[3H]Ouabain Binding of Red Blood Cells in
Whites and Blacks
LASZLO HOPP, NORMAN LASKER, SUSAN GROSSMAN,
RONI BAMFORTH, AND ABRAHAM AVIV
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SUMMARY In a previous study, we demonstrated that the red blood cell Na+ concentration and
Na + ,K + -ATPase activity are sex-dependent and race-dependent: a higher intracellular Na+ concentration in blacks and men was associated with a lower Na + ,K + -ATPase activity. To examine whether
the low Na + ,K + -ATPase activity is due to a decreased number of enzyme units, altered structure of
the enzyme, or the presence of an endogenous digoxinlike substance, ouabain binding studies were
performed on the same subject group. The measurements included displacement of [-'HJouabain from
its specific binding sites by unlabeled ouabain or potassium. The results demonstrate that groups with
lower enzyme activity manifest lower numbers of total specific ouabain binding sites on the surface of
the red blood cell (mean ± SD: blacks, 654 ± 24.4; whites, 806 ± 18.3; women, 806 ± 26.9; men,
728 ± 21.2). Other kinetic parameters of [3H]ouabain displacement appear to be the same among the
groups. The respective red blood cell Na+ and K + concentrations were negatively and positively
correlated with the number of ouabain binding sites. Our findings suggest that the lower activity of red
blood cell Na + ,K + -ATPase in blacks and men is a function of a lower number of Na + -K + pump units.
The results also indicate that sex and race should be considered when red blood cell ouabain binding is
examined. (Hypertension 8: 1050-1057, 1986)
KEY WORDS
• sodium
• potassium
• Na + ,K + -ATPase
I
• blacks • whites
men have a higher intracellular Na + concentration than
that of RBCs of whites and women. A lower Na + ,K + ATPase activity appeared to be at least partly responsible for the differences. The lower activity of the enzyme may reflect intrinsic, qualitative or quantitative
differences in the Na + ,K + -ATPase system or the presence of plasma modulators. Analysis of ouabain binding to its specific cellular receptors can be used to
determine the number of Na + ,K + -ATPase units. It
also provides information about differences in enzyme
structure and the presence of certain humoral modulators. The main purpose of the present investigation was
to delineate, by analysis of ouabain binding, the underlying mechanism for the lower Na + ,K + -ATPase activity in RBCs of blacks and men as compared with
whites and women.
NTRACELLULAR Na + appears to be a regulatory factor in the contraction of vascular smooth
muscle cells.1"3 Despite some controversy (for
review, see Reference 4), a substantial number of reports5"10 have demonstrated higher intracellular Na +
levels in human essential hypertension and genetic hypertension in rats (for review, see Reference 11). Normotensive offspring of patients with essential hypertension, a group predisposed to high blood pressure
later in life, also manifest a higher Na + concentration
in their red and white blood cells.10-12~14 Blacks and
men have a higher incidence of essential hypertension
than whites and women." 116 In a previous study,17 we
demonstrated that red blood cells (RBCs) of blacks and
From the Hypertension Research Unit and the Division of Nephrology, New Jersey Medical School, Newark, New Jersey.
Supported by grants from the National Heart, Lung, and Blood
Institute (R23HL29221) and the Foundation of the University of
Medicine and Dentistry of New Jersey. Dr. Hopp is a recipient of a
research fellowship from the New Jersey Affiliate of the American
Heart Association.
Address for reprints: Abraham Aviv, M.D., Director, Hypertension Research Unit (Room F578-MSB), New Jersey Medical
School, 100 Bergen Street, Newark, NJ 07103.
Received July 7, 1985; accepted June 11, 1986.
Subjects and Methods
This study was initiated 6 months after the completion of an investigation devoted to studying racial and
sex-related differences in RBC Na + ,K + -ATPase. 17
The subjects recruited were the same as those in our
previous study with the exception of one black woman
1050
RACE- AND SEX-DEPENDENT [3H]OUABAIN BINDING/Hopp et al.
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and one black man who could not be recalled. The
remaining 8 black women, 8 black men, 9 white women, and 10 white men were members of the faculty,
student body, and resident and nursing staffs of the
University of Medicine and Dentistry of New Jersey.
