Potassium Relaxation of Vascular Smooth Muscle
from DOCA Hypertensive Pigs
R. CLINTON W E B B , P H . D .
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SUMMARY This study was designed to characterize potassium-induced relaxation in vascular
smooth muscle during the development of deoxycorticosterone acetate (DOCA) hypertension. Pigs
were implanted subcutaneously with 100 mg/kg DOCA. Mean arterial pressure in the DOCA-treated
pigs reached levels approximately 37% greater than controls. In some pigs, the left hindlimb vascular
bed was "protected" from the rise in arterial pressure by ligation of the iliac artery. Arterial strips
from DOCA hypertensive and normotensive pigs relaxed in response to potassium after contraction
induced by norepinephrine in potassium-free solution. Arterial strips from DOCA hypertensive pigs
showed greater relaxation than did those from normotensive pigs. The magnitude of relaxation in
femoral arteries from "protected" hindlimbs was similar to that in arteries from the contralateral
unoccluded limb. Potassium-induced relaxation in tail arteries from DOCA hypertensive pigs was
more sensitive to ouabain inhibition than that from normotensive pigs. Relaxation induced by potassium varied with: 1) length of incubation in potassium-free solution; 2) concentration of added
potassium; and 3) concentration of norepinephrine added during the potassium-free interval. The
amplitude of potassium-induced relaxation is believed to be a functional index of the activity of the
electrogenic sodium-potassium transport system. These experiments support the hypothesis that
vascular smooth muscle from DOCA hypertensive animals has increased electrogenic sodium pump
activity. The development of this vascular change parallels the increase in blood pressure induced by
mineralocorticoid excess. (Hypertension 4: 609-619, 1982)
KEY WORDS • electrogenic pump * ouabain
• femoral artery • renal artery
A
sodium
• norepinephrine
• tail artery
sodium pump has a similar temperature dependence7
being inactivated by temperatures around 20°C. Other
experimental conditions (potassium, sodium, and
magnesium concentration, ouabain, monovalent ion
specificity) also alter the magnitude of potassium-induced relaxation in exactly the same manner as that
predicted from the literature dealing with that variable
on the sodium pump.
In hypertensive humans and animal models, vasodilator responses to potassium in intact vascular beds are
attenuated.2 8>9 These observations led to the hypothesis that the activity of the electrogenic sodium pump
in vascular smooth muscle is depressed during hypertension. In contrast, recent observations in this laboratory indicated that isolated vascular segments from rats
with genetic hypertension consistently show greater
relaxation in response to potassium,10 suggesting an
enhanced pump activity. The current study was intended to add evidence bearing on the controversial reports
assessing potassium-induced relaxation of vascular
smooth muscle in hypertension and to further characterize vascular changes in mineralocorticoid-induced
hypertension.
N appropriate increase in the extracellular
concentration of potassium ion produces va. sodilation in intact vascular beds' 2 and relaxation of isolated blood vessel segments.3-4 This action
of potassium is believed to be the result of activation of
the electrogenic sodium pump producing membrane
hyperpolarization which causes the relaxation of the
vascular smooth muscle.3"5
Recent observations6 suggest that potassium-induced relaxation of isolated vascular segments may be
used as a functional indicator of electrogenic sodium
pump activity. For instance, the magnitude of potassium-induced relaxation in rat tail artery strips decreases to zero as the temperature of the bathing medium is reduced from 37° to 20°C. The electrogenic
From the Department of Physiology, University of Michigan
Medical School, Ann Arbor, Michigan.
Supported by a grant from the Michigan Heart Association, by a
grant from the Michigan Memorial Phoenix Project, and by Grant
HL-18575 from the National Institutes of Health. Dr. Webb is a
recipient of Research Career Development Award HL-00813 from
the National Institutes of Health.
Received May 19, 1981; revision accepted February 1, 1982.
609
HYPERTENSION
610
Methods
Animals
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Young male feeder pigs (20-30 kg, Chester White
or Yorkshire) were purchased from local farmers. The
animals were housed in individual cages and given
Purina Pig Chow (Growena) and tap water ad libitum.
Chronic instrumentation for measurement of cardiac
output, aortic blood pressure, and femoral arterial
pressure was performed as described previously." 12
After 5 to 10 days of stable baseline measurements, the
pigs were anesthetized with thiamylal (Surital), and
underwent subcutaneous implantation (into the left
flank) of deoxycorticosterone acetate (DOCA) impregnated in Silastic rubber strips. This implant contained
100 mg/kg DOCA. Silastic rubber strips without
DOC A were implanted in control pigs. A recent review article13 details various changes (e.g., hormonal
effects, electrolyte balance) which characterize this
model of mineralocortocoid hypertension.
Tissue Preparation
Prior to and at intervals after implantation of the
Silastic strips, the pigs were anesthetized with thiamylal and a segment of the tail (4—6 cm) was removed.
