Kinetics of Active Sodium Transport in Aortas from
Control and Deoxycorticosterone Hypertensive Rats
ALLAN W. JONES, PH.D.
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SUMMARY The efflux of sodium (MNa) was measured in the aortas from control and deoxycorticosterone
(DOCA) hypertensive rats. Measurements were also made of aortic water content, extracellular space, and
electrolyte contents. The objective was to determine the kinetics for potassium (K)-stimulated (active)
transport of sodium and to determine which, if any, kinetic parameters are altered during DOCA hypertension. The K-stimulated efflux of MNa (about 50% of the total) exhibited a sigmoidal dependence on extracellular K ([K]o], and agreed well with a model based on the cooperative interaction between Na and K at
transport sites. Half saturation was achieved at [K]<, - 1.7 to 2.8 mM for the various groups. The maximum
efflux was increased 40% to 50% by DOCA (p < 0.01). Aortas from control and DOCA-treated rats had
similar [Nab,, (11 mM/llter cell H,O, for physiological solution) under conditions of partial or complete Na
loading. Active transport of Na showed a sigmoidal dependence on [NaJc,,. Half saturation was achieved at
[Na]o,,i = 20 to 24 mM/liter cell H,0 in control and DOCA aortas. The maximum efflux was elevated 46% by
DOCA treatment (p < 0.01). It is concluded that DOCA hypertension is associated with an increased leak of
Na into vascular smooth muscle and increased active transport of Na. This is accomplished primarily by an upward shift of the transport curves as a result of increased maximal transport The net effect is to maintain
[Nalo.,1 relatively constant in DOCA hypertensives under physiological conditions.
(Hypertension 3: 631-640, 1981)
KEY WORDS
radioisotopes
vascular smooth muscle
"Na efflux • cell-Na
electrolyte contents
K-stimulation
A
extracellular space
A corollary to this hypothesis is that active transport of sodium is proportionately elevated to the inward leak in vascular smooth muscle from DOCAtreated rats and SHR. This was based initially on the
observation that cellular concentrations of sodium did
not increase in the presence of increased permeability.*1 8> ••10 Increased uptake of potassium and extrusion of sodium from sodium-loaded tail arteries has
also been reported.9-10 Measures of electrical and
mechanical responses have also indicated increased
active transport in tail arteries from SHR.n> u Additions of potassium after a potassium-free incubation
caused a greater hyperpolarization,11 as well as increased rate of relaxation to a contractile response to
norepinephrine.11 One dissenting study on DOCA
hypertensives reported a decrease in ouabain sensitive
(active) uptake of "rubidium by tail arteries.1' This
was taken to support the hypothesis that active transport is suppressed in volume-expanded hypertension. "• "
The objective of this study was to determine the
kinetics for K-stimulated (active) transport of sodium
in aortic smooth muscle, and to determine which, if
any, of the kinetic parameters were altered during
DOCA hypertension. These experiments were designed on the rationale that the study of transport
properties over a wide range of conditions would more
growing body of evidence has linked altered
ionic transport by vascular smooth muscle
to experimental hypertension. Radioisotopic studies from our laboratory have shown increased turnover of sodium, potassium, and chloride
in aortas from spontaneously hypertensive rats
(SHR).1"* These observations were later extended to
rats made hypertensive with deoxycorticosterone acetate (DOCA).4> • The ability of norepinephrine to increase ionic fluxes also showed a supersensitivity.1-9
Friedman7-• has demonstrated an increased exchange
of sodium and potassium for lithium in tail arteries
from DOCA-treated rats and SHR. These exchanges
were mainly diffusional movements. Evidence from
two laboratories using different methods therefore
support the hypothesis that increased membrane
permeability to small ions accompany DOCA and
spontaneous hypertension in the rat.
From the Department of Physiology, University of Missouri,
Columbia, Missouri.
Supported by United States Public Health Service Grant HL15852. Dr. Jones is an Established Investigator of the American
Heart Association.
Address for reprints: Dr. Allan W. Jones, Department of Physiology, University of Missouri, Columbia, Missouri 65212.
Received July 16, 1980; revision accepted April 27, 1981.
631
HYPERTENSION
632
clearly identify the specific transport alterations. The
principal change found in the aortas from DOCAtreated rats is an increased maximal rate for active
transport. A preliminary report has appeared previously.15
Methods
Animals and Tissues
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Male Wistar rats weighing 200 g were subjected to a
left nephrectomy. The control rats were placed on 1%
wt/vol saline to drink while the DOCA-treated rats
received in addition K G (0.2% wt/vol) in the saline.
Six mg of DOCA suspended in sesame oil was injected intramuscularly twice per week for 6 weeks. 4 '"
Systolic blood pressure was measured by a tail cuff
technique.' All hypertensives had systolic pressures
above 180 mm Hg (200 mm Hg average) as compared
to 130 mm Hg in the controls. The rats were killed
with a blow to the head. The thoracic aorta was
removed, trimmed, and cut along its length while incubating in a dissection solution at 37°C. This strip in
turn was cut into three segments, and each was
mounted on a stainless steel holder.
