Cadmium Effect on the Na,K-ATPase System in
Cultured Vascular Smooth Muscle Cells
AIJIROH TOKUSHIGE, M.D.,
HIROKAZU TAMURA, M.D.,
HIROHIKO HIGASHINO, M.D.,
MINORU K I N O , M.D.,
AND ABRAHAM AVIV,
BERNARD M.
SEARLE, P H . D . ,
P H . D . , JOHN D. BOGDEN, P H . D . ,
M.D.
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SUMMARY The present study focuses on the interaction between cadmium (Cd) and the Na, KATPase system in in vitro grown vascular smooth muscle cells (VSMCs) derived from the rat carotid
artery. In disrupted VSMCs rendered permeable by osmotic shock, Cd inhibited Na, K-ATPase; I50
was reached at 10"5M Cd. Mg-ATPase was also inhibited by Cd; 1^ was attained at concentrations of 10~4
M Cd. Cd inhibition of Na,K-ATPase in the VSMCs was noncompetitive with respect to Na, K, and ATP.
Rubidium transport experiments performed with intact VSMCs demonstrated that within an incubation
period of 150 minutes, a concentration of 10~4 M Cd in the extracellular fluid exerted no acute effect on the
Na-K pump. Within this time interval, intracellular Cd attained a concentration eightfold higher than the
extracellular Cd concentration. Thus, it appears that under acute conditions Cd exerts its inhibitory effect
on Na, K-ATPase only in disrupted VSMCs. The data further suggest that, in the VSMC, conditions under
which Cd inhibits Na, K-ATPase are consistent with inhibition from the cytoplasmic side of the cell
membrane. (Hypertension 6: 20-26, 1984)
KEY WORDS
• heavy metals • Mg-ATPase • rubidium uptake • Na-K pump
N
A, K-ATPase (the enzymatic correlate of the
electrogenic Na-K pump) is an essential factor for the regulation of vascular tone.'" 3
Cadmium (Cd) has been reported to inhibit Na, KATPase in homogenates and subcellular fractions in a
variety of organs and tissues originating from different
animal species.6~'° Several studies have shown that
hypertension can be induced by chronic ingestion of
Cd or by acute systemic administration of this element. "~14 However, a number of other investigators
have not been able to induce hypertension in laboratory
animals exposed to Cd.15"17 It is possible that, if it
occurs, hypertension induced by Cd results from inhibition of the vascular smooth muscle cell (VSMC)
Na,K-ATPase. Thus, knowledge pertaining to the
interaction between Cd and the VSMC Na, K-ATPase
could be of substantial importance.
In the present study we have investigated the impact
of Cd on the Na, K-ATPase system in in vitro grown
VSMCs by examining its uptake by VSMCs and by
observing its effect on the following cellular modalities: 1) the specific activity and kinetics of Na, KATPase as assayed by the ouabain sensitive hydrolysis
of ATP; and 2) the operation of the Na-K pump as
measured by the ouabain-sensitive rudibium (^Rb) uptake by VSMCs. The data reported herein demonstrate
thatCd exerts a substantial effect on the VSMC Na, KATPase only in disrupted VSMCs but has no demonstrable acute effect on the Na-K pump in intact
VSMCs.
From the Division of Pediatric Nephrology and Human Genetics,
and the Department of Preventive Medicine and Community
Health, University of Medicine and Dentistry of New Jersey, New
Jersey Medical School, Newark. New Jersey.
A portion of this work was presented at the 15th annual meeting
of the American Society of Nephrology (1982) and was published in
an abstract form.
Supported in part by Grant 1 R23 HL-29221 from the National
Heart, Lung, and Blood Institute, and by a grant from the American
Heart Association. New Jersey Affiliate.
Dr. Tokushige is a Research Fellow of the American Heart Association, New Jersey Affiliate. Dr. Kino is a Research Fellow of the
National Kidney Foundation.
Current address for Dr. Higashino: Department of Pediatrics,
Kansai Medical University. Osaka, Japan.
Address for reprints: Abraham Aviv. M.D., Division of Pediatric
Nephrology. University of Medicine and Dentistry of New Jersey,
New Jersey Medical School, 100 Bergen Street. Newark, New
Jersey 07103.
Received March 8. 1983; revision accepted August 4, 1983.
