Red Cell Lithium-Sodium Countertransport and
Sodium-Potassium Cotransport in Patients with
Essential Hypertension
NORMA C. ADRAGNA, P H . D . , MITZY L. CANESSA, P H . D . , HAROLD SOLOMON,
EVE SLATER, M.D.,
AND DANIEL C. TOSTESON,
M.D.,
M.D.
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SUMMARY Alterations in sodium countertransport and cotransport have been reported in red
cells of patients with essential hypertension. We have investigated the relationship between these two
systems by performing simultaneous measurements of the maximal rates of lithium-sodium (Li,-Na0)
countertransport and outward sodium-potassium (Na-K) cotransport in red cells from normotensive
and hypertensive subjects. Lii-Nao countertransport was assayed by measuring the Nao-stimulated Li
efflux from cells loaded to contain 10 mmoles Li per liter of cells by incubation in isotonic LiCI. Na-K
cotransport was assayed by measuring the furosemide-sensitive component of Na and K efflux into
magnesium-sucrose medium from cells loaded by the p-chloromercuribenzene sulfonic acid (PCMBS)
procedure to obtain 50 mmoles of both ions per liter of cells. The mean values (± SE) for 16
normotensives and 22 hypertensives were (mmole/liter cells x hour): Na countertransport = 0.29 ±
0.02 vs 0.51 ± 0.03 (p < 0.001); Na cotransport = 0.30 ± 0.03 vs 0.51 ± 0.05 (p < 0.005); and K
cotransport = 0.34 ± 0.03 vs 0.60 ± 0.04 (p < 0.005). Li,-Na0 countertransport correlated significantly with Na cotransport (r = 0.50, n = 38, p < 0.005) and K cotransport (r = 0.57, p < 0.005).
This observation suggests that both transport systems are somehow regulated to be more active in this
group of hypertensive patients. The increased cotransport in hypertensive patients is also apparent
from two other measurements of Na and K fluxes in red cells suspended in Na medium. First, the
furosemide-sensitive net Na efflux into Na medium was (mean ± SE) 0.25 ± 0.05 in 10 normotensive
subjects and 0.50 ± 0.09 in 12 hypertensive patients. Second, the furosemide-sensitive net K efflux
into Na medium was (mean ± SE) 0.25 ± 0.04 in 13 normotensive subjects and 0.43 ± 0.04 in 16
hypertensive patients (p < 0.005). We conclude that mean values for both Na countertransport and
Na-K cotransport are significantly higher in the group of hypertensives than in the group of normal
control subjects. (Hypertension 4: 795-804, 1982)
KEY WORDS • cotransport
• countertransport
T
• hypertension
sion.2-3 A reduced net Na extrusion in the presence of
ouabain and a reduction in the maximal rate of sodiumpotassium (Na-K) cotransport system have been reported by Garay et al.4"* in the red cells of hypertensive
patients.
In this paper, we report simultaneous measurements
of the maximal rates of Li,-Na0 countertransport and of
outward Na-K cotransport as well as net movements of
Na and K in Na medium in red cells of normotensive
controls and essential hypertensive patients. We confirm our earlier observation that the maximal rate of
Li,-Nao countertransport is significantly higher in hypertensive patients. Strikingly, we also found an elevated Na-K cotransport in the red cells of patients with
elevated countertransport. Thus, despite the many differences between Li-Na countertransport and Na-K cotransport leading to the inference that they are separate
pathways for Na movement,7 both are somehow regu-
WO types of ouabain-resistant sodium (Na)
transport, Na countertransport, and cotransport have been reported to be altered in the red
cells of patients with essential hypertension.'"* The
maximum rate of lithium-sodium (Li,-Nao) countertransport is increased in the red cells of some patients
with established essential hypertension, but not in patients with secondary hypertension.2 Countertransport
was also found elevated in red cells from the firstdegree relatives of patients with essential hypertenFrom the Department of Physiology and Biophysics, Harvard
Medical School, Boston, Massachusetts.
Supported by grants GM-25686 and HL-25064 from the National
Institutes of Health.
Address for reprints: Dr. Norma C. Adragna, Department of
Physiology, Duke University Medical Center, Box 3709, Durham,
North Carolina 27710.
Received November 23, 1981; revision accepted April 26, 1982.
795
796
HYPERTENSION
lated to be more active in the red cells of this group of
patients with essential hypertension than in normal
control subjects. The origin(s) of the differences between our results showing increased cotransport and
the observations of others4"* showing decreased cotransport in the red cells of hypertensive patients is not
clear at this time. Partial results were communicated at
the IV Scientific Meeting of the InterAmerican Society
of Hypertension and at the VIII Scientific Meeting of
the International Society of Hypertension. 89
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Materials and Methods
We studied three groups of subjects, all of whom
were Caucasian. The control group was composed of
16 normotensive subjects (nine women and seven
men) with a diastolic blood pressure under 90 mm Hg
and no personal or family history of hypertension.
They ate a normal diet with salt ad libitum and were
medically normal at the time of the study. A second
group of normotensive subjects (two women and six
men) had a family history of hypertension.