The subjects had no history of hypertension, renal disease, hemolytic or sickle cell anemia, or thyroid or
neurological disease. No dietary restrictions were applied. No significant weight difference or changes in
the subjects' dietary habits were noted as compared
with the initial study." The subjects were not taking
diuretics, antihypertensive or thyroid medications,
oral contraceptives, or estrogens. Pregnancy, obesity
(Quetelet index [lb/in2 X 100] greater than 5), 18 systolic blood pressure (BP) greater than 140 mm Hg, or
diastolic BP exceeding 90 mm Hg excluded potential
subjects from the study. Subjects' BP was the average
taken in both arms in a sitting position, using the Korotkoff first and fifth components.
Blood was drawn the morning after an overnight
fast, while the subjects were in a sitting position after
they had ambulated for 1 hour. For plasma renin activity (PRA) assay, the blood was taken into ethylenediaminetetraacetic acid, while heparin (20 U/ml) was the
anticoagulant for all the other determinations. Measurements of RBC Na + and K + were performed as
previously described.17 The Na + and K + concentrations were determined by flame photometry. Creatinine was determined by a modified picrate method,19
and PRA was measured with the Squibb radioimmunoassay kit (Princeton, NJ, USA).
The ouabain binding studies were conducted as follows. First, 8 ml of blood was centrifuged at 270 g for
10 minutes, and the platelet-rich plasma was aspirated.
The RBC pellet was suspended in a 10 mM Na + phosphate buffer solution (1 mM MgCl2, 150 mM NaCl,
pH 7.42) and centrifuged at 1100^ for 10 minutes. The
supernatant was discarded, and the washing procedure
was repeated two more times. The cells were resuspended in the Na + phosphate buffer solution at a 20 to
25% hematocrit. Aliquots of 0.5 ml of this cell suspension were added to tubes that contained 8.34 X 10~8 M
[3H]ouabain (27-33 Ci/mmol; Amersham, Arlington
Heights, IL, USA) and various concentrations of unlabeled ouabain or K + in 0.5 ml of buffer solution. In
preliminary studies, it was demonstrated that the
steady state for ouabain binding was achieved within
60 minutes of incubation at 37 °C. In subsequent experiments, the test tubes were incubated with gentle
agitation for 1 hour in a water bath. At the end of this
period, 100-pil aliquots of the cell suspension were
removed and added to 2 ml of ice-cold phosphate buffer solution. Cells were centrifuged at 3000 g for 2
minutes, and the supernatant was discarded. Two additional washings followed in 2 ml of buffer solution (the
third washing solution was free of any radioactivity),
and 0.2 ml of 10% perchloric acid was added to the cell
pellet to extract the bound, labeled ouabain. Following
centrifugation, the supernatant was placed into 10-ml
scintillation fluid (Hydroflour). To ensure complete
removal of bound ouabain, the extraction procedure
1051
was repeated two more times. The efficiency of the
radioactive counting was 22%. Cell number was determined by a Coulter counter (Coulter Electronics, Hialeah, FL, USA).
The derivation of the ouabain binding parameters is
described in detail in our previous publication.20 The
binding parameters depend on the radiochemical
specifications of the labeled ligand (specific activity
and radioactive concentration). We, therefore,
checked the accuracy of the manufacturer's specifications. Briefly, the displacement of the labeled ouabain
from its specific binding sites by unlabeled ouabain
can be expressed as:
J_
B
fl+ L flm X L
xc
(1)
where B = specific ouabain, Bm = maximum specific
ouabain binding, Ka = equilibrium dissociation constant for ouabain binding, L = concentration of the
labeled ouabain (kept constant), and c = concentration
of the unlabeled ouabain (variable). Therefore:
fl
=
(2)
bXL
(3)
L
where a = the y axis intercept of the straight line that is
described by Equation 1 and b = the slope of the
straight line. Two straight lines can be obtained by
keeping L constant at two different concentrations and
by varying c. The x coordinate of the intercept of the
two straight lines with each other corresponds to (-)^ d .