Solutions
The physiologic solution has the following composition, mM: N a \ 146.2; K + , 5.0; Mg»\ 1.2; Ca f + , 2.5;
Cl", 143.9; HCOi, 13.5; H.POi, 1.2 and glucose, 5.7.
Solutions were gassed with 97% O, and 3% CO, at
37°C, which yielded a pH of 7.4. The dissection solution was K-free and contained reduced Ca 2+ , 0.2 mM.
This allowed reversible depletion of K and the loading of tissue with Na during the \Vi hour dissection.
This was necessary to achieve equilibration of cellular
Na and K with solutions of varying K + concentrations ([K]o). Variations in [K]o were achieved by adding or omitting the appropriate amount of KC1. Ouabain (Sigma Corporation) solutions of 2 mM were
made by dissolving the glycoside in half the final
volume before adding the salts.
Isotope Fluxes
The procedure has been used and evaluated previously.8- '••17 The strips were incubated at 37°C for 3
hours in a physiologic or modified solution containing
"Na (20-30 /iCi/ml, University of Missouri Research
Reactor). The cellular level of Na, [Na] raM , was controlled by varying [K]o. K-free solution was used to
achieve maximal [Na]c, n ; [K]o was equal to 5 mM for
physiologic levels (low [ N a ^ n ) and [K]o equaled 1 to
1.3 mM for an intermediate [Najdi. The final 8
minutes of incubation in 24Na took place at 1°C, and
the washout was begun at 1°C as the tissues were
passed through a series of vigorously gassed solutions
that contained the desired [K]o. This technique provides a rapid dilution of the isotope into the nonlabeled solution, thus allowing efflux components with
low temperature coefficients to be readily separated
from those that are highly sensitive.17 At the same
time, the desired [K]o can be established while active
VOL 3, No 6, NOVEMBER-DECEMBER
1981
transport is under cold inhibition.17 After 40 minutes,
the strips were quickly (about 1 to 2 seconds) transferred to tubes at 37°C, and the washout was continued until greater than 90% of the slowly exchanging counts were removed.
Liquid scintillation spectroscopy was used to measure " N a activity. The residual counts were extracted
from the weighed tissues into 5 ml of a standard electrolyte diluent; (0.1 N HNO,, 10 mM La s + , and 15
mM LiNO8) for 10 to 15 minutes, then neutralized
with 0.5 ml of 1 N NaOH. The tissues, washout tubes,
and standards from the " N a loading solutions were
mixed with 10 ml of scintillation fluid (3A70B),
Research Products International) and counted.
Quenching as monitored by a channel ratio method
was uniform for the samples. The counting of/3 activity yielded a combination of high efficiency (almost
100%), low background, and sufficient resolution to
detect changes of 25% in effluxes from components
which, under some conditions, contained only 1% of
the tissue sodium.
The washout curves were derived by sequentially
adding the counts in the tissue and tubes (corrected for
background and decay) followed by division of the
counts remaining after each period by the counts at
time equal zero. The rate constants (k, min"1) for
isotope loss were also computed for each period, and
the constant for the initial period at 37°C was used as
the measure of Na transport. This provided a consistent time for comparing the fluxes under a wide
range of conditions, e.g., more than 10-fold difference
in [Na]Mn. The slowly exchanging Na was estimated
from the tissue counts (cps) that remained after 10
minutes washout at 1°C. The product of the specific
activity in the loading solution (mmole Na/cps "Na)
and cps " N a / k g wet wt of tissue yielded the content of
slowly exchanging Na, mmole/kg wet wt. The [Na]Mn
was calculated by dividing slowly exchanging Na by
the estimated cell water, liter or kg/kg wet wt. The
efflux of sodium, mmole/liter/min, was calculated
from the product, k X [Na]c, u . A cell volume to area
ratio of 0.6 X 10^ cm can be assumed to convert the
flux basis to membrane area. These procedures have
been routinely applied previously.*• "• l "
One difficulty with the approach is that nonlabeled
Na is slowly taken up by low Na cells during the
washout at 1°C.17 This tends to dilute the cellular
"Na, which will slow the rate of loss of counts at
37°C. The elevated [Najcen, on the other hand, would
stimulate the transport processes. These changes tend
to cancel each other but limit (± 25%) the detailed
analysis for low [Najcn. This factor would be negligible, however, for Na-loaded tissues where no net uptake occurs.
Extracellular Space and Electrolyte
Tissues were maintained at 37°C for 3 hours in the
desired incubation media, followed by 15 minutes in a
similar solution with labeled "CoEDTA (0.5 MCi/ml)
and nonlabeled CoEDTA (2 mM). 4 ' " The strips were
then blotted and weighed in plastic tubes. Water con-
SODIUM TRANSPORT AND DOCA HYPERTENSION/Zones
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tents were determined by weight difference after drying (20 hours at 93°C). The dry specimens were
counted in a gamma counter, and the "CoEDTA
space was calculated from the ratio of counts/sec in
the strip to counts/sec in a weighed sample of the incubation media. This was represented in terms of kg
HjO/kg dry weight. "Cell" water was taken as the
difference between total water and "CoEDTA space.