Twenty-two established essential hypertensive patients (nine women and 13 men) had no evidence of
renal or heart failure or severe retinopathy. The duration of known hypertension ranged from 2 months to
10 years. Five hypertensive patients were not on treatment and two were studied after a 48-hour interruption
of treatment; 15 patients were receiving one or another
of the following drugs when the blood was sampled:
chlorthalidone, propranolol, hydrochlorothiazide, methoprolol, Dyazide R, nadolol, and Aldactazide R.
Preparation of Red Cells
Blood was drawn from donors into heparin vacuum
tubes and processed within 1-2 hours. Plasma and
buffy coat were removed by centrifugation for 10 minutes at 6000 X g at 4°C in a Sorvall RC-6B centrifuge
(Dupont-Sorvall Instruments, Newtown, Connecticut). We separated 2 ml of cells for lithium loading,
and washed the remainder 4 times with a washing
solution (WS) containing (mM): 75 MgCl,, 95 sucrose, 10 Tris-MOPS, pH 7.4 at 4°C. Then 2 ml of
packed red cells was separated into two tubes for pchloromercuribenzene sulfonic acid (PCMBS) loading; and the rest of the pellet was washed twice and
suspended at approximately 50% hematocrit in WS.
To measure dry weight in triplicate, as previously described,2 100 (j.\ of packed red cells were used. The
hemoglobin per liter and the cation composition of the
initial cell suspension were determined by analyzing a
1/50 dilution in 0.02% Acationox.
Measurements of the Maximal Activation of Outward
Sodium-Potassium Cotransport
PCMBS Loading Procedure
Two ml of washed red cells were suspended (4%
hematocrit) in a solution containing (mM): 120 NaCl,
30 KC1, 1 MgCL,, 2.5 Na, phosphate buffer (pH 7.2
at 4°C), 1 Tris-EGTA, and 0.02 freshly-prepared
VOL 4, No 6, NOVEMBER-DECEMBER 1982
PCMBS. The cells were incubated in the cold for 20
hours with mild agitation and a change in loading solution after 6 hours. The cells were collected by centrifugation at 6000 x g for 10 minutes, and the supernatant
fluid was removed. Failure to remove all of the loading
solution impaired the recovery of normal cell permeability.
Sealing Step
The cells were resealed by incubation (1 hour at
37°C) in a recovery medium containing (mM): 2 adenine, 3 inosine, 4 cysteine, 10 glucose, 2.5 Na-PO4
buffer (pH 7.2 at 37°C), 5 K.C1, 145 NaCl, and 1 TrisEGTA. Afterward, the cells were collected and
washed 6 times with another washing solution (WSO)
containing (mM): 75 MgCl,, 95 sucrose, 10 TrisMOPS (pH 7.4 at 4°C), and 0.1 ouabain. A cell suspension in WSO (approximately 50% hematocrit) was
prepared for efflux measurements and determinations
of cation and hemoglobin contents. The Na and K.
concentrations of the suspension medium was less
than 5
Cation Efflux Measurement
Furosemide-sensitive Na and K effluxes were measured into Na-free and K-free Mg medium containing
(mM): 75 MgCl2, 85 sucrose, 10 Tris-MOPS (pH 7.4
at 37°C), 10 glucose, and 0.1 ouabain. Furosemide (20
mM) was freshly prepared by titration to pH 7.4 with
Tris base and added to the Mg medium to a final
concentration of 1 mM. Washed cells suspended at
50% hematocrit were diluted with the cold medium to
a final hematocrit of 4-5%. Triplicate tubes containing
2.0 ml of the flux suspension were incubated for 30,
60, and 90 minutes at 37°C. To stop the reaction, tubes
were transferred to 4°C for 1 minute and then centrifuged for 5 minutes at 6000 x g. The supernatant
fluids were transferred with plastic syringes into plastic tubes for cation content analysis.
Na and K concentrations were measured in a Perkin
Elmer atomic absorption spectrophotometer (Model
5000) using standards of Na and K in water (10-200
£iM). The efflux was calculated from the slope of the
line relating the external cation concentration with
time in the nine samples. The slope and its standard
deviation were converted into flux units of mmole/liter
cells x hour using appropriate factors derived from
the hemoglobin and measured hematocrit of the fresh
and loaded cells. The furosemide-sensitive component
was calculated from the difference between the cation
fluxes in the presence and absence of the inhibitor.
Flux measurements in the PCMBS-loaded cells were
not made when the cells were swollen (i.e., when the
water content of the loaded cells was more than 4%
greater than the water content of cells prior to loading.)
Measurements of the Maximal Rate of Li,-Nao
Countertransport
Lithium Loading Procedure
Two ml of washed red cells were incubated (at 20%
hematocrit) for 3 hours at 37°C in a solution contain-
COUNTERTRANSPORT AND COTRANSPORT IN ESSENTIAL HYPERTENSlOWAdragna
ing: 150LiCl, lOTris-MOPS (pH 7.4 at 37°C), and 10
glucose. Li was eliminated by 6 washes in WSO. A
50% cell suspension in WSO was used for measurements of hematocrit, hemoglobin (1/50 dilution), cation composition, and efflux measurements.