The tail artery (0.8 to 1.0 mm o.d.; ventral caudal
artery) was rapidly excised and transferred to a dissection dish containing physiological salt solution (PSS).
The vessel was cut helically into strips (0.8 — 1,0 x
10 mm) under a dissecting microscope. The strips were
mounted vertically on a glass holder in a tissue bath
containing 50 ml of PSS. The upper end of each strip
was connected to a force transducer (Grass FT.03) and
the resting tension of each strip was adjusted so that it
produced maximum active force in response to a standard dose of norepinephrine (10~7 g/ml). Before the
start of experiments, the strips were allowed to equilibrate for 90 to 120 minutes in PSS. The bathing medium was maintained at 37°C and aerated with a mixture
of 95% O2 and 5% CO 2 . The pH of the PSS was 7.4 and
the composition (mmole/liter) was as follows: NaCl,
130; KC1, 4.7; KH2PO4, 1.18; MgSO4 • 7H2O, 1.17;
CaCl2 • 2H2O, 1.6; NaHCO3, 14.9; dextrose, 5.5;
CaNa2 EDTA, 0.03. The potassium concentration in
the bath was varied without compensating for changes
in tonicity.
Additional experiments were performed on helical
strips (1.0 x 10.0 mm) of renal arteries and femoral
arteries removed from the pigs when the study was
terminated. The femoral arteries were obtained from
pigs in whom the left iliac artery had been ligated to
protect the distal vascular bed from the increase in
arterial pressure which accompanied the implantation
of DOCA.12
The results of these experiments were analyzed by a
variety of statistical procedures. Dose-response curves
were calculated as geometrical means. Paired and unpaired t tests and curve fitting analyses (probit transformation) were performed. A p value less than 0.05 was
considered to be statistically significant.
VOL 4, No 5, SEPTEMBER-OCTOBER
1982
Drugs used were: norepinephrine (Levophed bitartrate, Winthrop Laboratories, New York, New York),
deoxycorticosterone acetate (Sigma Chemical Company, St. Louis, Missouri) and ouabain (Nutritional
Biochemical Corporation, Cleveland, Ohio).
Results
Animals
A total of 29 pigs were used in these experiments: 14
received DOCA in Silastic implants, 11 received Silastic implants without DOCA, and four received no implant (table 1). All pigs implanted with DOCA demonstrated elevations in mean aortic blood pressure within
the first 3 to 5 days. At 55 ± 6 days postimplantation,
the mean aortic pressure reached a plateau at approximately 37% above the preimplant values. Pigs that
received Silastic implants without DOCA showed no
significant changes in mean aortic blood pressure from
preimplant values.
Ligation of the left iliac artery was performed in
three DOCA-treated pigs and four normotensive control pigs (Silastic implant without DOCA). This arterial ligation reduced the arterial pressure in the femoral
artery distal to the ligation. The pressure gradient between the aorta and the left femoral aratery was approximately 27 mm Hg in the DOCA-treated pigs and
approximately 10 mm Hg in the normotensive pigs
when the study was terminated.
It was evident from the body weights that the pigs
were growing rapidly. There was no significant difference in weight gain between the normotensive pigs and
the DOCA-hypertensive pigs.
TABLE I.
Blood Pressures and Body Weights
Femoral
Mean
arterial
aortic
pressure
pressure
(mm Hg) (mm Hg)
Preimplant pigs
(n = 29)
Body
weight
(kg)
100 ± 2
—
34.2 ± 1.8
DOCA 3-5 days postimplant (n = 14)
113 ± 3*t
—
38.4 ± 2.4*
DOCA 55 + 6 days
postimplant (n = 14)
137 ± 2*t 110 ± 4
65.4 ± 3.3*
(n = 3)
Silastic 3-5 days postimplant (n = 11)
Silastic 47 ± 7 days
postimplant (n = 11)
98 ± 2
105 ± 2
—
36.0 ± 3.0*
95 ± 3* 62.2 ± 5.8*
(n = 4)
'Significant difference between preimplant and postimplant values (p < 0.05).
tSignificant difference between DOCA-implanted pigs and Silastic-implanted control pigs at 3 to 5 days post implant (p < 0.05).
tSignificant difference between chronic DOCA-implanted pigs
and chronic control pigs (p < 0.05).
POTASSIUM RELAXATION IN DOCA-HYPERTENS1VE P\GS/Webb
611
I mm
4 0 0 mg
Pre-implant
I mm
800 mj J
Silostic
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400
DOCA
Hypertensive
Potassium-free
PSS, 17 min
I0' 7 g/ml
Norepinephrine
15 mM KCI
FIGURE 1. Relaxation induced by potassium. Helical strips of tail arteries from DOCA hypertensive and
normotensive pigs relaxed in response to potassium (I5.0mM) after contraction induced by norepinephrine (10~7
g/ml) in potassium-free solution. Tail artery strips from DOCA hypertensive pigs consistently showed greater
relaxation in response to potassium than those from normolensive pigs. Following the increase in mechanical
reponse which occurred after several minutes of relaxation, the strips were returned to normal PSS (5.0 mM
potassium).