Methods
The VSMCs in these experiments originated from
carotid arteries of male adult Sprague-Dawley rats.
The cells were grown in Dulbecco's Modified Eagle
Medium (DMEM, Gibco 320-1885) plus 292 /xg/ml of
L-glutamine, antibiotics (50 /ng penicillin, 150 fig
streptomycin, and 150 /xg neomycin per ml) and 17%
heat-inactivated fetal calf serum (FCS). The techniques for growing the cells and their enrichment have
already been described.18
20
CADMIUM AND VASCULAR SMOOTH MUSCLE CELL NA, K-ATPASEJTokushige et al.
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The acute effect of the Cd on Na,K-ATPase in the
VSMCs was examined as follows. Aliquots consisting
of 0.8 to 1.0 X 105 cells were inoculated into each well
of Costar 24-well plates. DMEM plus 17% FCS without antibiotics was used to grow trie cells in each well.
The effect of Cd on Na, K-ATPase activity was measured 48 hours after inoculation of the cells when the
number in each well was approximately 2.5 x 105.
The techniques for in situ assays of VSMC Na, KATPase have been described.18 Briefly, the medium
was aspirated from each well and the cell layers
washed twice with 150 raM Tris-HCl (pH 7.4). The
VSMCs permeability to the reactants (used in the substrate solutions for the enzymatic assays) was then
increased by adding distilled water to the wells and
placing the plates in dry ice for 30 minutes. Thereafter,
the cells were thawed at 37°C.
The substrate solution used to measure Na, KATPase consisted of the following, in mM: NaCl 100,
KC1 10, MgCl2 5, imidazole-HCl 100, ATP 3 (pH
7.4). Ouabain (10~3 M) was added and KC1 omitted to
inhibit Na, K-ATPase activity. Variable concentrations of ATP, Na, and K were used for the enzyme
kinetic analyses. Blank wells containing cells treated
prior to incubation with 30% trichloroacetic acid
(TCA) were used for measurements of the nonenzymatic hydrolysis of ATP. The reaction in all other wells
was terminated after the incubation period at 37°C for
30 minutes by placing the plates on ice and by the
addition of 250 pil ice cold 30% TCA.
The inorganic phosphate (P,) generated was measured using the Fiske and Subbarow method,19 protein
was determined by the Lowry et al. method,20 and the
cell number was counted by a Coulter Counter (Model
ZBI). The specific activities of Na, K-ATPase and the
ouabain insensitive Mg-ATPase were expressed as
/nmol P, generated per mg cell protein or per 10* cells
per hour. In preliminary experiments, we demonstrated no effect of different concentrations of Cd (in the
form of CdCl2) on the activity of Na, K-ATPase when
it was mixed in the substrate solutions used to assay the
enzyme. The most likely reason for this finding was
the binding of Cd to imidazole, which was used as the
buffer system in the substrate solutions. The inhibition
by Cd of Na, K-ATPase in the VSMCs could, however, be easily demonstrated when Cd was added during
the process of disrupting the cells prior to the introduction of the substrate solutions. Thus, prior to the enzymatic assays, the cells were preincubated in the presence or absence of Cd for 20 minutes at 37°C.
The effect of Cd on Rb uptake by intact VSMCs was
measured as follows. The medium containing FCS was
aspirated and fresh DMEM (without FCS) containing
approximately 1 /xCi/ml of **Rb and 0.1 mM RbCl was
added to the wells. When appropriate, CdCl, at a concentration of 10~4 M was also added. The K and Na
concentrations in the DMEM were 7.0 and 140 mEq/
liter, respectively. The cells were incubated at 37° C
and 5% CO2-air for various time intervals. The uptake
experiments were stopped by aspiration of the medium
and rapidly washing the cells four times with ice cold
21
0.01 M MgCl2. The cells were then extracted for 1
hour with 5% TCA: aliquots were added to Aquasol-2
(New England Nuclear, Billerica, Massachusetts) and
measured in a liquid scintillation counter.