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Efflux Measurements
Li efflux was measured in Mg and Na medium (4%
to 5% hematocrit). Na medium contained (mM): 150
Na, 10 glucose, 10 Tris-MOPS (pH 7.4 at 37°C), and
0.1 ouabain. The 50% cell suspension was diluted in
cold (4°C) Mg and Na media to make suspensions with
a hematocrit of 4-5%. Keeping this ratio yields about 4
mM Mg in Na medium. These diluted cell suspensions
were divided into triplicate tubes containing 2.0 ml
flux media, which were then incubated for 30, 60, and
90 minutes at 37°C in a shaker bath. Subsequently, we
followed the procedure above for Na-K cotransport.
The Li concentration in Mg medium was determined
with standards containing 75 mM MgCl2. Li standards
in water were used for Na medium since no interference was detected by our instrument. The Li standard
was prepared by weighing dried LiCl (Mallinckrodt,
Inc., St. Louis, Missouri) and checked with commercial standard (Alfa Division, Ventron Corporation,
Danvers, Massachusetts).
Measurement of Net Cation Movement
Two ml of the PCMBS-loaded cells, prepared for
measurements of outward cotransport as described
above for sodium-potassium cotransport, were incubated at 4% to 5% hematocrit in Na medium in the
presence and absence of furosemide. Quadruplicate
samples were taken after 5 hours of incubation at 37°C.
The cells were washed 3 times in WSO and lysed in
appropriate volumes of Acationox (0.02%; Scientific
Products Corporation, Bedford, Massachusetts) for
hemoglobin and electrolyte measurements.
Reproducibility of the Transport Measurements
The experimental procedure used to determine LiNa countertransport was slightly modified.2 The 50%
cell suspension was added to cold medium, and the
efflux was started by incubation at 37°C. In the former
method, the cell suspension was added to prewarmed
media. No differences were found between the two
procedures.
Table 1 gives the reproducibility of Li,-Nao countertransport, which has a coefficient of variation of 10%.
Na-K cotransport has a larger variation in a given subject (table 2). An increase of 4% or higher in water
content of the PCMBS-loaded cells was associated
with low cotransport. Since we previously showed that
a 4% increase in normal cell volume inhibits the Na-K
cotransport,10 we did not include in this report flux
measurements for swollen cells.
All the solutions used in these experiments were
adjusted to 300 ± 5 mOsM. Concentrations of MgCl2
and sucrose stock solutions were calculated from the
measured osmotic pressure and osmotic coefficient."
Reproducibility of the cotransport measurements was
et al.
797
improved by: 1) the addition of 1 mM EGTA to the
loading and sealing solutions (suggested by Dr. R.P.
Garay, Hopital Necker, Paris, France); 2) recovery of
normal cell volume after PCMBS treatment; 3) control
of osmotic pressure of the solutions; and 4) rigorously
maintaining hematocrit of the efflux media between
4% and 5%.
Chemicals
KC1, NaCl, LiCl, and MgCL, were purchased from
Mallinckrodt, Inc. (St. Louis, Missouri). Tris,
PCMBS, cysteine, adenine, and ouabain were purchased from Sigma Chemical Company (St. Louis,
Missouri). Furosemide was a gift of Hoechst Roussel
Pharmaceuticals, Inc. (Somerville, New Jersey). All
solutions were made in deionized and double-distilled
water.
TABLE 1. Reproducibility of Lithium-Sodium (LirNao)
transport Measurements
Exp.
no.
Sex
Year
10
102
GH
M
1979
1980
66
46
53
75
80
90
NA
F
1979
1980
1980
1980
1980
1981
90
LD
F
1979
1980
89
56
GD Jr.
M
1979
1980
88
KD
F
1979
1980
GD Sr.
M
1979
1980
85
59
Ch 0 Sr.
M
1979
1980
51
76
HS
M
1979
1980
MC
F
1980
1981
1981
1981
1981
1981
1981
1981
1981
AS
F
1981
1981
1981
1981
1981
1981
Donor
57
87
58
126
129
Coef var = coefficient of variation.
Counter-
Na-Stimulated
Li efflux
Coef
(mmole/liter
var
cells x hr)
(%)
0.66
0.67
0.21
0.19
0.20
0.18
0.21
0.19
0.16
0.23
0.41
0.44
0.29
0.23
0.42
0.47
0.58
0.63
0 27
0.27
0.29
0.30
0.38
0.37
0.36
0.33
0.34
0.36
0.32
0.40
0.39
0.35
0.37
0.20
0.35
1
6
25
5
16
8
6
0
9
18
798
HYPERTENSION
TABLE 2. Reproducibility of Sodium-Potassium
Cells in the Presence of EGTA)
Exp.
No.