Relaxation Induced by Potassium
The tracings in figure 1 illustrate the procedure used
to evaluate potassium-induced relaxation in isolated
vascular segments from pigs. Helical strips of tail artery were placed in a potassium-free solution for 17
minutes. At 14 minutes into this interval, norepinephrine (10~7 g/ml) was added to the muscle bath. Three
minutes later, when the contractile response to norepinephrine had reached a plateau, the bath concentration of potassim was increased to 15.0 mM and an
abrupt relaxation occurred. The resultant relaxation
was quantified as a percentage of the total contraction
that existed just before the potassium was added. Tail
artery strips from DOCA-hypertensive pigs relaxed to
a greater percentage of their norepinephrine contraction than those from normotensive control pigs. Following several minutes of relaxation, a spontaneous
and abrupt increase in mechanical response was observed in arterial strips from both DOCA-hypertensive
and normotensive pigs. Following the increase in mechanical response, the arterial strips were returned to
normal PSS.
The magnitude and duration of the relaxation induced by potassium varied with the time of exposure to
potassium-free solution (fig. 2). When the interval of
time was short, the magnitude of the relaxation was
small (fig. 2 left) and the duration of the relaxation was
short (fig. 2 right). Increasing the interval caused the
potassium-induced relaxation to be greater in magnitude and in duration. Helical strips of tail artery from
DOCA-hypertensive pigs showed greater relaxation in
response to the addition of potassium than did those
from normotensive pigs (preimplant or chronic pigs).
The duration of the relaxation tended to be longer in
tail artery strips from DOCA-hypertensive pigs.
Magnitude of Contraction to Norepinephrine
and Potassium-Induced Relaxation
To determine whether the magnitude of the norepinephrine response was responsible for the difference in
relaxation between DOCA-hypertensive and normotensive pigs, norepinephrine concentrations of 10~9 to
10~5 g/ml were used to produce variations in the magni-
HYPERTENSION
612
VOL 4, No 5, SEPTEMBER-OCTOBER 1982
10
I 2Q
Silgstic (N=5)
I
I 40
^
I
Pre-imp|ant (N=8)
8
6
DOCA
60
DOCA (N=6)
Silastic
£ 80
Pre-implant
10
100
20
30
10
Minutes in Potassium-free PSS
20
30
Minutes in Potassium-free
PSS
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FIGURE 2. Potassium-induced relaxation following different intervals in potassium-free PSS. Potassium-induced relaxation of tail
artery strips was performed as described in figure 1 except that the period of time the arterial strips were exposed to potassium-free
solution was varied. The magnitude (leftj and the duration fright] of the relaxation varied with the time of exposure to potassium-free
solution. Values are the means ± standard error of the mean (SEM). * = statistically significant difference between DOCAhypertensive and Silastic-implanted normotensive pigs (p < 0.05). t = statistically significant difference between DOCA-hypertensive and preimplant pigs (p < 0.05). Values in parentheses indicate the number of pigs.
tude of contraction (fig. 3 left). The percent relaxation
induced by potassium was much greater when a lower
concentration of norepinephrine was used in all arterial
strips. Tail artery strips from DOCA-hypertensive pigs
relaxed to a greater percentage of their norepinephrine
contraction at all concentrations of norepinephrine
(fig. 3 right) as compared to those from normotensive
pigs (either preimplant or with elastic implant). The
concentration of norepinephrine at which there was a
half-maximal contraction was lower in tail arteries
from DOCA-hypertensive pigs (table 2). The maximal
contractile responses of tail artery strips from DOCA-
0
100
1
Silastic
DOCA (N=4)
80
20
1
40
40
$
60
I.
<**
80
I
60
Pre-implant
y/f
't
Pre-implant (N=6)
?
20
/ / /
1
lO" 9
100
I0- 7
[Norepinephrine]
KT
(g/rnl)
D
O
C
A
}*t
10-9
1a5
I0- 7
[Norepinephrine]
(g/ml)
FIGURE 3. Influence of contractile magnitude on potassium-induced relaxation. Potassium-induced relaxation of tail artery strips
was performed as described in figure 1 except that the contractile state of the strips was altered by changing the concentration of
norepinephrine added during the potassium-free cycle. The magnitude of the potassium-induced relaxation (rightj varied inverse!}1
with the magnitude of the norepinephrine contraction (left,). Values are the mean ± SEM. * = statistically significant difference
between DOCA-hypertensive and normotensive control pigs (p < 0.05). t = statistically significant difference between DOCAhypertensive and preimplant pigs (p < 0.05). The values in parentheses are the number of pigs.