Similarly, the cell electrolyte content was estimated to
be the difference between the total electrolyte content
and that dissolved in the "CoEDTA space. No correction for adsorption was employed.
The tissues undergoing electrolyte analysis were
ashed at 85°C in H,O, (30% w/v) containing AgNO,
to precipitate the Cl". The ash was dissolved in a solution containing 0.1 N HNO,, 10 mM La' + , and 15
mM Li in the NO, form. The Mg, Ca, and Ag were
analyzed on an atomic absorption spectrometer
(Instrumentation Laboratory, IL 453) and Na and K
on a flame photometer (IL 153). Cl was calculated
from the difference between total Ag added and final
Ag in the extract.19 Contents are presented in terms of
mmoles/kg dry weight.
Data Analysis
Significance was determined by Student's / test. A
curve-fitting routine was used to derive the parameters for the flux model. This was based on the GaussNewton algorithm for nonlinear least squares fit and
was performed on a digital computer (PDP 11/60
Control Data Equipment Corporation). Details of the
analysis and the application to several models appear
elsewhere.20' " The automated curve fit has the advantage of using all the data points to derive all the
parameters in the model; this reduces investigator
bias, which can occur when curves are fitted by hand
methods.
Results
About 1% to 2% of the "Na washed out slowly at
1°C in aortas that had been previously equilibrated in
physiological solution at 37°C (fig. 1). When the tissues were equilibrated in K-free solution, the percent
of "Na that washed out slowly increased about 10-fold
(fig. 2). Upon return of the aorta to 37°C, the efflux of
"Na proceeded 25 to 90 times faster in both K-loaded
and Na-loaded tissues (fig. 1, fig. 2, and table 1). The
efflux of "Na into K-free solutions followed a uniform (single) exponential loss from at least 90% of the
slow component. A paired comparison was made
between the efflux of "Na from K-loaded control aortas into either K-free solutions or [K]o = 5 mM solutions containing 2 mM ouabain. The rate constants of
0.148 ±0.016 (5) and 0.130 ± 0.008 (5) min"1 respectively were not significantly different. K-containing
solutions (without ouabain) doubled the efflux at 37°C
(p < 0.01) and caused some nonlinear behavior in the
later stages of the washout. Differences between the
efflux in K-containing and K-free solution are termed
633
10
oo
8
o0.5
2.0.1
0
5
10
20
30
40
50
60
Minutes
FIGURE 1. Washout ofuNafrom
control rat aortas in the
presence of10 mM of potassium (Kj( • ) or K-free solution
(o). Tissues were equilibrated in physiological solution at
37°C. The arrow indicates the change in temperature during the washout. The logarithm of the percent of counts remaining is plotted vs time. Each point is the average of six
rats ± standard error of mean.
"K-stimulated transport." This is operationally
defined as active transport" and represents approximately 50% of the efflux.
The rate constants and concentration of the slowly
exchanging "Na in cell water, [Na]Mll, are given in
table 1 for control and DOCA-treated rats. The
[Na]c,ii was not significantly changed by DOCA treatment, but important differences were found in the rate
constants at 37°C. Rate constants for aortas that were
equilibrated in [K]o = 5 mM showed a 40% to 50% increase in the DOCA group both with K present or absent in the washout solution. The K-stimulated rate
100
50
o
1 C
° 20
.£
| 10
37"C
1
D—D
\°
3
° 5
5
0.5
u
0
5 10
20
30
Minutes
50
60
FIGURE 2. Washout of uNa from control rat aortas in the
presence of 10 mM K ( • ) or K-free solution (o). Tissues
were equilibrated in K-free solution at 37°C. The data are
plotted as in figure 1 with each point being the average of
seven rats.
HYPERTENSION
634
VOL 3, No
6, NOVEMBER-DECEMBER
1981
TABLE 1. Rate Constants (min'1) and Cell Sodium Concentration in Rat Aorta Incubated Under Various Equilibration and
Washout Conditions
Equilibration, K-free
Equilibration, [K]0 = 5mM
Control
Rate constant
Wash solution:
[K],, = 10 mM
Wash solution:
K-free
Wash solution:
K-stimulated
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Sodium concentration:
[NaLji mmole/liter
cellHijO
DOCA
Control
DOCA
1°C
37° C
1°C
37° C
1°C
37° C
1°C
37° C
0.0034
±0.0007
(6)
0.0021
±0.0003
(5)
0.0013
±0.0008
(6)
0.314
± 0.013
(6)
0.126
±0.004
(5)
0.188
± 0.014
(6)
0.0040
±0.0005
(6)
0.0025
±0.0001
(5)
0.0015
±0.0005
(6)
0.445*
± 0.020
"(6)
0.193*
±0.005
(5)
0.252f
± 0.021
(6)
0.0012
± 0.00017
(7)
0.0014
± 0.0001
(5)
-0.0002
±0.0001
(7)
0.117
±0.002
(7)
0.062
±0.006
(5)
0.055
±0.006
(7)
0.0028*
±0.0003
(7)
0.0027f
± 0.0004
(6)
0.0001
±0.0005
(7)
0.150*
±0.006
(7)
0.066
±0.001
(6)
0.0841
±0.006
(6)
11 ±0.5
(14)
154 ± 8
(6)
11 ±0.3
(19)
147 ± 6
(8)
Values are means ± standard error of the mean. Parentheses indicate the number of rats.