VOL 4, No 6, NOVEMBER-DECEMBER 1982
(Na-K) Cotransport Measurements (Assay Performed by Loading
Furosemide-Sensitive Efflux
(mmole/liter cells x hr)
Donor
79
139
Sex
MC
175
AK
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75
80
89
134
138
24
NA
AS
M
Date
Sodium
Potassium
Sodium
Potassium
1980
1981
1981
1981
1981
1981
1981
1981
1981
1980
1980
1980
1981
1981
1981
1981
1981
1981
1981
1981
0.29
0.14
0.45
0.27
1.08
0.97
0.82
0.85
0.81
0.26
0.21
0.32
0.31
0.34
0.45
0.43
0.46
0.31
0.39
0.34
0.30
0.27
0.41
0.20
44
30
1.30
1.04
0.67
0.75
0.83
0.35
0.34
0.46
0.31
0.31
0.40
0.25
13
28
26
20
13
28
Results
Lithium-Sodium Countertransport in Red Cells of
Normotensive Subjects and Hypertensive Patients
Table 3 shows the cation and water content of fresh
and Li-loaded cells from normotensive and hypertensive subjects. There was no significant difference between the two groups in red cell ion and water content
before and after Li loading.
Table 4 shows mean values of Li,-Na0 countertransport in red cells of normotensive and hypertensive subjects. The mean value of Li,-Na0 countertransport of
the hypertensives was significantly higher than that of
normotensives. The normotensive group with a family
history of hypertension showed no significant difference in the Na-stimulated Li efflux with respect to the
control group. However, three of these subjects had
TABLE 3.
Coefficient of Variation
0.57
0.40
0.45
0.52
values over 0.4 mmole/liter cells X hour. One essential hypertensive patient with elevated plasma renin
activity had an elevated countertransport. This finding
contrasts with our previous observation of normal
countertransport in five patients with high renin
levels.3
Table 5 shows Li-Na countertransport and diastolic
blood pressure of untreated and treated patients. These
data indicate that Na-stimulated Li efflux is increased
in both subgroups of hypertensive patients.
It has been suggested that hypokalemia produced by
treatment with diuretics alters the rate of Li-Na countertransport in human red cells.12 Accordingly, we report a few observations on the relationship between
plasma potassium levels and Li,-Nao countertransport
in these hypertensive patients. Of 11 treated hyperten-
Red Cell Electrolytes and Water Content in Normotensive Subjects and Essential Hypertensive
Sodium
(mmole/liter cells)
Potassium
(mmole/liter cells)
Initial
8.6±0.5 (14)
100.8± 1.0 (16)
Lithium-loaded
4 . 6 ± 0 . 4 (16)
93.8 + 0.8 (16)
Initial
8.1 ± 0 . 4 (19)
1 0 1 . 4 ± l , 2 (18)
Lithium-loaded
3 . 6 ± 0 3 (20)
91.8 ± 1 . 0 (20)
Group
Lithium
(mmole/liter cells)
Patients
Water content
(% w/w)
Normotensive
6 3 . 1 + 0 . 3 (14)
8.9 + 0.4 (16)
64.0 + 0.3 (13)
Hypertensive
62.4 + 0.3 (14)
8.5 ±0.3 (20)
63.7 + 0.3 (14)
Values expressed as means ± standard error. Numbers in parentheses indicate the number of subjects, w/w = weight
by weight.
COUNTERTRANSPORT AND COTRANSPORT IN ESSENTIAL H YPERTENSIONMrfragna et al.
799
TABLE 4. Lithium-Sodium (LirNa0) Countertransport in Red Cells of Normotensive Subjects and Essential Hypertensive Patients
Group
Normotensive
(-)FHH
( + )FHH
Hypertensive
Established
No. of
Subjects
Age
(yrs)
16
8
37±3
28±4
Blood pressure
Diastolic
Systolic
(mm Hg)
(mm Hg)
74±2
75 ±2
124±3
121+3
Lithium efflux
Na-free
Medium
Na Medium
(mmole/liter cells x hour)
Countertransport
(mmole/liter
cells x hr)
O.I5±O.OI
0.23 + 0.08
0.29 ±0.02
O.32±O.O3
0.44±0.03
0.55 ±0.08
22
90±2
0.20 ±0.02 0.71 ±0.04* 0.51 ±0.03*
45 ± 3
151±5
Values expressed as means ± standard error. FHH = family history of hypertension; ( — ) = negative FHH, ( + )
positive FHH.
*p < 0.001. p values calculated vs the mean value of normotensives without a FHH.
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sive patients, only two had hypokalemia (2.9 and 3.4
mEq/liter, as compared with normal values of 3.5 to
5.2 mEq/liter). Li r Na 0 countertransport was 0.60 and
0.76 mmole/liter cells X hour, respectively, in these
two patients. These values are not significantly different from the mean (0.51 ± 0.03 mmole/liter cells X
hour) and range (0.24 to 0.81) observed in hypertensive subjects with normal plasma potassium concentrations.
Sodium-Potassium Cotransport in Normotensive Subjects
and Hypertensive Patients
Table 6 shows the Na, K, and water content of red
cells prepared for cotransport measurements from normotensive subjects and hypertensive patients. Kinetic
studies on the red cells of five normotensive subjects
have revealed that the maximal activation of outward
cotransport is achieved at internal Na concentrations
higher than 25 mmoles/liter cells." To ensure saturation of internal sites, cells were loaded to contain approximately (mmoles/liter cells) 50 Na and 60 K.
