of stimulation. It is noteworthy, however, that the compounds
partially purified from toad skin,30 human cerebrospinal fluid,31 and sheep brain30 also increase the contractile force of heart muscle. The inotropic effect of the
toad skin OLC was characterized in detail and was
found to be almost identical to that obtained with cardiac glycosides (unpublished observation). Since all the
compounds in these studies were separated using almost identical methods, the similarity of the toad skin
compound to digitalis may also apply to the OLCs
obtained from other sources. The presence of such
compounds in mammalian blood raises the possibility
that they might participate in the regulation of cardiac
contractility under physiological conditions.
In spite of extensive investigation, the structure of
these compounds is unknown. Partial characterization
of the OLC obtained from different sources14"24 indicates that it is of low molecular weight ( < 1000 daltons). The elucidation of its structure is clearly an
important challenge, and studies are in progress to
achieve this goal.
In the present study, while the difference in blood
pressure between SBH and SBN was highly significant, no difference in OLC levels was detected. The
moderate difference in systolic blood pressure reported
here was invariably associated with a measurable degree of cardiac hypertrophy in the SBH. For example,
the heart weight of adult rats corrected for 100 g body
weight averaged 268 ± 8 mg in SBH compared with
247 ± 4 m g i n S B N ( / > < 0 . 0 1 ) . Previous studies have
shown that the two strains are dissimilar in several
other physiological parameters. For example, compared with SBN, the SBH are heavier, have lower fluid
intake and urinary output, and have markedly elevated
urine osmolality.28 These important differences have
no apparent effect on the activity of the renal Na, KATPase, which is comparable in isolated nephron segments.32 The lack of difference in OLC levels (as estimated by inhibition of ouabain binding in rat brain
synaptosomes), in conjunction with results in isolated
nephron, does not suggest a correlation between high
blood pressure and alterations in pump activity in the
SBH. These results are at variance with two recent
studies in humans in which a significant difference
between hypertensive and normotensive subjects was
observed.17-l8
The large variation of OLC level in our rats may
reflect individual variability of undefined physiological parameters that affect the metabolism of the compound. Alternatively, it may be due to technical prob-
OUABAINLIKE COMPOUND IN HYPERTENSION/L/ctoem et al.
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lems such as variation in the recovery of the
chromatographic system used for the separation of the
compound. Another factor could be the time interval
from the extraction of the material until its determination, which ranged from 1 week up to 3 months in this
study. Since the long-term stability of the compound is
still unknown, the length of storage may have affected
the results.
Finally, since the OLC was determined on deproteinized plasmas, the effect of denaturation of plasma
proteins should be considered. It is possible that a large
portion of the compound, like steroid hormones, is
bound to plasma proteins. Assuming that only the free
fraction is physiologically active and relevant to hypertension, a putative strain difference would be obliterated by the current method in which the total plasma
OLC values were determined.
20.
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D Lichtstein, D Minc, Y Shimoni, J Deutsch, J Mekler and D Ben-Ishay
Hypertension. 1985;7:729-733
doi: 10.1161/01.HYP.7.5.729
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