613
POTASSIUM RELAXATION IN DOCA-HYPERTENSIVE PIGS/Webb
hypertensive pigs were less (1908 ± 4 1 1 mg) than
those from Silastic implanted pigs (3100 ± 3 2 1 mg)
but not those from preimplant pigs (1677 ± 196 mg).
Effect of Ouabain on Potassium-Induced Relaxation
Ouabain, an inhibitor of the electrogenic sodiumpotassium pump, 7 decreased the amplitude of potassium-induced relaxation in tail arteries from all pigs
(fig. 4). However, tail arteries from DOCA-hypertensive pigs were more sensitive to inhibition by ouabain
than were those from normotensive pigs (preimplant or
with elastic implant). This was evident when the responses in the presence of ouabain were normalized to
their respective control responses (table 2, fig. 4). The
magnitudes of the relaxation prior to ouabain treatment
were: 1) DOCA-hypertensive pigs = 60.0 ± 3.5%; 2)
preimplant normotensive pigs =; 40.2 ± 1.7%; and 3)
Silastic-irhpiant normotensive pigs = 45.1 ± 2.6%,
At the highest concentrations of ouabain (10"6 and 10*5
M), tail artery strips from DOCAj-hypertensive pigs
contracted when potassium (15.0 mM)Avas readmitted
to. the muscle bath, whereas those from norrnotensiye
pigs did not contract.
Concentration-Response to Potassium in Arteries
from Different Anatomical Locations
In the preceding experiments, potassium was added
back to the bath to attain a final concentration 15.0
mM. Figure 5 depicts the effect of potassium concentration on the amplitude of potassium-induced relaxation in tail arteries (fig. 5 upper left), renal arteries
(fig. 5 upper right) and femoral arteries (fig. 5 lower
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TABLE 2. Concentrations of Norepinephrine, Oiiahain and Potassium to Produce Threshold and Half-Maximal
Responses
Treatment
Artery
Preimplant
DOCA hypertensive pigs
(55 ± 6 days
postimplant)
Normotensive
control pigs
(Silastic
implant; 47 ± 7
days postimplant)
5.6 x i o - " t
g/ml
(n = 4)
30.0 x 10-»
g/ml
(n = 5)
Norcpincphrine
concentration at
which there was a
half-maximal contraction (fig. 3 left)
tail.
artery
17.0 x 10-**
g/ml
(n = 6)
Norcpinephrine
concentration at
which there was a 50%
relaxation (fig. 3 right)
tail
artery
4.8 x I 0 - "
g/ml
(n = 6)
38.0 x l 0 - « * t
g/ml
(n = 4)
10.0 x 0-«
g/ml
(n = 5)
Ouabain
concentration at which
there was half-maximal
inhibition (fig. 4)
tail
artery
44.0 x lO-* M
(n = 6)
7.9 x \0-l> M*t
(n = 4)
69.0 x I 0 " 9 M
(n = 4)
Potassium concentration at which
there was a half-maximal
relaxation
(fig. 5)
tail
artery
2.88 mM
(n = 5)
2.10 mMt
(n = 5)
3.68 mM
(n = 4)
—
2.20 mMt
(n = 4)
4.32 mM
(n = 5)
femoral artery
A. protected
—
B. unprotected
—
1.89
(n =
1.87
(n =
2.52
(n =
2.96
(n =
renal
artery
tail
Potassium concentraartery
tion at which there was
a 10% maximal response —
threshold response
renal
artery
(fig. 5)
femoral artery
A. protected
B. unprotected
1.24 mM
(n = 5)
—
—
—
mM
3)
mMt
4)
mM
4)
mM
5)
0.66 m M ' t
(n = 5)
1.30 mM
(n = 4)
0.62 mMt
(n = 5)
1.71 mM
(n = 4)
0.53
(n =
0.61
(n =
1.07
(n =
1.10
(n =
mMt
3)
mMt
4)
mM
4)
mM
5)
•Significant difference between preimplant and postimplant values (p < 0.05).
tSignificant difference between DOCA-hypertensive pigs and Silastic-implanted control pigs (p < 0.05). The values
in parentheses are the number of pigs.
HYPERTENSION
614
V 6 L 4, No 5, SEPTEMBER-OCTOBER
1982
140
120
FIGURE 4. Inhibition by ouabain. Potassium-induced relaxation of tail artery strips was performed as described in figure
I. Ouabain (10~m to 10~5 M) was added to the bath 5 minutes
(N=6) after the beginning of exposure to potassium-free solution (12
minutes prior to the addition of potassium to the bath). The
responses of tail artery strips from DOCA-hypertensive pigs
(N = 4)
were more sensitive to inhibition by ouabain than those from
preimplant pigs or chronic normotensive pigs. Values are the
mean ± SEM. * = statistically significant difference between
DOCA-hypertensive and normotensive control pigs (p <
0.05). t = statistically significant difference between DOCAhypertensive and preimplant pigs (p < 0.05). The wlues in
parentheses are the number of pigs.