Since Na-K cotransport is inhibited by cell swelling,10
the water content of the red cells prior to efflux measurements was determined by measuring the percentage
of dry weight. Table 6 shows that the red cells from
normotensive subjects and hypertensive patients recovered normal volume after the PCMBS loading
procedure.
Table 7 summarizes values of the maximal velocity
of furosemide-sensitive Na and K effluxes into Mg
TABLE 5. Sodium-Lithium (Nu-Li) Countertransport and Sodium-Potassium (Na-K) Colransport in Treated and Untreated Patients with Essential Hypertension
Na-stimulated
Furosemide-sensitive
1 i cfllnic
Diastolic
(mmole/liter
Na efflux
K efflux
BP
Treatcells x hr)
ment
No.
(mmole/liter cells x hr)
(mm Hg)
0.57 + 0.12
0.51+0.05
0.46 ±0 07
l00±2
5
(0
39-1.03)
(96-104)
(0.29-0.67)
(0.32-0.74)
+
17
87 ±2
(65-100)
0.51 ±0.04
(0.24-0.81)
0.52±006
(0.09-1.1)
0.60±0.05
(0.26-1.03)
Range is given in parentheses.
TABLE 6. Cation and Water Content of PCMBS-Loaded Cells from Normotensive Subjects and Essential Hypertensive
Patients
Water Content
Sodium
Potassium
After PCMBS
(mmolc/litcr
(mmole/liter
Initial
treatment
cells)
(% w/w)
(% w/w)
cells)
A
Group
50.6±2.8 (16)
59.0 + 2.9 (16)
63.2±0.2 (14)
64.7±0.2 (13)
Normotensive
49 6 ±2.7 (20)
62.8±2.8 (20)
62.7±0.4 (15)
Hypertensive
64.1 ±0.3 (16)
Values are means ± standard error. Number of patients given in parentheses.
PCMBS = p-chloromercuribenzene sulfonic acid.
1 5±0.2 (13)
1 1 ±0 2 (14)
HYPERTENSION
800
TABLE 7. Sodium-Potassium Cotransport in Red Cells from Normotensive Subjects and Essential Hypertensive Patients
Group
Normotensive
(-)FHH
( + )FHH
Hypertensive
Established
No. of .
subjects
Furosemide-sensitive
(mmole/liter cells x hr)
Na efflux
VOL 4, No 6, NOVEMBER-DECEMBER 1982
TABLE 8. Furosemide-Sensitive Potassium Efflux into 150 mM
NaCl Medium from Red Cells Loaded for Cotransport Measurements in Normotensive and Hypertensive Subjects
16
8
0.30 ±0.03
0.53 + 0.11*
0.34 ±0.03
O.55±O.O8t
22
0.51 ±0.05*
0.60±0.04t
Values are means ± standard error.
*p < 0.05.
ip < 0.025.
Xp < 0.005; p values were calculated vs the mean value of the
normotensive population with no family history of hypertension
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medium in the three groups of subjects described
above. The outward cotransport of Na and K was significantly greater in the red cells of patients with essential hypertension than in the red cells of the control
group without a family history of hypertension. The
group of hypertensives includes untreated (n = 5) and
treated (n = 17) patients. Table 5 summarizes the
values of Na and K cotransport in treated and untreated
hypertensive patients. These values were not significantly different in either group, suggesting, again, that
both transport systems, Na-Li countertransport and
Na-K cotransport, are not affected by chronic administration of /3-blockers or diuretics. The group of normotensives with a family history of hypertension also
showed an increased cotransport.
In 11 of the treated patients, the Na, but not the K,
cotransport correlated slightly with plasma potassium
levels (r = 0.58, p < 0.05). The Na cotransport
values of the two patients with hypokalemia (one with
3.4 and one with 2.9 mEq/liter) were 0.42 and 0.89.
mmole/liter cell per hour, respectively. The elevated
value of K cotransport in hypertensive patients was
also observed when measuring the cation efflux into
Na medium in the presence and absence of furosemide
(table 8).
Relationship Between Countertransport and Cotransport
Simultaneous measurements of the Li,-Nao countertransport and Na-K cotransport were performed in normotensive subjects and established essential hypertensive patients. Figure 1 shows a scatter diagram of the
maximal rate of both transport systems in the two
groups of subjects. As previously shown,2 most patients with essential hypertension have a red cell maximum Li,-Na0 countertransport above 0.4 mmole/liter
cells X hour, while most normal subjects have values
below this level. In contrast, the variation of Na and K
cotransport was larger in our normotensive control
group and, consequently, the overlap was greater. As
seen in countertransport measurements, the mean value of cotransport was significantly higher (p <
0.0005) in the group of hypertensive patients.
Figure 2 shows the relationship between Li-Na
countertransport and Na cotransport (left) and K co-
K efflux
(mmole/liter cells X hr)
Group
No.
of
subjects
Without
furosemide
With
furosemide
Furosemide
sensitive
Normotensive
(-)FHH
( + )FHH
Hypertensive
13
7
16
1.35±0.13
2.21 ±0.46
1.70±0.15
1.09±0.12
1.68 ±0.44
l.27±0.15
0.25 ±0.04
0.52±0.06*
0.43 ±0.04*
K efflux
*p < 0.005.