*p < 0.001; TP < 0.01; \p < 0.05; DOCA vs control.
constant was similarly elevated. The hypertensive
arteries that were equilibrated in K-free solution
showed a 25% increase in rate when washed out in the
presence of K, but the rate constant was unchanged in
K-free washout solution when compared to control
aortas. The K-stimulated rate was therefore significantly elevated. The K-stimulated rates at 1°C
were less than 1% those at 37°C for all conditions.
The washout of "Na was systematically studied at
intermediate [K]o to gain more detailed knowledge of
the kinetics. The rate constants are shown in figure 3
0.5
r
for aortas that were equilibrated at normal [K]o. The
rates for the total exchange were consistently higher
for the DOCA hypertensives (p < 0.001). Kstimulated rates for the DOCA-treated group were
significantly elevated at [K]o = 5 mM (p < 0.025) and
10 mM (p < 0.05).
The rate constants appear infigure4 for aortas that
were equilibrated in K-free solution. The total exchange was elevated in the DOCA aortas at [K]o = 2
mM (p < 0.05) and 10 mM (p < 0.01). The K-stimulated components increased in a sigmoidal fashion
0.15 -
DOCA
f 0.4
Total
8.
Total
c
E
S> 0.10
c
o
6 0.3
•5
CD
§0.2
0.05
c
o
u
o
0.1
0
1
FIGURE 3. Rate constants for uNa efflux at 37°C from
control and DOCA aortas measured at various [K]o. Tissues were equilibrated in physiological solutions at 37°C.
Constants for the efflux in the presence of[K]o are joined by
solid straight lines for control ( •) and DOCA (o). The
effects of K-stimulation (total — rate in K-free solution) are
joined by dashed lines for control (k) and DOCA (A). Each
point is the average of five to eight rats ± standard error of
the mean.
0
1
10
[Kl o ,mM
FIGURE 4. Rate constants for "Na efflux at 37"C from
control and DOCA aortas measured at various [K]o. Tissues were equilibrated in K-free solution at 37°C. Data are
plotted as in figure 3. Each point is the average of four to
eight rats ± standard error of mean.
635
SODIUM TRANSPORT AND DOCA HYPERTENSION//onw
with increasing [K]o. DOCA hypertensives were consistently elevated, but only the comparison at [K]o =
10 mM was significant (p < 0.01).
The K-stimulated exchange of M Na was subjected
to kinetic analysis to determine the parameters most
subject to change during DOCA hypertension. A
model was applied that describes both a hyperbolic
dependence on [K]o or a sigmoidal dependence (fig. 4).
This behavior depends on the value for a parameter of
nearest neighbor interaction, n. This and other kinetic
models are discussed in a review." The model takes
the form:
.g
112
.2
110
X
I8
z
6
"8 4
10
[K] o , mM
l+
I"*.-
(1)
[Na].
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[Nat,
K . - l J +4
FIGURE 5. K-stimulated "Na efflux from control (k) and
DOCA (A) aortas equilibrated in K-free solution; and from
control ( • ) and DOCA (o) aorta equilibrated in physiological solution. The solid lines were computed from equation 1 with the parameters in table 2. Each point is the
average of four to eight rats ± standard error of mean, except where the symbol size exceeds the error.
where T N a is the K-stimulated (active) efflux of Na,
mmole/liter cell H f O/min; T£; x is the maximum
efflux; K0 is the selectivity of the outside transport sites
for K over Na. If n > 1, there is, according to this approach, an autocooperative interaction between membrane transport sites. Uptake of K onto one site reinforces the uptake of K onto a neighboring site and so
on. If n = 1, no interaction takes place, and equation
1 reduces to the Michaelis-Menton model.18
The K-stimulated effluxes of M Na are given in figure
5. The effluxes were computed from the product of the
K-stimulated rate constants (figs. 3 and 4) and the appropriate [Na]Mii (table 1). The smooth lines result
from the curve-fitting routine, and good agreement is
seen with the predictions of equation 1. Table 2 contains the parameters used for the computations. T£; x
was increased 40% to 50% in the DOCA aortas under
conditions of both high and low [NaJdi. This is significant at the 1% level if it is assumed that the standard error associated with TflJ, is similar to that for
T N a at [K]o = 10 mM (fig. 5). The potassium concentrations required for half maximal transport, [K]S",
increased 20% to 40% during DOCA hypertension.