This KA value is independent of the manufacturer's
specifications, as is the alb ratio in Equation 3. Thus,
by substituting the real KA value in Equation 3, the real
L value can be calculated. We checked two different
batches of the Amersham [3H]ouabain preparation
(Batches 20 and 21) and found L values 80 and 51%
higher than those calculated on the basis of the manufacturer's specifications (L = radioactive concentration/specific activity). The two curves, the intercept of
which was used for testing Batch 21 (specific activity = 27 Ci/mmol), are presented in Figure 1. In all
further calculations, we used the corrected L values.
Discrepancies between the concentration of labeled
ouabain and that claimed by the supplier have also
been reported by Akera and Cheng.21
Statistical evaluations consisted of weighted linear
regression analysis of the pooled, unlabeled ouabain
displacement curves. Two-parameter nonlinear regression analysis was used for the K+ displacement curves:
(4)
KA\ + —
+L
where i = K + concentration, Kt = apparent equilibrium dissociation constant for K + , and n = Hill coefficient analogue. The values of Bm and Ka were obtained
from the unlabeled ouabain displacement studies and
substituted into Equation 4. One-way analysis of vari-
1052
HYPERTENSION
FIGURE 1. Dixon transformation of [3H]ouabain displacement from human red blood
cells by unlabeled ouabain. Each point represents the mean of two observations.
7/B = reciprocal of specific [3H]ouabain binding. [3H]-ouabain concentrations (based
on specifications by the manufacturer):
L, = 3.6x 10-8M;L2 =
i.2xl0-?M.The
real Kd derived from the intercept of the two
straight lines is 2.97 x 10'8 M. The Kd calculated from the straight line ofL, according to
Equation 3 in Methods is 4.57 x 10'' M,
while the Kd calculated from the straight line of
L? is 1.Ox 10~7 M. The L values calculated
from the real YLd using Equation 3 in Methods
are5.3 x 10'8MforL,
and 1.9 x 10~7Mfor
VOL 8, No 11, NOVEMBER
1986
280
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O.I
0.2
0.3
0.4
0.5
0.6
0.7
0.8
UNLABELLEO OUflBAIN (lO^M)
ance was used for comparisons between the four basic
groups (black women and men, white women and
men); when appropriate, this was complemented by
Duncan's test. Interaction tests were performed to examine sex and race differences. Calculation of correlation coefficients also was performed. To obtain individual Bm values for the correlations, simple linear
regression analysis was employed for each subject.
The means of the parameter obtained in this way were
not significantly different from those derived from
weighted linear regression analysis of the pooled data.
The calculations were performed on an IBM 3033-U
computer (Boca Raton, FL, USA) using the Statistical
Analysis System program library.22 Data are presented
as means ± SD.
TABLE 1.
Results
Age, systolic and diastolic BP values, Quetelet index, and plasma Na + , K + , and creatinine concentrations are presented in Table 1. The white female group
manifested a relatively low systolic and diastolic BP.
Plasma creatinine concentration, as expected, was
lower in women than in men. The RBC Na+ and K+
concentrations and PRA values are presented in Table
2. Duncan's test revealed that black men had the highest RBC Na+ concentration and Na+/K+ ratio. The
interaction test disclosed strong race and sex interrelations with the RBC Na+ concentration and the RBC
Na+/K+ ratio. The latter values are not shown in Table
2{p = 0.016 and p = 0.032 for race and sex, respectively). Blacks and men exhibited higher values for
General Clinical and Laboratory Data
Plasma
DBP
(mm Hg)
Quetelet index
(lb/in2 x 100)
(mEq/L)
[K + ]
(mEq/L)
Creatinine
(mg/dl)
[Na + ]
Age (yr)
SBP
(mm Hg)
Women
32.6±7.1
113.0 + 6.0
74.0±5.8
3.29 ±0.49
139.8± 1.4
4.0±0.2
0.95 ±0.12
Men
28.1 +; 1.4
118.6 + 9.9
78.5±5.8
3.42 + 0.39
141.1 ± 2 . 2
4.2±0.4
1.08±0.16
Women
31.0±3.4
108.9 + 7.8*t
70.1 ±3.5t
3.17 + 0.25
140.6+ 1.5
4.2±0.3
0.83+0.15
Men
29.6±3.6
120.1 ±8.7
75.7±4.9
3.41 ±0.30
140.6±2.0
4.2±0.5
1.05 ±0.12*
All blacks
30.3 ±5.5
115.8 + 8.4
76.3 + 6.0
3.35+0.43
140.4 ± 1 . 9
4.1±0.3
1.02 ±0.16
All whites
30.3 + 3.5
114.8 + 9.9
73.4 + 4.8
3.30 + 0.30
140.6+ 1.7
4.2±0.4
0.95 ±0.17
All women
31.8 + 5.4
110.8±7.I
72.4±4.8
3.23±0.38
140.1 ± 1 . 5
4.1 ± 0 . 2
0.88±0.14
All men
28.9±2.9
119.4±9.0§
76.9 ± 5.3||
3.41 ±0.33
140.8 + 2.0
4.2±0.4
1.06 ±0.14^
Group
Black
White
Values are means ± SD. SBP = systolic blood pressure; DBP = diastolic blood pressure.