The uptake of Cd by the VSMCs was measured
using the following method. DMEM (without FCS)
containing 10"4 M CdCl2 with approximately 0.4 /xCi
l09
Cd/ml was added to the wells. The cells were incubated for various time intervals, at 37° C and 5% CO2air, and then rapidly washed four times with a solution
of 150 mM NaCl with/without 10" 4 EDTA. The
VSMCs were then subjected to either one of the following treatments: extraction with 5% TCA, or trypsinization (0.01% trypsin in phosphate buffer). Aliquots of these preparations were counted in a gamma
counter. In this set of experiments we also examined
the intracellular water space using 3-0-methyl-D-glucose, as described by Kletzien et al. 2 ' for liver parenchymal cells and Brock and Smith22 for cultured
VSMCs. Briefly, the cells were incubated for various
time intervals (2 to 20 minutes) in phosphate buffer
containing 1-2 /u.Ci/ml of 3-0 [methyl-l4C]-D-glucose
and either 2 or 10 mM of unlabeled 3-0 methyl-Dglucose. The equilibrium distribution of 3-0-methylD-glucose was reached after 10 minutes of incubation.
Following this period, the cells were washed four
times with an ice cold phosphate buffer containing 1
mM phloretin. The cells were then either extracted
with 5% TCA or trypsinized. Aliquots of the preparation were counted in a liquid scintillation counter using
Aquasol-2 as the scintillation fluid. There were no
differences between the activities of the trypsinized
samples and samples extracted with TCA. The availability of the measurements of the intracellular water
space made it possible to express the amount of Cd
gaining access to the interior of the VSMCs as concentration per unit of cellular water volume. Data are
presented as means ± SEM. Statistical analyses have
utilized the Student's t test.
Results
The specific activity of Na, K-ATPase in the
VSMCs was 2.37 ± 0.05 /xmol P/mg protein/hr or
0.82 ± 0.02 /xmol P/lO^ell/hr. The specific activity
of Mg-ATPase was 2.44 ± 0.06 /xmol P/mg protein/
hr or 0.82 ± 0.02 /u.Mol P/10 6 cells/hr. These results
are quite similar to those observed in our previous
studies.18 Cd exerted a potent inhibitory effect on the
VSMC Na, K-ATPase. Figure 1 depicts the dose response curve for Cd inhibition of the VSMC Na, KATPase. The I,,, (50% inhibition) by Cd was reached at
concentrations of approximately 10" 5 M and almost
complete inhibition of the enzyme was evident at 10~3
M. Mg-ATPase was approximately 10-fold more resistant to inhibition by Cd than the Na, K-ATPase
(Figure 1).
Enzyme kinetic experiments (Figure 2) demonstrated that inhibition of the VSMC Na, K-ATPase by Cd at
concentrations of 10~3 M resulted from an acute reduction in the V ^ (maximal rate of the enzymatic reac-
22
HYPERTENSION
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tion) rather than as a consequence of alteration in the
apparent K^ (criterion for affinity) of the enzyme to K,
Na, or ATP. When measuring the effect of Cd on the
ATP kinetics, the specific activity of Na, K-ATPase in
the VSMCs was somewhat lower than in the other
kinetic experiments. However, the inhibitory effect of
Cd on the enzyme was quite apparent. At ATP concentrations of 7 mM, there was a marked decline in the
specific activity of the enzyme in the presence or absence of Cd. This was observed previously by us and
others.1823 This decline in the activity of the enzyme at
higher ATP concentrations is likely to be due to substrate inhibition.
The capacity of the chelating agent EDTA to prevent
or reverse Cd inhibition of the VSMC Na, K-ATPase
and Mg-ATPase is shown in Figure 3. EDTA was
partially effective in preventing the inhibitory effect of
IO-5and l(T 4 MCdwhen 10"4 M of the chelator was
introduced into the medium concurrently with Cd.
However, the addition of 10~4 M EDTA after 20 minutes of preincubation with 10~5 M Cd (third, crosshatched, bar in Figure 3 top) was less effective in
reversing the inhibitory effect of the element than when
introduced concurrently with Cd. The addition of
EDTA at 10"4 M concentrations after 20 minutes incubation with 10"4 Cd (sixth, cross-hatched, bar in Figure 3) was ineffective in reversing Cd inhibition of the
enzyme. Similar observations were noted in regard to
the effect of Cd on Mg-ATPase. EDTA at a concentration of 10~4Mhad no effect on the basal activity of Na,
K-ATPase.
100
80
i
VOL
1, JANUARY-FEBRUARY
1984
20
(mEq/L)
B
a.