The associated KO from the relation:
tively unchanged by treatment with DOCA. A rigorous
statistical test was not available for these parameters,
however. A 13-fold increase in [Na]cn, from 11 to 150
mmole/liter cell H , 0 increased TflJ, fourfold (table
2). Sodium loading was associated with lesser increases in KO and n, which falls within the experimental
error.
K-stimulated transport was further studied at an intermediate [NaJcn and [K]o = 10 mM. A sigmoidal
dependence on [Najd, is seen in figure 6. Equivalent
[Na] Mll was achieved by controls and DOCA aorta for
the same equilibration conditions. The parameters for
equation 1 appear in table 3. TSl, exhibits a 46% increase for the DOCA group (p < 0.01). The difference
in sodium concentration required for half maximal
transport, [Na]?.-?,, and the associated selectivity of the
transport system on the inside of the membrane, « ln ,
are likely within the experimental error. «,„ was
calculated from the relation:
(2)
(3)
correspondingly decreased with DOCA. The parameter, n, for nearest neighbor interaction remained rela-
For the purpose of this analysis it was assumed [K] Mll
+ [NaJcn = constant = 160 m M . ' 1 " A higher value
[K]S«
TABLE
2. Kinetic Parameters for K-Stimulated uNa Efflux from Rat Aorta
[ N a ] ^ •11 mmole/liter cell H2O
[NaJcn- 150 mmole/liter cell H2O
mmole/liter
cell H 2 0/min
Control
2.5
DOCA
3.4
Rat
[KB-6
mM
K0
n
2.3
2.8
65
53
1.1
1.3
Parameters were derived from equation 1.
mmole/liter
cell H 2 0/min
8.9
13.9
[KB 6
mM
1.7
2.4
o
n
90
62
1.8
1.5
K
636
HYPERTENSION
112
DOCA
o 6
z
C DOCA
U
0 10
30
50
[Nalcell.mM
150
100
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FIGURE 6. Dependence of K-stimulated uNa efflux on
[Na/aU. Control (•) and DOCA (o) aortas were equilibrated in solutions of varying [K]a to achieve the 3 levels of
/Wa/di. The solid lines were computed from equation 1 with
the parameters in table 3. The arrows indicate the [Nafiin.
Each point is the average of six to eight rats ± standard
error of mean. Note both abscissa and ordinate exhibit
significant variation.
TABLE 3. Kinetic Parameters for
[Na]ceu-Stimulated
24
Na Efflux from Rat Aorta at [#]<, = 10 mM
Rat
Control
DOCA
mmole/liter mmole/liter
cell HaO/min cell H2O
8.5
12.4
24
6, NOVEMBER-DECEMBER
n
1.8
1.4
Parameters were derived from equation 1, with [ K ] ^ and
[Nalceii substituted for [Na],, and [K],, respectively. [Na]nu
was measured and [ K ] ^ was calculated from the relation;
a^u = constant = 160 mM.
for cell cation concentration would increase the
calculated *cln. The relatively constant concentration
of Na + K in cell water (table 4) supports the general
assumption made in calculating « ln . The parameter, n,
was not changed by more than the experimental error
of about 20%.
Analyses of water and electrolyte contents were performed on aortas incubated in [K]o = 0, 1, or 10 mM
(table 4). DOCA hypertensives showed significant elevations in total and cellular water contents. This was
also reflected by the increased contents of potassium
and magnesium over the entire range of [K]o. Fewer
significant differences were observed in Na and Cl
contents. Elevated Na contents were observed when
the total was corrected for the contents of the
"CoEDTA space. No significant changes in Ca contents were noted. Normalization of Na and K in terms
of cell water eliminated the differences observed on the
dry weight basis. As expected, lowered [K]o reduced
cellular K concentration while raising that for Na. The
sum remained relatively constant. It is of interest that
cell water was not increased in K-free solution
although active transport was inhibited. The concentrations of Na in cell water (table 4) exceeded the estimate that was based on the efflux of " N a (table 1).
This most likely resulted from the contribution of Na
adsorbed to connective tissue,1*1 " which was not subtracted from the total contents. This adsorptive
process also involves Ca and could explain the high
levels of Ca in excess of that in the "CoEDTA space.18
Kinetic Parameters
An increased TJSS, represents the major change
found in active transport during DOCA-salt hypertension. This was observed when [K]o was varied at
constant cell sodium, and when [ N a ^ n was varied at
constant [K]o. Several factors may underlie the in-
TABLE 4. Water and electrolyte distribution in rat aorta incubated in solutions of various potassium
Rat
(no.)