*p < 0.05 (Duncan's test) vs white men, tvs black men, tvs white women.
§p = 0.005, ||p = 0.01, Ijp = 0.001 (interaction test), vs all women.
RACE- AND SEX-DEPENDENT [3H]OUABAIN BINDING/Hopp et al.
TABLE 3. Parameters of [3H]Ouabain Displacement by Unlabeled Ouabain (B m , Kj) andK+ (K,, n) from Red Blood Cells
TABLE 2. Blood Chemistry Values
RBC
[Na + ]
(mEq/L)
Group
Black
Women
7.45 ±1.78
9.76 ±2.59*
Men
White
Women
1053
[K + ]
(mEq/L)
88.2±9.3
88.2±7.0
PRA
(ng ANG I/ml/hr)
Group
Black
Women
1.59 ±1.57
2.55 ±1.57
Men
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2.79±1.74
91.2±17.1
Men
3.37±1.58
93.4±5.6
All blacks
2.07 ±1.94
88.2±7.9
8.6O±2.6t
All whites
7.07 ±1.49
3.10±1.64*
92.3±12.1
All women
6.9O±1.58
89.8±13.6
2.23 ±1.73
All men
8.59 + 2.255
91.1+6.6
3.00+1.89
Values are means ± SD. RBC = red blood cell; PRA = plasma
renin activity; ANG I = angiotensin I.
*p < 0.05 (Duncan's test) vs black women, white women, and
white men.
tp = 0.016 vs all whites; %p = 0.10 vs all blacks; §p = 0.007 vs
all women.
6.42 ±1.29
7.66±1.48
these parameters than whites and women. The PRA
showed a similar pattern to that observed in our previous study,17 with whites demonstrating a mean value
50% higher than that in blacks. Because of the large
scatter of the data, however, only very weak racerelated interactions could be demonstrated.
The displacement of [5H]ouabain from its specific
binding sites by unlabeled ouabain is presented in Figure 2. The inset depicts the Dixon transformation of
(I/cell)
ATd(nM)
Jf,(raM)
693 ±30.8* 14.4±6.0 2.18 + 0.34
582 + 32.It 17.3 + 5.7 2.75 ±0.43
n
1.51 ±0.17
1.57±0.19
White
Women
25.3±4.3 2.81 ±0.32 1.49 ±0.14
877 ± 34$
761 ±18.9 16.8±3.8 2.06 ±0.24 1.40±0.11
All blacks 654±24.4§ 15.3 + 5.1 2.13±0.24 1.46±0.11
All whites 8O6±18.3 20.0±3.6 2.39±0.20 1.45 ±0.09
All women 806 ± 26.9|| 24.2±5.0 2.95±0.27 1.54±0.12
728±21.2 20.7 ±4.4 2.29±0.22 1.39±0.10
All men
Values are means ± SD. Mean values for the last four groups (all
blacks, all whites, all women, all men) were obtained by computing
the pooled data. The p values were obtained using Student's t test.