I
-A
-
6, No
j
20
No+
i
100
40
(mEq/L)
V
V—
<t
o
60
\
O
01
X
40
o
o
20
10,-7
1
10'r 6
1
D
10"
Cd(M)
1
10"
^ ^
10,-3
FIGURE 1. Dose response curve for the effect of cadmium
(Cd) on the VSMC Na, K-ATPase (•) and Mg-ATPase (A). The
y axis represents the percentage of activity of the enzyme with
respect to control. Each point represents se\>en to eight pairs of
wells. Horizontal bars represent SEM.
0
\
2
i
b
r
ATP (mM)
FIGURE 2. Kinetics of Na, K-ATPase under control conditions (•), or in the presence of 10 5 M Cd (O). A. Potassium
kinetics. B. Sodium kinetics. C. ATP kinetics. Each point represents eight to 12 pairs of wells.
CADMIUM AND VASCULAR SMOOTH MUSCLE CELL NA, K-ATPASE/Tokushige et al.
I
100
23
led
80
60
y 40
o
P,
20
10" 5 Mol Cd
I0" 5 Mol Cd
Mol Cd
I 0 " 4 Mol Cd
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FIGURE 3. The effect of 10~4 M EDTA on cadmium (Cd) inhibition ofNa,K-ATPase (A) and Mg-ATPase (B) at / 0 " 5 M and IO~4 M
Cd concentrations. Cd + IO~4M EDTA represents a condition in which both Cd and 10 4 M EDTA were preincubated together with
theVSMCs. Cd-+IO~4 M EDTA represents experiments starting with preincubat ion with Cdfollowed by the addition of IO~4 M EDTA.
Each bar in A represents nine to 16 pairs of wells. Each bar in B represents II to 22 pairs of wells **Significant with respect to the
effect of Cd alone at p < 0.01.
Rb uptake experiments (Figure 4) demonstrated no
effect of 10~4 Cd on the rate of ouabain-sensitive Rb
uptake within the minimum incubation time of 150
minutes. There was also no effect of Cd on the ouabain-insensitive uptake of Rb (not shown in figure 4).
Despite the presence of Cd in the medium, the pump
demonstrated a full capacity to increase its activity in
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FIGURE 4. Ouabain-sensitive Rb uptake by intact VSMCs as a
function of time. (•) control, (O) IO~4 M Cd, (A) 5 fig/ml
monensin, (A; IO'4 M Cd + 5 uglml monensin. Each point
represents six to 12 pairs of wells.
£
response to an increase in the intracellular Na concentration, which was induced by introducing 5.0 /xg/ml
of monensin. As shown, Rb uptake by the VSMCs was
increased to the same extent in the presence or absence
of Cd in the medium when the ionophore was added
into the medium. After 150 minutes of incubation, the
cumulative uptake of Rb by the VSMC was 9.6 ± 0.5
nmol/mg protein. Addition of monensin to the medium
resulted in almost doubling the rate of ouabain-sensitive Rb uptake to 18.5 ± 1.4 nmol/mg protein in 150
minutes. Cd did not alter this response.
Cd uptake by the VSMCs is presented in Figure 5.
Approximately 50% of the Cd taken up by the cells
was chdated by EDTA when the latter was added to
the washing solutions. After 150 minutes of incubaf
ion, cells washed with 150 mM NaCl and extracted
with 5% TCA showed a cumulative uptake of 3.50 ±
0.20 nmol Cd/mg protein or 1.30 ± 0.01 nmolCd/10 6
cells. Cells washed with 150 mM NaCl + 10" 4 M
EDTA and then extracted with TCA demonstrated Cd
uptake of 1.70 ± 0.07 nmol/mg protein or 0.66 ±
0.02 nmol Cd/106 cells in 150 minutes of incubation.
The most likely explanation for this discrepancy is that
a substantial percentage of Cd was bound to the plasma
side of the cellular membrane and that this bound Cd
was chelated by the EDTA. There were no statistically
significant differences in cellular Cd values obtained
from measurements of Cd by its extraction from the
cells with TCA or by measurements of total Cd in the
trypsinized cells (Figure 5). These observations suggest that within the experimental time period Cd gaining access into the cell interior was reversibly or loosely bound to cellular elements.