Sec table 6 for cation composition of cells.
FHH = family history of hypertension.
transport (right) in red cells of normotensive subjects
and hypertensive patients. A significant correlation coefficient was found between the rate of Li,-Na0 countertransport and Na-K cotransport (n = 38, r = 0.53, p
< 0.0005).
Li - Na
Countertransport
Na
K
Cotransport
FIGURE 1. Scatter diagram of Li-Na countertransport, Na
and K cotransport (mmole/liter cells X hour) performed simultaneously in red cells of normotensive subjects (O) and hypertensive patients (*).
COUNTERTRANSPORT AND COTRANSPORT IN ESSENTIAL HYPERTENSION/Adragmj et al.
801
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n .it
r : o so
p<o OOB
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n . s«
r 0 57
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Na Cotranapori, mmo(/l cell x hr
K Cotransport, mmol/1 eel x hr
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ra
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Furo sem
Net Sodium Extrusion Against a Sodium Gradient in Red
Cells of Normotensive and Hypertensive Subjects
The same Na-loaded cells shown in table 6 were
used for measurements of net Na and K fluxes after 5
hours of incubation in K-free medium containing 150
mM Na. The initial Na concentration was 76 and 72
mmoles/kg of water inside the red cells from normotensives and hypertensives, respectively. Under this
inward Na gradient, both types of cells gained Na
(table 9). The outward K gradient did not promote
uphill movement of Na in red cells of normotensive
subjects or hypertensive patients. However, in the
presence of furosemide, there was an increased net Na
gain and a reduced net K loss. The net furosemidesensitive Na efflux was calculated as the difference in
net Na flux in the presence and absence of furosemide.
It can be seen that the mean value of the furosemidesensitive loss of Na is significantly greater in the red
cells of hypertensive patients. For K, no significant
difference was found.
Figure 3 shows the relationship between initial rates
of outward Na cotransport into Mg medium and net
cation loss against a Na gradient measured simultaneously in 10 normotensive and 12 hypertensive sub-
OUJUJ
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FIGURE 2. Li-Na countertransport as a function ofNa cotransport (left) and K cotransport (right) (mmolelliter
cells X hour) in red cells of normotensive subjects (A, O) and hypertensive patients (A,*J.
T3
•4—
<D
— 0.6
o
cm
o
z
0.5
1.0
Na Cotransport, mmol/l cell x hr
FIGURE 3. Net furosemide-sensitive Na efflux andNa cotransport (mmolelliter cells X hour) in red cells of normotensive
subjects (O) and hypertensive (*) patients.
TABLE 9. Net Furosemide-Sensitive Sodium and Potassium Fluxes Against a Sodium Gradient in Red Blood Cells from Normotensive
Subjects and Hypertensive Patients
Net Na flux (mmole/liter cells x hr)
Net K flux (mmole/liter cells x hr)
With
Furosemide
Without
Furosemide
Without
With
furosemide
sensitive
Group
furosemide
furosemide
furosemide
sensitive
-0.22±0.04
-0.25±0.05
Normotensive
1.19±0.12
-1.16±0.19
0.95 ±0.11
-O.94±0.17
-O.5O±0.O9*
1.67 + 0.28
-0.35 ±0.12
Hypertensive
-1.22±0.22
1.19 + 0.26
-1.56±0.18
The minus sign indicates net cation loss from cells into 150 mM NaCl medium after 5 hours of incubation. Values are expressed as means
standard error. Normotensives, n = 10; hypertensives, n = 12.
*p < 0.025.
802
HYPERTENSION
TABLE 10. Rate Constants of Furosemide-lnsensitive Sodium
and Potassium Efflux in Red Cells Loaded for Cotransport Measurements in Normotensive Subjects and Hypertensive Patients
Determination
Normotensives
Hypertensives
Cellular Na
(mmole/liter cells)
Furosemide-insensitive
Na efflux
(mmolc/liter
cells x hr)
k
Na (hr)-i
46.6±2.l
49.6±2.7
0.74±0.06
0.90 ±0.08
0.0!6±0.00l
0.018±0.001
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Cellular K
63 4±3.2
62.3±3.O
(mmolc/liter cells)
Furosemide-insensitive
0.91 ±0.06
1.01 ±0.08
K efflux
(mmole/liter
cells x hr)
L
0.014±0.00l
Na (hr)- 1
0.017±0.OO2
Normotensives, n = 15; hypertensives, n = 22. Values are
means ± standard error. The incubation medium contained (mM):
75 MgCk 85 sucrose. lOTris-MOPS (pH 7.4at37°C). 10 glucose.
0.1 ouabain. and I furosemide.
jects. A correlation coefficient of 0.869 (p < 0.0005)
was obtained for Na fluxes in the red cells of hypertensive patients (fig. 3). Therefore, the increased furosemide-sensitive sodium extrusion in red cells from hypertensives is probably due to an increased rate of Na
cotransport.