Bm = maximum specific ouabain binding; KA = equilibrium
dissociation constant for ouabain binding; Kt = apparent equilibrium dissociation constant for K + ; n = Hill coefficient analogue.
*p < 0.05 vs black men and p < 0.001 vs white women.
tp < 0.001 vs white men; tp < 0.01 vs white men; §p < 0.001
vs all whites; \\p < 0.005 vs all men.
Men
the data, the weighted linear regressions of which were
used to compute the Bm and Kd values (Table 3). The
number of ouabain binding sites on the surface of the
RBC was significantly lower in blacks than in whites
and in men than in women. The Kd values were essentially the same for all groups. The Bm values were
correlated with the results of Na + ,K + -ATPase activity
900
800
- 700
S 600
I 500
cc
<3 Af.r
o
X
TJ
300
« 200
COi
0.06
0 '0
0 2C
0 4&
U N L A B E l l E D OUABAIN (IO" 6 M)
FIGURE 2. Specific [3HJouabain binding to human red blood cells as a function ofunlabeled ouabain concentration in black women (a), black men (•), white women (O), and white men (n). Vertical lines represent SD. Dixon
transformation of the data is depicted in the inset. 1/B = reciprocal of specific [3HJouabain binding in black
women (•— • —), black men (•— • —), white women (o—j, and white men (O
).
1054
HYPERTENSION
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at 6 and 10 mEq/L K + as well as at 20 and 100 mEq/L
Na + that were reported in our previous communication.17 The activity of RBC Na + , K + -ATPase at the
two indicated K + concentrations represents the maximal initial reaction velocity (V m J. The Na + ,K + ATPase activity at the lower Na + concentration was
the closest to the Vma, whereas the higher Na + concentration incorporated the inhibitory effect of the ion on
the enzyme. 17 The Na + ,K + -ATPase activity at the two
K + concentrations demonstrated a significant correlation with the Bm values (r = 0.451, p < 0 . 0 1 and
r = 0.430, p < 0.01 at 6 and 10 mEq/L K + , respectively). The ATPase activity at 20 mEq/L Na + showed a
significant correlation with the Bm (r = 0.509, p<
0.01). The correlation was much weaker when the
Na + ,K + -ATPase activity at 100 mEq/L Na + was compared with the Bm (r = 0.349, p < 0 . 0 5 ) . This finding
probably relates to the inhibitory effect of higher Na +
concentration on the enzyme, which may mask correlations between the Bm and the Vmm values.
The results of the K + displacement studies are presented in Figure 3. The figure inset demonstrates the
computer-predicted curves according to Equation 4.
The computed values of the apparent dissociation constant (K) and Hill-type coefficient (n) in Table 3 show
that the differences in the displacement curves among
the groups are primarily derived from the Bm values
and not from the Kt or n values.
Several correlation coefficients believed to be relevant to regulation of intracellular cation concentration
and BP are listed in Table 4. Most striking was the
overall negative correlation between the number of
ouabain binding sites (Na + -K + pump units) and either
VOL 8, No
11, NOVEMBER
1986
the intracellular Na + concentration (see Figure 4) or
the RBC Na + /K + ratio. Significant correlations between these parameters were also noted for several
groups. The slight positive correlations of the Bm with
RBC K + concentration is consistent with these findings. In the white group, renin was negatively related
to the Bm and, as a complementary finding, positively
related to the RBC Na + level and Na + /K + ratio. The
Quetelet index had a negative relation to the Bm. Supporting this finding was a positive correlation between
the Quetelet index and RBC Na + /K + ratio in several
groups. Age showed a negative effect on the RBC K +
concentration, particularly in women.
Discussion
That intracellular Na + has a role in the contractile
process of vascular smooth muscle cells has been suggested for many years.'• 2 An elevation of the intracellular Na + level favors the contraction of blood vessels
and results in vasoconstriction. Through a membrane
Na + -Ca 2+ exchange or by the mobilization of Ca 2+
from intracellular storage sites, 1 ' 3 Na + can regulate the
amount of cellular ionized Ca 2+ . Intracellular Na + also
may enhance the sensitivity of the contractile apparatus for Ca 2+ . 2 Our finding of a higher RBC Na +
concentration in blacks and men as compared with
whites and women is consistent with previous
studies. 1417 "" 25 If the same phenomenon exists in
vascular tissues, it may predispose blacks and men to
hypertension.