Control
(8),
DOCA
(8)
Control
(8)
DOCA
(8)
Control
(8)
DOCA
(8)
Water
eocoEDTA
Cell
kg H 2 O/kg dry wt
[K]o
mM
Total
0
2.35
±0.06
2.47
±0.08
2.25
±0.06
2.61 *
±0.06
2.28
±0.05
2.70 *
±0.08
0
1.0
1.0
10
10
1981
Discussion
7.0
5.5
20
VOL 3, No
1.48
±0.07
1.25f
±0.08
1.41
±0.07
1.46
±0.04
1.35
±0.05
1.49
±0.06
0.86
±0.03
1.22*
±0.04
0.84
±0.03
1.14*
±0.03
0.93
±0.01
1.28*
±0.06
Values are means ± standard error of mean.
*p < 0.001; fp < 0.01; %p < 0.05; DOCA vs control.
Na
K
412
±11
435
±20
313
±16
0.4
±0.2
1-3J
±0.3
78
±8
349
±7
285
±6
338*
±10
130*
±8
132
±9
181*
±4
Total electrolyte
Cl
mmole/kg dry wt
314
±14
317
±12
314
±14
316
±11
308
±7
344f
±7
concentrations
Ca
Mg
16.2
±1.4
18.6
±0.6
18.9
±1.5
18.0
±0.8
19.8
±2.4
17.3
±0.8
±0.7
16.5*
±0.4
11.9
±0.6
15.2j
±0.7
11.3
±0.4
13.9|
±0.6
11.4
637
SODIUM TRANSPORT AND DOCA HYPERTENSION//on«
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creased T£! x . The turnover of active transport sites
could be elevated under the influence of DOCA. On
the other hand, an increased number of transport sites
may exist, with each having essentially normal transport behavior. Studies of transport protein content
and inhibitor binding (ouabain) would be helpful in
sorting out the possibilities. These are technically
difficult to conduct in rat aorta, which is relatively insensitive to ouabain, however.
Selectivity of the transport sites on the outside of
the membrane was much greater for [K]o than [Na] 0 ,
while the sites on the inside of the membrane were
more selective to Na than to K. These selectivities
were slightly reduced in aortas from the DOCAtreated rats. It is not certain if these are significantly
different, or resulted from random error. The parameters for nearest neighbor interaction were not consistently changed. The lack of definitive alterations in
selectivity and nearest neighbor interaction would suggest that individual transport sites are exhibiting near
normal kinetics during DOCA hypertension, but this
needs further testing.
The parameters were derived for a model based on
cooperative interaction between transport sites.18 This
has its origins in the analysis of cooperative adsorption of ions to fixed charges," and is not put forth as a
unique model. Other multiparameter models predict
sigmoidal transport in red blood cells,**' " and could
fit well within the standard errors associated with the
"Na effluxes.
Studies of active transport in red blood cells have
provided a standard for those in mammalian tissues.
The [K] o stimulation of active transport at normal
[Na] 0 is sigmoidal in red blood cells28 and vascular
smooth muscle (fig. 5), and the [K]?-5 are also similar
at 2 to 3 mM. The stimulation of active transport by
[Na]ceii has a similar [Na]^5,) and follows sigmoidal
kinetics in the aorta (fig. 6) and human red blood
cells.14 The major difference between these cell types is
TABLE 4.
a 100-fold increase in TMJX for vascular smooth muscle. It would be of interest to determine whether this
correlates with a similar increase in the number of
transport sites, as reported for taenia coli.M The active
transport mechanism in vascular smooth muscle
shares a significant number of similar characteristics
with red blood cells, however. This has additional relevance for the study of hypertension. Recent investigations have shown transport abnormalities in red
blood cells from human essential hypertension." 1 " By
inference, alterations may also occur in human blood
vessels, but at present little information is available to
test this speculation.
Interpretation of Effluxes
Some comments are in order concerning interpretation of the " N a effluxes. The assumption was
made that the component remaining after 10 minutes
washout in the cold represents a uniform cellular pool.
The basis for this resides in: the high temperature
coefficient, sensitivity to [K]o, and a uniform rate constant for the washout of over 90% of the " N a pool into
a K-free solution. In addition, the amount of slow Na
increased predictably when the tissues were incubated
in K-free solutions. A recent study of M Na effluxes
from rat tail arteries identified two slowly exchanging
components.™ One has properties similar to those seen
in rat aorta, while a second slow component (approximately 15% of the slow M Na) exchanged at 10% of the
rate for vascular smooth muscle. The authors proposed that the slowest component was associated with
fibroblasts and endothelial cells.** Unlike the tail
artery, the rat aorta has a relatively thin adventitia
and high ratio of smooth muscle to endothelial cells.*0
It is therefore assumed the contribution of nonmuscle
components is negligible.