100
DOCA (N=4)
80
j?
4
60
40
20
0
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10".-6
l0 -e
10'
[Ouabain] (M)
Silastic (N<=4)
Pre-implant (N»5)
20
Silastic (N«5)
20
|
40
5 40
DOCA
I
DOCA (N=
1 60
80
100 l
'
0.5
I
25
5
[Potassium]
10
20
(mM)
I
Silastic-protected (N=4)
40
60
DOCA-unprotected * I \
(N=4)
80
I001-
DOCA-protected
(N=3)
\
J
l
0.5
2.5
[Potassium]
5
(mM)
80
100 _l
0.5
2.5
[Potassium]
Silastic-unprotected (NC5)
20
(N = 4)
10
20
5
10
20
(mM)
FIGURE 5. Concentration response to potassium and the independence of relaxation magnitude from femoral arterial pressure. Potassium-induced relaxation was performed as described in figure I except that the concentration of potassium
added back to the bath was varied from 0.5 to 20 mM. The
magnitude of potassium-induced relaxation was related to the
concentration of the added potassium in all arterial strips.
Upper Left: Tail arteries. Lower Left: Femoral arteries. Upper
Right: Renal arteries. Values are the means ± SEM. The values
in parentheses are the number of pigs. * = statistically significant difference between DOCA-hypertensive pigs and chronic
normotensive pigs (p < 0.05). t = statistically significant
difference betiveen DOCA-hypertensive pigs and preimplant
pigs (p < 0.05).
POTASSIUM RELAXATION IN DOCA-HYPERTENSIVE PIGS/Webb
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left) from DOCA-hypertensive and normotensive pigs
(preimplant and chronic pigs). The magnitude of relaxation increased as the concentration of potassium increased over a range of 0.5 to 20.0 mM in all arterial
strips. Arterial strips from DOCA-hypertensive pigs
relaxed to a greater extent than those from normotensive control pigs.
The magnitude of potassium-induced relaxation was
independent of the level of femoral arterial pressure.
Figure 5 lower left shows the dose-response curves for
potassium of femoral arteries isolated from "protected" and "unprotected" hindlimbs of DOCA-hypertensive and chronic normotensive pigs. The magnitude
of potassium-induced relaxation was similar in femoral arteries from "protected" hindlimbs and those
from the "unprotected" hindlimbs at all concentrations of potassium.
To determine if there was a change in the sensitivity
of the relaxation to potassium in arteries from DOCAhypertensive pigs, the data presented in figure 5 were
transformed to percent maximum relaxation and the
i!
i!
concentration at which half-maximal relaxation JQ
occurred was calculated by probit analysis (table 2).
The ED,,, for arterial strips from DOCA-hypertensive
pigs were significantly different from that for arterial
strips isolated from Silastic-implanted control pigs but
not from preimplant control pigs. The threshold concentration of potassium required to produce relaxation
(10% of maximum) was significantly lower in arterial
strips from DOCA hypertensive pigs as compared to
that in arterial strips from normotensive control pigs
(table 2).
Temporal Relationship Between the Rise in Blood Pressure
and the Magnitude of Potassium-Induced Relaxation
To determine the onset of changes in vascular responsiveness during the development of hypertension,
potassium-induced relaxation was performed in consecutive portions of the tail artery prior to and after
implantation in six control pigs (Silastic implant) and
in five DOCA-treated pigs (fig. 6). Each pig was anes-
140
140
120
120
100
"11 100
Si
80
615
0
80
Or
20
20
!
§
40
40
60
60
••o
80
40
Days
80
80
40
80
Days
FIGURE 6. Temporal relationships between the rise in blood pressure and the magnitude of potassium-induced relaxation. Potassium-induced relaxation of isolated tail arteries was performed as described in figure 1. Tail arteries were isolated before implantation
(day zero) of Silastic strips (Fig. 6 left graphs; n = 6) or before implantation of Silastic strips impregnated with DOCA (fig. 6 right
graphs; n = 5), at 3 to 5 days postimplantation, and at the time of termination of the pigs. In pigs implanted with Silastic there was no
significant change in the magnitude of potassium-induced relaxation, whereas pigs implanted with DOCA showed an increase in the
magnitude of potassium-induced relaxation at 3 to 5 days postimplant, and a further increase in the magnitude of potassium-induced
relaxation in DOCA-treated pigs paralleled the rise in mean arterial pressure.