In a previous work, we showed that the higher RBC
Na+ concentration in blacks and men is, at least partly,
related to lower Na + ,K + -ATPase activity.17 The pres-
S> 200
too -
(mM)
FIGURE 3. Specific [3H]ouabain binding to human red blood cells as a function of extracellular K+ concentration. See Figure 2 for explanation of symbols. Inset presents the computer-predicted specific binding curves
according to Equation 4 in Methods for black women (— —), black men (
) , white women (
) , and white
men (
).
RACE- AND SEX-DEPENDENT [3H]OUABAIN BWDING/Hopp et al.
TABLE 4.
16 r
Correlation Coefficients (r)
Parameter
Group
r
Bm vs RBC Na +
Overall
-0.657
P
0.0001
All blacks
-0.514
0.0420
Bm vs RBC Na+/K +
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flm vs RBC K +
All whites
-0.729
0.0004
All women
-0.621
0.0078
All men
-0.620
0.0061
White men
-0.720
0.0190
White women
-0.645
0.0610
Overall
-0.645
0.0001
All blacks
-0.496
0.0510
All whites
-0.706
0.0007
All women
-0.596
0.0120
AJ1 men
-0.630
0.0050
White men
-0.706
0.0220
White women
-0.675
0.0460
Overall
0.324
0.0580
All men
0.587
0.0104
Bm vs renin
All whites
-0.677
0.0015
White women
-0.807
0.0085
Renin vs RBC Na +
All whites
0.579
0.0094
White men
0.658
0.0390
Renin vs RBC Na+/K + All whites
0.608
0.0058
White men
Bm vs Quetelet index
Quetelet index vs
RBC Na+/K +
Age vs RBC K +
0.711
0.0210
Overall
-0.325
0.0570
All whites
-0.477
0.0390
All whites
0.441
0.0590
All women
0.504
0.0390
White women
0.685
0 0410
Overall
-0.412
0.0140
All blacks
-0.558
0.0250
All women
-0.625
0.0072
Black women
-0.828
0.0112
-0.700
0.0360
White women
1055
Bm = maximum specific ouabain binding; RBC = red blood
cell.
ent results substantiate these findings by the demonstration of a lower number of ouabain binding sites in
groups manifesting lower enzyme activity. The Bm values in the present study correlate well with the
Na + ,K + -ATPase activity measured six months earlier
in RBCs of the same subjects. In this regard, it has
been shown previously that the number of ouabain
binding sites is quite stable.26 A relative turnover rate
for adenosine 5'-triphosphate hydrolysis by the enzyme can be calculated using Vmtx values from the
previous study and the Bm values of the present work.
These relative turnover rates are quite similar. For
instance, when the V^ for K + activation is used, the
relative turnover rates (cell x mole of inorganic phosphate per hour per mg protein per Na + -K + pump unit)
are 0.278 ±0.015 for blacks, 0.269 ±0.017 for
3 14
E
r-IJH-0.0063>
r - -0.657
400
600
800 1000 1200 MOO 1600 1800
NUMBER Of OUABAIN BINDING SITES (I/cellI
FIGURE 4. Correlation between the number of ouabain binding sites and intracellular Na+ concentration in black women
(*), black men (*), white women (o), and white men (O).
whites, 0.275 ± 0.016 for women, and 0.243 ± 0.013
for men. These findings suggest that a lower number of
Na + ,K + -ATPase units of RBCs is responsible for the
decreased enzyme activity in blacks and men as compared with whites and women.
Differences in the enzyme structure among the
groups could also account for the unequal activity of
Na + ,K + -ATPase. From a functional perspective, this
explanation is not supported by the comparative equalities of the relative turnover rates. In addition, the
similarities in the ouabain and K+ binding parameters
{KA, Kt, ri) provide further, though not compelling,
support to the notion that the various enzymatic activities among the four groups are not the result of different enzymatic structures.