Interpretation of the " N a effluxes also depends on
the possible contribution of diffusional delays.11 These
(Continued)
Total — 6OC0EDTA
Na
K
191
0.3
±0.2
±8
248J
±22
102
±14
130
±7
82
±4
114*
±5
1.1J
±0.2
77
±8
128*
±8
118
±9
166*
±4
Cl
mmole/kg dry wt
Ca
Mg
Na
93
12.3
±1.3
15.4
±0.8
15.3
±1.4
14.1
±0.8
16.4
±2.4
13.6
±0.8
9.4
±0.6
218
±9
201
±17
122
±16
116
±6
130T
±7
102
±9
96
±7
105
±7
121 "
±6
15.0*
±0.5
10.1
±0.5
13.2$
±0.6
9.6
±0.4
12.0|
±0.6
±9
89
±5
95
±6
Total - 6OC0EDTA
Na + K
K
mmole/kg cell H2O
0.4
218
±9
±0.3
0.9
202
±0.1
±17
92
214
±9
113
±6
128
±10
137
±5
±18
229
±9
217
±13
232
±10
638
HYPERTENSION
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are most prominent in thicker tissues with high rates
of exchange such as those from DOCA-treated rats.
The most extreme condition in this study is the " N a
efflux from aortas that were equilibrated and washed
out in the presence of potassium (table 1). The ratio
between several kinetic parameters is important to the
estimation of diffusional delays. The diffusion constant, D, for Na in arteries is 0.6 X 10"' cma sec"1.11
The wall thickness for the aorta from DOCA hypertensives is 0.19 cm8, which results in a diffusion path,
1, of 0.0095 cm. The ratio R of cellular to extracellular Na in K-loaded aorta is 0.02, and the maximum rate constant associated with this component is
0.445 min"1 (table 1) or 0.74 X 1 0 l sec'1. Tables were
derived for the relation between the ratio of these
parameters and the ratio of the actual rate constant to
the measured one.81 For the above data, kl'R/D
equals 0.002 which yields a ratio of 1.15 (table 1 in ref.
31). The measured rate is therefore about 15% less
than the rate in the absence of any diffusional delay.
The estimated delays were about 5% or less in the control aortas and in Na-loaded aortas from DOCAtreated rats. The diffusional delays would tend to
reduce the increased " N a effluxes observed in aortas
from DOCA-treated rats, and are not a likely source
for the elevated T£;».
VOL 3, No
6, NOVEMBER-DECEMBER
1981
concentrations in vivo where neural and endocrine factors are acutely active. The reduced active uptake of
M
Rb by arteries incubated in the plasma from renal
hypertensive dogs may represent the action of an
acutely active factor that could be washed out under
steady-state in vitro conditions, as shown by the borderline statistical significance associated with that
group. The decrease in active uptake of " R b by tail
arteries from DOCA-treated rats under standard in
vitro conditions (no plasma) 1 ' is at variance with the
observations in this and other studies.'- B The different
protocols used to assay active transport may have led
to some of the apparent differences. For instance, the
M
Rb uptake was measured at [Rb] o = 2 mM, which
would tend to minimize any increase in T m , x and
would exaggerate the effects of a shift in K0. The Rb
uptake was also measured relatively soon after removing the tissue from the animal, and the cellular Na was
high but uncontrolled.1' Some residual hormonal activity could be present, which would be washed out
during the extended protocols of our study. These and
other factors need to be evaluated in future work to
clarify the apparent discrepancies between the studies.
Effects of Altered Transport
The [Na]CTn was similar in DOCA and controls over
a wide range of conditions (fig. 6 and table 4). This
does not offer direct support for the hypothesis that increased [ N a ^ n during hypertension will increase cell
calcium and contraction via the mass action of a NaCa exchange mechanism." The energy for the exchange diffusion of 3 Na is required to remove one Ca
from the cell,18- *a hence the transmembrane ratio for
calcium depends on [Na]'. A 10% increase in [Na] M n
would increase [Cajcn by 33%. If the cells were
operating well below the [ C A ^ n for initiating tension
(10~7 molar)," this increase would be of little consequence. It could have a significant mechanical effect,
however, if the smooth muscle operates near this concentration. Unfortunately, the resolution of the " N a
efflux measurements is not sufficient to detect 10%
alterations in [Na] Mll . A 25% change should be detectable, which would put a two-fold limit on the increase in [Ca] Mn via altered Na-Ca exchange in
DOCA. More information is needed concerning the
normal operating point for [Ca]cn and the kinetics for
Na-Ca exchange before fitting this mechanism into a
causal role in DOCA hypertension. Some evidence for
altered transport of Ca has been found in microsomes
prepared from mesenteric arteries of DOCA hypertensive rats." The DOCA-treated group showed a
reduced ATP dependent accumulation of "Ca into a
fraction that was enriched in plasma membranes.
Such fractions are proposed to contain an ATPdependent transport system for Ca, and reduced activity may be a factor in altered excitation-contraction coupling." At this time little information is
available on fluxes of " C a in intact cells during
DOCA hypertension.