616
HYPERTENSION
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thetized with thiamylal at three separate times in the
course of treatment with either Silastic with DOCA
(DOCA pigs) or Silastic without DOCA (normotensive pigs): 1) 1 to 5 days preimplantation (designated
as 0 days in fig. 6); 2) 3 to 5 days postimplaritation
(designated as 5 days in fig. 6); 3) 10 to 90 days
postimplantation (designated as the actual day postimplant in fig. 6). At each time point, a 3 to 4 cm section
of the tail was surgically removed and the tail artery
was isolated.
Potassium-induced relaxation in tail arteries from
normotensive pigs (fig. 6 upper and lower left) did not
change significantly from preirriplant values (day
zero). At 3 to 5 days postimplantation of DOCA there
was a 37% increase in the magnitude of potassiuminduced relaxation compared to preimplant values; and
at the time Of termination the magnitude of potassiuminduced relaxation had increased to 64% above the
preimplant values. The change in vascular responsiveness to potassium paralleled the rise in mean arterial
pressure in DOCA-treated pigs (compare top and bottom panels of fig. 6 upper and lower right).
Discussion
Potassium-induced relaxation in intact vascular beds
of renal hypertensive animals, essential hypertensive
humans, and genetically hypertensive rats (New Zealand strain) has been reported to be decreased, 28 - 9
whereas potassium-induced relaxation in isolated vascular segments from genetically hypertensive rats
(spontaneously hypertensive rats (SHR), Japanese
strain) has been observed to be increased as compared
to normotensive animals.10 The results of the current
study provide evidence that relaxation in response to
potassium is exaggerated in the vasculature of DOCAhypertensive pigs.
The cellular process responsible for potassium-induced relaxation is the electrogenic pumping of sodium and potassium by sodium-potassium adenosine triphosphatase.6 Bonaccorsi etal? observed that isolated
rat tail artery strips, made to contract in response to
serotonin in potassium-free solution, relaxed when potassium was returned to the bathing medium. They
suggested that during incubation in potassium-free solution, the activity of the sodium pump is eliminated,
and therefore sodium accumulates intracellularly.
When potassium is returned to the bathing medium,
the sodium pump is activated due to the high intracellular concentration of sodium. This results in hyperpolarization which decreases membrane excitability and
causes relaxation. Once the pump reduces the intracellular concentration of sodium toward normal, its activity decreases and the membrane potential returns toward normal. The presence of serotonin then caused
excitation of the smooth muscle and an increase in
mechanical response was observed. These interpretations were supported by membrane potential measurements and by the observation that ouabain, an inhibitor
of the sodium pump, 7 eliminated the potassium-induced relaxation. Other investigators have suggested
VOL 4, No 5, SEPTEMBER-OCTOBER
1982
the same mechanism for potassium-induced relaxation
of vascular smooth muscle based on similar experimental observations.6
The observations of the current study support the
hypothesis that vascular smooth muscle from DOCAhypertensive animals has increased electrogenic
sodium pump activity.14"17 Although the precise
mechanism responsible for this increased activity is
unknown, it may be that Vascular smooth muscle from
DOCA-hypertensive animals has either an increased
intrinsic electrogenic sodium pump or an electrogenic
pump that has been stimulated to a greater degree by ati
elevated intracellulaT sodium concentration. Evidence
in support of the latter conclusion is that the magnitude
and duration of potassium-induced relaxation is greater in tail artery strips from DOCA-hypertensive pigs as
compared to that from normotensive pigs when the
duration of the exposure of the vascular strip to potassium-free solution is increased (fig. 2). Intracellular
sodium accumulates under potassium-free conditions14 " and longer exposures would therefore lead to
a greater sodium accumulation producing an increasing magnitude of relaxation following readmission of
potassium.
Friedman and Friedman14 observed that the intracellular concentration of sodium, measured in normal
PSS, is less in tail arteries from DOCA-treated rats and
SHR as compared to their respective controls. Incubation of the arteries in potassium-free solution resulted
in an increase in the intracellular concentration of sodium (and, conversely, potassium lost), and the amount
gained was greater in the arteries from the hypertensive
rats. The increased cellular sodium in hypertensive
arteries, under these conditions is probably due to a
greater membrane leak of the cation as established in
studies of radioactive ion fluxes.1617 An increased
membrane permeability to sodium would also explain
the observation that the duration of relaxation was
longer in arterial strips from DOCA-hypertensive pigs.
The termination of the relaxation is believed to indicate
that the intracellular sodium concentration has returned toward normal, and therefore the activity of the
electrogenic sodium pump has returned the membrane
potential toward normal. 3 The presence of norepinephrine may then again cause excitation of the cell and the
mechanical response returns to normal. Recent observations by Jones and coworkers18 demonstrate that the
passive ionic permeability for sodium is 1.4- to 2.0fold higher in aortic smooth muscle from DOCA-hypertensive rats than in that from normotensive controls
even when the active transport of sodium is stimulated
by high extracellular potassium (10.0 mM). Thus, it
may be that it requires more time for the sodium pump
to return the membrane potential toward normal in
hypertensive arteries resulting in a longer duration of
relaxation. Alternatively, the transition from the relaxed to the contracted state involves other factors (calcium metabolism, excitation-contraction coupling)
which account for the difference in the duration of
relaxation between arterial strips from hypertensive
and normotensive pigs.