Rate Constants of Furosemide-insensitive Cation Efflux in
Red Cells
Table 10 summarizes values for the rate constants of
furosemide-resistant Na and K efflux measured in cells
loaded by the PCMBS procedure. This parameter is a
good approximation of the passive permeability. The
rate constant (hr~') was calculated as the ratio between
furosemide-resistant fluxes and the cell cation concentration. No significant differences between red cells of
normotensive and hypertensive subjects were observed. The rate constants of Li and K were also estimated in cells loaded for countertransport (table 11).
The rate constant was calculated as the ratio between
VOL 4, No 6, NOVEMBER-DECEMBER 1982
cation fluxes into Mg medium and the cellular Li and K
concentrations. For K, no significant differences were
found between the cells of normotensive and hypertensive subjects. In the case of Li, the rate constant increased in red cells from hypertensives as compared to
normotensives.
This result disagrees with the previous observations
reported by our laboratory.2 In our previous study, Li
efflux into Mg medium was determined by taking duplicate samples at 20 and 40 minutes, while in the
present study, triplicate samples were taken at 30, 60,
and 90 minutes. The smaller standard deviation of
these flux measurements apparently revealed the differences between normotensive and hypertensive subjects. A further possible explanation for this result
could be the presence in this group of a larger number
of hypertensive subjects with increased outward Li
cotransport. We have recently shown that Li can replace Na in the outward cotransport.7 Even though the
internal Li concentration required for maximal outward Li-K cotransport is high (60 mmoles/liter cells)
as compared to the concentration of Li in cells used for
assay of countertransport (10 mmoles/liter cells), it is
possible that a small contribution to the total outward
Li movement into a Mg medium could occur by this
pathway, thus leading to a falsely high estimate of
passive permeability. This conclusion is supported by
the significant correlation (r = 0.65, n = 19, p <
0.005) between the rate constant of Li efflux into Mg
medium and the maximal rate of Na-K cotransport in
hypertensive patients. Since outward Li-K cotransport
is inhibited by furosemide, measurements of Li outward movement in the presence of this inhibitor should
settle the point.
Discussion
The main objective of this study was to determine
the relation between red cell Li-Na countertransport
and Na-K cotransport in normotensive and hypertensive subjects. We confirm our previous observation
that Li.-Na,, countertransport is elevated in the red cells
of many, but not all, patients with essential hypertension. In the first sample, 90% of the hypertensive patients had countertransport over 0.4 mmole/liter cells
x hour, while in the present paper, 77% of these
patients were over 0.4% and 9 1 % over 0.35. In subjects with diagnoses of labile and mild hypertension.
Rate Constants of Lithiumand Potassium Efflux in Red Cells Loaded for Countertransport Measurementsin Normotensive
Subjects andHypertensive Patients
Li efflux
Internal Li
K efflux
Internal K
in Mg medium
in Mg medium
concentration
concentration
(mmole/liter
(mmolc/liter
(mmole/liter
(mmolc/liter
cells x hr)
cells x hr)
(hr-i)
Group
(hr- 1 )
cells)
cells)
1.23 + 0.07
8.9±0.4
Normotensive
0.15 ±0.01
0 .016 + 0.001
0.013 ±0.001
94.0±0.8
(n = 16)
0 .023 ± 0 002*
0.012±0.0O4
Hypertensive
O.2O±O.O2
9I.9±I.O
8.5±0.3
I.15±O.O4
(n = 22)
The incubation medium was the same as that described in the legend to table 10.
*p < 0.005.
TABLE 11.
COUNTERTRANSPORT AND COTRANSPORT IN ESSENTIAL HYPERTENSION/zWragna et al.
803
TABLE 12. Lithium-Sodium Countertransport in Red Cells of Normotensive Subjects, Labile, Mild, and Established
Hypertensive Patients
Li-Na
Countertransport
Blood Pressure (mm Hg)
Age
No. of
(mmole/liter
subjects
Diastolic
Group
Systolic
cells X hr)
(yrs)
Normotensive
(-)FHH
( + )FHH
36
16
35±2
30±4
7I±1
75 ± 2
124 ± 2
124±2
0.27±0.01
O.28±O.O3
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Hypertensive
89±2*t
39 ± 3
24
0.36±0.02*
141 ± 3 *
Labile + mild
96±l*t
45 ± 2
O.53±O.O2*t
158±3*t
49
Established
*p < 0.005; p values were calculated vs the mean value of the normotensive population with no family history of
hypertension ((-)FHH).
tp < 0.005; p values were calculated vs the mean value of the labile + mild hypertensive population.