24
HYPERTENSION
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FIGURE 5. Cadmium (Cd) uptake bx intact VSMCs as a function of time. (Oj cells washed with 150 mM NaCl and extracted
with 5% TCA; (A) cells washed with 150 mM NaCl and then
trypsinized; (•) cells washed with ISO mM NaCl + 10~4 M
EDTA and extracted with 5% TCA; (A) cells washed with ISO
mM NaCl + IO~4 EDTA and then trypsinized. Each point
represents four to six pairs of wells.
Using the measurements of the 3-0-methyl-D-glucose, we determined that the intracellular water space
of the VSMCs was 0.81 ± 0.01 /xl/IO6 cells. Therefore, the concentration of Cd gaining access to the
intracellular space after 150 minutes' incubation was
8.10 ± 0.26 x 10"4 M, a concentration substantially
higher than that of the extracellular Cd concentration.
Discussion
It is well recognized that some of the heavy metals
inhibit Na, K-ATPase in homogenates and subcellular
fractions derived from a variety of tissues. 6 ^ 24 25
Brain and kidney exhibit high activity of Na, KATPase. Thus, investigators have often used tissues
obtained from these organs as model systems to study
the interaction between heavy metals and Na, KATPase. Homogenates and subcellular fractions of
blood vessels demonstrate low specific activity of ouabain-sensitive Na, K-ATPase. In addition to VSMCs,
blood vessels consist of connective tissue and other
nonmuscle elements. Thus, the data generated by investigations examining Na, K-ATPase in homogenates
or subcellular fractions of blood vessels are not due
only to this enzyme in VSMCs but are rather a function
of a myriad of cellular and extracellular components of
blood vessels. Tissue culture preparations can provide
a useful model to study the effect of various compounds on the VSMC Na, K-ATPase. In vitro preparations of VSMCs are devoid of influences of nonVSMCs and extracellular factors. Therefore, such
preparations are ideally suited to study the effects of
agents on the VSMC Na, K-ATPase. We recently de-
VOL 6, No
1, JANUARY-FEBRUARY
1984
veloped methods to assay Na, K-ATPase in preparations of in vitro grown VSMCs.18 These techniques
have made it possible to examine, among other factors,
the effect of metal ions on the VSMC Na, K-ATPase
system. Our initial investigations have focused primarily on Cd because divalent cadmium has been reported
to produce hypertension in laboratory animals."" 14
This effect could be mediated via inhibition of VSMC
Na, K-ATPase.
The present study demonstrates that VSMC Na, KATPase is quite sensitive to Cd inhibition. The 1^ for
Cd inhibition of the enzyme in our experiments was
reached at approximately 10~5 M. Other investigators
reported 1^ values of 10" 6 M for Cd inhibition of Na,
K-ATPase in microsomal preparations of the rat kidney and rat brain synaptosomes. 8 ' l0 Na, K-ATPase
preparations derived from the human or canine kidneys
are more resistant to Cd inhibition with reported 1^
values being attained at 10~4 M.6-26 Our observation
that Mg-ATPase is more resistant to Cd inhibition than
Na, K-ATPase concurs with previous findings demonstrating that Mg-ATPase derived from the brain and
kidney was 10- to 1000-fold more resistant to Cd inhibition than Na, K-ATPase.6-8-10-26
It is very unlikely that the inhibitory effect of Cd on
the VSMC Na, K-ATPase is specific to this enzyme
system. Heavy metals exert their toxicity at the cellular
level by several mechanisms that may result in altering
the activities of a variety of enzymes.27 Perhaps the
most likely mode of inhibition of VSMC Na, KATPase relates to the high affinity of Cd and other
heavy metals for sulfhydryl groups.27 Na, K-ATPase is
rich in sulfhydryl groups, and sulfhydryl reagents inhibit the enzyme.28 x We have shown that other heavy
metals such as lead and mercury also inhibit the VSMC
Na, K-ATPase.30 Similar observations were reported
by others who examined the effect of heavy metals on
Na, K-ATPase in a variety of tissues. The inhibitory
effect of Cd on the VSMC Na, K-ATPase was probably mediated via toxic damage to the enzyme units, as
evidenced by the following observations: 1) the inhibition of the enzyme by Cd was exerted by alterations of
the v
™ ; 2) at I0- 5 M Cd, 10"4 M EDTA was only
partially capable in reversing the inhibitory effect of
the element; and 3) at 10"4M Cd, 10~4 M EDTA was
ineffective in reversing Cd inhibition. In the disrupted
VSMCs, reversal of Cd inhibition by EDTA would
have been expected had Cd not exerted irreversible
toxic effect on the Na, K-ATPase system.