POTASSIUM RELAXATION IN DOCA-HYPERTENSIVE PIGS/Webb
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It is possible that an increased intrinsic electrogenic
pump produces the greater relaxation response to potassium in hypertensive arteries. Knox and Sen19 have
observed that one of the proteins synthesized in increased amounts in response to the mineralocorticoid,
aldosterone, is sodium-potassium ATPase. DeLuise et
al.20 have shown a positive correlation between the
amount of radioactive ouabain binding and the cation
transport activity of the pump (as measured by radioactive rubidium uptake) in erythrocytes from normal and
obese persons. Red blood cells from normal individuals had increased cation transport activity in parallel
with a greater ouabain binding suggesting a greater
number of pump units in these cells as compared to
those from obese subjects. Thus, the greater magnitude of potassium-induced relaxation in vascular
smooth muscle from DOCA-hypertensive pigs may
reflect an increase in the number of pump sites as
compared to that in normotensive pigs.
An interesting observation of this study is that relaxation in response to potassium from hypertensive pigs
was more sensitive to the inhibitory effects of ouabain
than those from normotensive animals. Similarly,
Webb and Bohr10 observed that there was a significant
shift to the left in the dose-response curve for ouabain
inhibition of potassium-induced relaxation in tail arteries from SHR compared to those from normotensive
rats. Gothberg et al.21 have shown that ouabain produces a leftward shift in the vascular resistance doseresponse curves to norepinephrine in the hindquarters
of SHR and normotensive rats. The shift in the curve
produced by ouabain was greater in SHR as compared
to normotensive rats. Similar results were obtained
when vasopressin or barium were used as the vasoconstrictor agonists. It is difficult to choose among the
several possibilities that might explain this increased
sensitivity to ouabain in hypertension. For example, a
difference in the stability of the pump-glycoside complex could contribute to differences in ouabain inhibition. Alternatively, a difference in intracellular adenosine triphosphate or sodium levels could influence
ouabain inhibition, since the glycoside binds to a sodium-activated, phosphorylated intermediate of sodiumpotassium ATPase. 7
Tail artery strips from DOCA-hypertensive pigs contracted when potassium was readmitted to the bathing
medium in the presence of high concentrations of ouabain
whereas those from normotensive pigs did not. The reason for this unusual difference in response to potassium is
unclear. It may be that vascular smooth muscle from
hypertensive pigs may depolarize to a greater extent
during ouabain treatment that alters membrane
conductance.22
The magnitude of potassium-induced relaxation increased as the concentration of added potassium was
raised from 0.5 to 20.0 mM. The magnitude of responses in arterial strips from DOCA-hypertensive
pigs was observed to be greater at both low and high
concentrations. This relationship to potassium concentration was independent of the anatomical location of
the blood vessel, suggesting that increased potassium-
617
induced relaxation may be a generalized change in
vascular function. When the data were normalized to
the maximum response, arterial strips from DOCAhypertensive pigs showed lower thresholds and EDJO
levels than those from Silastic control pigs. The concentration of potassium required to produce a threshold
response was lower in strips from DOCA-hypertensive
pigs as compared to those from preimplant normotensive pigs, whereas the concentration of potassium required to produce half-maximal relaxation was not statistically different between these two groups of
animals. The reason for a change in threshold without
a concomitant shift in the EDj,, is not apparent, but
suggests that the electrogenic pump in tail arteries from
DOCA-hypertensive pigs is only slightly more sensitive to potassium than those from preimplant pigs. It
may be that the proper control group for the DOCAhypertensive pigs (55 ± 6 days postimplant) is the
Silastic-implanted pigs (47 ± 7 days postimplant)
since these animals are more closely age-matched to
the hypertensive animals than the preimplant pigs.
The magnitude of potassium-induced relaxation was
not dependent on the level of arterial pressure. Femoral
arterial strips from "protected" and "unprotected"
limbs of DOCA-hypertensive pigs showed increased
relaxation in response to potassium as compared to
those from chronic normotensive pigs. Although the
reduction in mean arterial preessure in the so-called
"protected" limb was small, it is unlikely that the
level of arterial pressure is an important determinant of
the relaxation response, since changes in threshold and
ED,,, did not occur in arterial strips from the "protected" limbs. The results do suggest that a functional
change in vascular smooth muscle occurs in DOCA
hypertension regardless of whether the vascular bed is
protected from the increased arterial pressure.