In addition to the patients described in this paper, the table includes patients studied in a previously published sample2
in which countertransport but not cotransport was measured. These individuals are included to allow comparison of
countertransport values in mild and severe forms of essential hypertension. The four groups include: normotensives
without family history of hypertension. 24 women and 12 men; normotensives with family history of hypertension, 6
women and 10 men; labile and mild hypertensives, 11 women and 13 men (22 of these 24 patients were untreated);
established hypertensives, 13 women and 36 men (24 of these patients were untreated). Labile hypertensives were
subjects whose diastolic blood pressures oscillated between 80 and 90 mm Hg, while mild hypertensives had sustained
values between 90 and 95 mm Hg. Established hypertensives had sustained diastolic blood pressure over 95 mm Hg.
only 33% of the patients had elevated countertransport
(table 12). The maximal rate of outward Na-K cotransport was also increased in many, but not all, of the
established hypertensive patients. Even though the
range of values of red cell Na-K cotransport in the
control and hypertensive groups overlapped, the mean
value of red cell Na-K cotransport was significantly
higher in the hypertensive than in the normotensive
group. It was also found that elevated countertransport
correlated significantly with elevated cotransport. The
mean value of Na cotransport in normotensive subjects
reported in this paper (0.30 ± 0.03) is in agreement
with the mean value obtained in 32 normotensive subjects from the Blood Bank (0.38 ± 0.8, ranging from
0.15 to 0.8 mmole/liter cells X hour). Our measurements of elevated Na-K cotransport in patients with
essential hypertension were confirmed by simultaneous measurements of net Na extrusion against a Na
gradient from cells containing approximately 50
mmoles/liter of Na and K. Similarly, the furosemidesensitive K efflux into 150 mM NaCl medium was
significantly increased in the hypertensive patients.
Our finding of elevated Na-K cotransport in patients
with essential hypertension does not confirm the report
of Garay et al.5-6i '4 that there is a reduction in outward
Na-K cotransport in hypertensive patients. The contradictory findings may, in part, be accounted for by
differences in the methods and, in part, by differences
in the red cell transport properties in the different populations of subjects studied.
We have found reduced Na-K cotransport associated with the swelling of Na-loaded cells.1" Therefore,
the measurements of Na-K cotransport reported in this
paper comprise only those red cells that recovered normal volume after the PCMBS-loading procedure. Cell
volume was directly determined by measurements of
dry weight and wet weight rather than indirectly
through measurements of hemoglobin per liter of cells.
Since Garay et al. 2 - l4 do not report direct measurements of cell volume in their assays of cotransport, it is
possible that some of their observations of reduced
outward Na cotransport were made on swollen cells
undetected as such by hemoglobin measurements.
More likely, the differences between our results and
those of Garay et al.3 could be due to differences in the
concentrations of Na and K in the red cells used to
assay outward cotransport. We have used cells containing (mmoles/liter cells) 50 Na and 60 K, while
Garay et al. have used cells containing 20 to 25 Na and
20 to 25 K. Since the concentration of Na required to
half-activate the cotransport is about 13 mmoles/liter
cells, it is probable that the procedure of Garay et al.
detects changes not only in the maximum rate of cotransport but also in the affinity of the system for internal Na. By using a higher internal Na concentration,
our method probably measures only the maximal rate
of cotransport. Indeed, Garay et al.15 have recently
reported that the affinity for internal Na is reduced in
outward cotransport in the red cells of some subjects
with essential hypertension.
Another possible explanation for the differences between Paris and Boston is that the populations of patients studied are in fact different. For example, table
12 summarizes measurements of blood pressure and
maximum rate of Li-Na countertransport not only in
the subjects reported in this paper but also in subjects
studied earlier2 when countertransport but not cotransport was measured. The subjects are considered in four
groups; normotensives without and with a family history of hypertension, labile and mild hypertensives,
804
HYPERTENSION
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and established hypertensives. Li-Na countertransport
was significantly lower in the group of mile and labile
hypertensives than in the group of established hypertensives. It is possible that the group of patients studied
in our laboratory, in Paris, and in other laboratories in
different parts of the world contain varying populations
of mild and labile as compared with established hypertensives. Further studies are required to establish
whether the simultaneous measurements of countertransport and Na-K cotransport can distinguish between mild and severe forms of essential hypertension.
It is also possible that the different groups of patients
studied contain varying subpopulations of essential hypertensives of differing etiologies and differing relations to red cell cation transport. Indeed, Li-Na countertransport has been found to be normal or only
slightly increased in a group of hypertensive patients
with reduced outward Na transport.16 l7 This finding
indicates that at least two types of abnormalities of red
cell Na transport can be found in individuals with essential hypertension: elevated countertransport with
elevated cotransport, and normal countertransport with
low cotransport.
The positive correlation between the maximum rates
of Li-Na countertransport and Na-K cotransport suggests that the two systems are somehow related. However, many kinetic properties of the two pathways are
different.7 For example, the affinity of cotransport for
internal Li is lower while its sensitivity to furosemide
is much greater than is the case for countertransport.
Moreover, the two systems can apparently operate at
maximum rates simultaneously. Further research is
necessary to clarify the relationship between these two
systems.
Acknowledgments
We arc endebted lo Mariamta Sanchez, for her secretarial work.
References
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Red cell lithium-sodium countertransport and sodium-potassium cotransport in patients
with essential hypertension.
N C Adragna, M L Canessa, H Solomon, E Slater and D C Tosteson
Hypertension. 1982;4:795-804
doi: 10.1161/01.HYP.4.6.795
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