Substantial effort has been made to relate the lower
activity of Na + ,K + -ATPase to circulating inhibitors.27"30 Several reports have indicated that at least one
of these inhibitors is a digoxinlike substance and that
its level is elevated in extracellular volume expansion.28"30 Blacks appear to have several characteristics
that can be associated with expanded extracellular
fluid volume and increased levels of a circulating
Na + ,K + -ATPase inhibitor.31 Strong binding of such a
factor could result in reduced Na + ,K + -ATPase activity
even when measured in vitro. This concept is not supported by the present study, as no between-group differences in the Kd values were demonstrated. Such
differences would be expected if various concentrations of an endogenous digitalislike factor compete
with the exogenous ouabain for its binding sites. Our
experiments cannot entirely exclude the existence of
an endogenous digitalislike factor. However, the binding affinity of such a factor should be of a magnitude
that eludes the detection of its dissociation within the
1-hour incubation period. Such binding should be considered irreversible and, under such circumstances, the
physiological role of an endogenous digitalislike factor
would be highly questionable.
The numbers of ouabain binding sites in this investi-
1056
HYPERTENSION
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gation correspond to those in several previous studies,32"34 but they are substantially higher than those
reported in others.35"38 The good correlations among
the calculated number of ouabain binding sites, the
previously measured Na+,K+-ATPase activity,17 and
the intracellular Na+ concentration or Na+/K+ ratio
indicate that the majority of these sites are functional
Na+-K+ pump units. The observation of less ouabain
binding in some previous studies could be due to an
inadequate extraction technique of the bound [3H]ouabain.32 Our observation of 50 to 80% higher labeled
ouabain concentration than that stated by the supplier
suggests another possibility for differences among laboratories. These results also emphasize the importance
of considering sex and race when comparing the number of RBC ouabain binding sites.
The correlation of the number of RBC Na+-K+
pump units with several other parameters may support
mechanistic links between hypertension and cellular
Na+ regulation. Potassium has been suggested to have
a protective role against elevated BP.39"11 In view of the
age-dependent rise in BP, it is of interest that the RBC
K+ concentration in our subjects decreased with age.
This finding is in agreement with a previous report on
total body K+ loss with aging.42 The mechanism of K+
protection against BP elevation is not clear at present;
natriuresis, decreased sympathetic activity, and inhibition of the renin-angiotensin system are among the
many causes that have been suggested.39"43
The negative correlation between the number of
ouabain binding sites and PRA in the white group is a
new finding, the significance of which is not clear at
this time. Based on recent reports pertaining to the
cellular uptake of renin and the presence of angiotensin
II receptors in intracellular organelles, such as nuclei,44
a question can be raised as to the possible role of
the renin-angiotensin system in the regulation of
Na+,K+-ATPase synthesis.
Low Na+,K+-ATPase activity has been related
to the pathogenesis of obesity,3745^7 although this
hypothesis has been challenged.48-49 The slightly negative correlation between the maximal ouabain binding
and the Quetelet index in the whole sample population
and in the white group agrees with findings obtained in
normal and overweight subjects.374549
In conclusion, our main findings demonstrate that a
lower number of Na+-K+ pump units on the surface of
the RBC is the reason for the lower Na+,K+-ATPase
activity in blacks and men as compared with whites
and women. These differences may contribute to the
higher intracellular Na+ concentration in the former
two groups. Such a phenomenon, if it occurs in vascular smooth muscle cells, may predispose to vasoconstriction and elevated BP. Plasma renin activity, age,
and Quetelet index appear to be related to RBC Na+K+ homeostasis. These factors should be evaluated in
relation to sex and race. Our results indicate the need
for more extensive, wide-scale epidemiological studies of intracellular Na+ homeostasis that would include
both normotensive and hypertensive groups.
VOL 8, No 11, NOVEMBER 1986
Acknowledgments
We thank Martin Feureman for the statistical analyses, Patricia
Mayo and Carol Sulinski for their technical help, and Eileen
Holmes for her secretarial skills.
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Hypertension. 1986;8:1050-1057
doi: 10.1161/01.HYP.8.11.1050
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