The development of an increased response to potassium paralleled the development of increased arterial
pressure in DOCA-treated pigs. Tail artery segments
isolated at 3 to 5 days postimplantation of DOCA demonstrated an increased magnitude of potassium-induced relaxation and the mean arterial pressure in these
pigs was already significantly increased above preimplant values. The early increase in potassium relaxation again suggests that this change is not secondary
to a prolonged period of increased wall stress.
The magnitude of potassium-induced relaxation in
tail artery strips decreased as the concentration of norepinephrine added during the potassium-free cycle increased, demonstrating that the level of contraction
influences the resultant relaxation caused by the cation. Since the level of the contractile responses
depends on a number of interacting variables (intracellular calcium concentration, coupling between
membrane potential and contraction, cyclic nucleotides, etc.),23-24 these observations demonstrate that the
comparison of potassium-induced relaxation in arteries
from DOCA-hypertensive pigs and those from normotensive pigs may be an oversimplified measure of sodium pump activity. For instance, the concentration of
norepinephrine at which there was a half-maximal con-
618
HYPERTENSION
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traction was lower in arterial strips from DOCA-hypertensive pigs. This effect probably reflects the increased
sensitivity of vascular smooth muscle to vasoactive
agents during the development of DOCA hypertension."- 12 This change in sensitivity to norepinephrine
may be caused by any of several processes which determine sensitivity to an agonist.21 However, the magnitude of potassium-induced relaxation was greater in
arteries of hypertensive pigs than in those of normotensive pigs, regardless of norepinephrine concentration,
suggesting that the increased vasoconstrictor sensitivity does not mask the increased activity of the sodium
pump in DOCA-hypertensive arteries under these experimental conditions.
Other cellular processes that may influence the level
of potassium-induced relaxation include: an alteration
in the sodium-calcium exchange mechanism; and altered calcium efflux and/or sequestration.24 It is doubtful that the sodium-calcium exchange mechanism
plays an important role in the relaxation for two reasons. First, according to this hypothesis, calcium entry
occurs when intracellular sodium is high. Thus, potassium-free conditions (which lead to increased intracellular sodium) should cause contraction. This is not the
case in the tail arteries of rats and pigs. The small
contraction that occurs during the potassium-free incubation (evident in fig. 1) is due to norepinephrine released from adrenergic nerve endings in the vessel wall
(unpublished observations).25 Second, it is doubtful
that an increase in potassium outside the cell could
alter the intracellular sodium concentration fast
enough to cause the relaxation by influencing a sodium-calcium exchange. Altered calcium efflux through
the cellular membrane and changes in the sequestration
of calcium of the subcellular level could also alter the
potassium-induced relaxation. Experimental evidence
suggests, however, that both of these processes are
decreased in arteries of hypertensive animals leading
to elevated levels of activator calcium.24 Thus, an increase in intracellular calcium by these two latter processes would be predicted to reduce the level of potassium-induced relaxation rather than increase the
response relative to the normotensive controls.
Many studies have been performed in an effort to
define electrogenic transport of monovalent ions in
vascular smooth muscle from hypertensive animals.24- M The techniques used to evaluate electrogenic
transport include: 1) potassium-induced relaxation; 2)
transmembrane electrical potential; 3) ion flux measurements; and 4) measurement of sodium-potassium
ATPase activity. The observations of these studies
have yielded opposing views concerning the activity of
the electrogenic sodium pump in hypertension. Interestingly, the observations of several different laboratories have indicated that electrogenic transport of sodium and potassium is increased in vascular smooth
muscle from animals with genetic hypertension. In
contrast, the electrogenic transport system has been
observed to be either increased or decreased16 24 in
animals with hypertension induced by experimental
intervention (renal hypertension or mineralocorticoid
VOL 4, No 5, SEPTEMBER-OCTOBER
1982
hypertension). Recent observations by Pamnani and
associates27 demonstrate that these differences in measured pump activity in hypertensive arteries may be due
to the washout of a humoral factor which inhibits the
electrogenic sodium pump (present in the plasma of
hypertensive animals). These investigators observed
that tail arteries from DOCA hypertensive rats showed
decreased ouabain-sensitive rubidium uptake relative
to controls when the arteries were studied immediately
after removal from the animal. When the arteries were
first incubated in PSS for 4.5 hours, the ouabain-sensitive rubidium uptake was increased in the hypertensive
arteries compared to the controls suggesting washout
of a factor which suppresses pump activity. The precise chemical nature of this humoral factor which inhibits the sodium pump is unknown. Although the
current evidence does not resolve the apparent conflict
concerning measured pump activity in hypertension it
adds breath of evidence in support of the conclusion
that electrogenic transport is elevated in mineralocorticoid-induced hypertension. This vascular change parallels the development of hypertension.
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Potassium relaxation of vascular smooth muscle from DOCA hypertensive pigs.
R C Webb
Hypertension. 1982;4:609-619
doi: 10.1161/01.HYP.4.5.609
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