Na, K-ATPase is an enzyme that is primarily associated with the plasma cell membrane. Its inhibition can
be exerted from the extracellular (plasma) side of the
membrane or from the intracellular (cytoplasmic) aspect of the membrane. Ouabain and vanadate, two
inhibitors of the enzyme, exert their effect from the
extracellular and intracellular sides of the cell membrane, respectively. Using our methods to assay the
enzyme in disrupted VSMCs made it impossible to
determine whether Cd inhibition of Na, K-ATPase in
the VSMC was exerted from the cytoplasmic or plasma
sides of the membrane. To measure the hydrolysis of
CADMIUM AND VASCULAR SMOOTH MUSCLE CELL NA, K-ATPASE/Tokushige el al.
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ATP by Na, K-ATPase, we subjected the cells to an
osmotic shock prior to the enzymatic assays, causing
cellular disruption. This procedure was necessary in
order to make ATP and other reactants in the substrate
solutions freely accessible to the interior of the cell.
The increased VSMC permeability was also likely to
alter cellular mechanisms and to render the vast extracellular Cd pool readily accessible to the cell interior.
Studying Rb uptake by the intact VSMCs concurrently
with the enzymatic assays enhanced our understanding
of the mode of Cd interaction with the VSMC Na, KATPase.
Since Cd in the extracellular fluid of the intact
VSMCs appeared to exert no effect on the Na-K pump,
it was essential to measure the uptake of Cd by the
intact VSMCs within the given experimental time intervals. It has been shown that EDTA does not cross the
skeletal muscle cell membrane.31 The same is likely to
be the case with respect to the VSMC. Thus, our observations that washing the VSMCs with solutions containing EDTA resulted in less Cd presence in these
cells suggest that the Cd removed by the EDTA was
bound primarily to the exterior of these cells.
The Rb uptake experiments clearly showed that, at
10~4 M concentrations in the medium, Cd exerted no
detectable inhibition of the activity of the Na-K pump.
(At this concentration of Cd in the medium, more than
50% of Na, K-ATPase was inhibited when the cells
were rendered permeable by osmotic shock). That the
Na-K pump was operating normally in the presence of
Cd in the extracellular fluid was demonstrated by the
response of the VSMCs to the introduction of monensin to the medium. It has been shown previously that
monensin rapidly increases the intracellular sodium
and that associated with this increase was an augmented activity of the Na-K pump.32 Our data demonstrate
that Cd did not alter the Na, K-ATPase response to
monensin. In other experiments we showed that vanadate was a potent inhibitor of Na, K-ATPase in VSMC
preparations rendered permeable by osmotic shock.33
The I,,, for vanadate inhibition was reached at 10~7 M,
and almost complete inhibition was achieved at 10"5
M concentrations of vanadate. However, in a manner
similar to the present experiments on Cd, vanadate at
10"5 M in the extracellular fluid concentration did not
alter Rb transport by the intact VSMCs.
The most likely explanation for the inability of Cd to
inhibit the Na-K pump in intact VSMCs, despite its
high intracellular concentration, is its binding to other
intracellular proteins and/or its sequestration in subcellular organelles. These may be the reasons for the
intracellular accumulation of Cd to concentrations
higher than its extracellular levels. Another less likely
possibility is that a specific pump actively transporting
Cd against its concentration gradient exists in the
VSMC membrane.
The present study does not attempt to support or
refute any of the opposing observations relating to the
in vivo effect of Cd on blood pressure because it is
difficult to extrapolate in vitro findings of the acute
effect of Cd on VSMC ATPase to the in vivo effects of
25
this heavy metal. Our data demonstrate, however, that
Cd exerts an acute inhibitory effect on Na, K-ATPase
in disrupted VSMCs and that the Na-K pump is not
inhibited in intact VSMCs by Cd concentrations that
generally inhibit Na, K-ATPase in disrupted VSMCs
or in homogenates and subcellular fractions derived
from a variety of organs and tissues. Finally, the study
underscores the necessity to assess the function of the
Na, K-ATPase (Na-K pump) by independent techniques.
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Hypertension. 1984;6:20-26
doi: 10.1161/01.HYP.6.1.20
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