The net effect of the increased active transport in
aortas from DOCA-treated rats is to maintain a constant [NaJcen in the presence of increased permeability to ions. 5 ' 7 These observations were made under
in vitro conditions and do not rule out altered ionic
Evidence was found for increased Na efflux from
DOCA aorta into K-free solution at low [Na]cen but
not at high [Na]^,,. The efflux under these conditions
is a mixture of several components including a passive
leak and exchange diffusion.1' Systematic studies that
Differences in membrane potentials under conditions of varying [K]o and [Najcn may contribute to
the "Na efflux parameters. Little information is available for smooth muscle on which to base quantitative
predictions, however. Since the principal thrust of this
study was to compare the behavior of the controls and
DOCA rats, a primary question is whether the membrane potential has a differential effect on the " N a
effluxes. Hermsmeyer11 observed that the smooth
muscle in the tail artery from SHR depolarized more
than controls when acutely transferred to a K-free
solution, and the SHR artery repolarized to a greater
extent when high [K]o was readmitted. Based on electrochemical considerations, K-free solutions would
tend to increase passive " N a efflux, whereas the
hyperpolarization would decrease it. 1 ' The difference
between these two conditions (K-stimulated Na efflux)
would be reduced in the hypertensives as compared to
the controls if the DOCA group behaves like the SHR
and the electrochemical gradient prevailed. The observations of increased K-stimulated " N a efflux in
DOCA-treated aortas would not appear to result from
such differential effects of [K]o on membrane potential. These considerations underscore the need for
more information on the relation between the membrane potential and ionic fluxes, however, so that
more precise conclusions can be made about cause and
effect relations.
SODIUM TRANSPORT AND DOCA HYPERTENSION//O71M
include sodium substitutes will be needed to separate
the contribution of individual K-independent flux
components to altered transport during DOCA
hypertension.
Cause and Effect Relations
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The cause and effect relations between DOCA-salt
treatment, Na-transport, and blood pressure are not
clearly defined at this time. Elevations in " K efflux
from aorta occur before the development of DOCA
hypertension,4 and changes in contractile activity are
seen in femoral arteries protected from the effects of
high blood pressure by iliac artery ligation." These
findings indicate responses to DOCA-salt treatment
take place independently of a rise in blood pressure
and may be causal to the development of increased
peripheral resistance. A recent study in our laboratory confirmed an elevated U K turnover in the
protected femoral artery of DOCA hypertensives, but
the largest changes took place in the vessels exposed to
the high pressure." Studies need to be done to determine whether the changes in active transport of Na occur independently of rises in blood pressure or are secondary to a rise in pressure and wall tension.
It is also unknown how DOCA (or aldosterone) induces the altered ion transport in blood vessels. Efforts
to determine by what molecular mechanism mineralcorticoids induce changes in nephrons and epithelial
models have not yielded a uniform picture. Evidence
has been reviewed which indicates specific proteins are
synthesized in response to the binding of aldosterone
to receptors.*•• " Increased membrane permeability to
Na and enhanced synthesis of ATP comprise at least
two of the cellular responses to this process.**1" Recent evidence also implicates increased Na-K ATPase
activity in rabbit nephrons during exposure to aldosterone." Both increased permeability and active
transport are consistent with our findings on Na transport in aorta. Insufficient evidence is available to conclude whether these changes are a direct result of
DOCA-salt treatment or whether other factors
produced in the animal act directly on the vascular
wall.
Cell Water
It was confirmed that the cell water per dry weight
was increased in the DOCA aorta. 4 Furthermore, the
elevation was observed in the presence and absence of
K-stimulated Na efflux. As shown in previous studies,
a constant cell volume can be maintained in smooth
muscle during inhibition of active transport which
leads to the loss of more than 99% of the cell K." The
maintained volume indicates that increased cell water
in aortas from DOCA-treated rats does not result
directly from inhibition of K-stimulated transport of
Na, but follows a more complex series of events including synthesis of vessel wall proteins.4' *° Increased
content of Mg in the DOCA-treated group (table 4) is
consistent with the concept of increased cellular protein since Mg is largely adsorbed to ani6nic charges in
the cells.18
639
This study has confirmed that a major alteration in
vascular Na metabolism accompanies the development of DOCA-salt hypertension. An increase in TJS;,
for active transport represents a major change in Na
efflux. This has the overall effect of balancing the increased leak into the cell such that [Najcen is maintained at near normal levels. The involvement of other
Na efflux mechanisms and of the coupling ratio
between active Na efflux and K influx needs to be
systematically investigated for their role in DOCA
hypertension. At present, however, it appears that increased permeability to small ions is more closely
related to the increased responses of vascular smooth
muscle to neural and endocrine stimulation.
Acknowledgments
The author is grateful to Dr. Francis Heidlage who processed the
efflux data through the curve fitting routine that he derived and programmed. I would like to thank Janet Schnauss Cohen and Fumiko
Suzuki for able assistance.
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hypertensive rats.
A W Jones
Hypertension. 1981;3:631-640
doi: 10.1161/01.HYP.3.6.631
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