687
Potassium Channels and Vascular Reactivity
in Genetically Hypertensive Rats
Philip B. Furspan and R. Clinton Webb
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In hypertension, membrane potassium permeability and vascular reactivity are increased. This
study characterizes a potassium-selective channel and contractions to barium, a potassium
channel inhibitor, in vascular smooth muscle (tail artery) from spontaneously hypertensive
stroke-prone rats (SHRSP) and normotensive Wistar-Kyoto (WKY) rats. Smooth muscle cells
were isolated by enzymatic digestion, and potassium channel activity was characterized by
using patch-clamp technique (inside-out configuration). Isometric contractile activity was
evaluated in helically cut arterial strips by using standard muscle bath methodology. In
membrane patches, a voltage-gated, calcium-insensitive, potassium-selective channel of large
conductance (200 picosiemens) was observed. The channel did not conduct sodium or
rubidium. Barium (10~6 to 10"4 M) produced a dose-dependent blockade of channel activity.
These channel characteristics did not differ in SHRSP and WKY rat cells. After treatment with
35 mM KCI, barium (10~5 to 10~3 M) caused greater contractions in SHRSP arteries compared
with arteries in WKY rats. The contractions to barium were markedly attenuated in calciumfree solution, and nifedipine and verapamil abolished contractions induced by barium in
depolarizing solution. We conclude that increased vascular reactivity to barium in SHRSP
arteries is not due to an alteration in the biophysical properties of the potassium channel
studied. (Hypertension 1990;15:687-691)
xtensive evidence indicates that K+ conductance is partly responsible for modulating
cellular excitability in vascular smooth
muscle.1"2 Electrophysiological studies with patchclamp technique have demonstrated the presence of
both voltage-dependent and Ca2+-dependent K+
channels in vascular tissue. Differences in the mechanisms of activation of these channels probably relate
to their functional roles, with some channels playing
a part in the maintenance of resting tone and others
limiting or terminating contraction induced by physiological agonists.
In hypertension, it has been demonstrated that
vascular reactivity and membrane K+ permeability
are both increased.34 Furthermore, several antihypertensive drugs that relax vascular smooth muscle
appear to do so by opening membrane K+ channels.2
The present study characterizes a K+-selective channel by using patch-clamp technique in arterial
smooth muscle cells isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto (WKY) rats. The inhibitory actions
E
From the Department of Physiology, University of Michigan,
Ann Arbor, Michigan.
Supported by grants HL-27020 and HL-18575 from the National
Institutes of Health.
Address for correspondence: Dr. Philip B. Furspan, Department of Physiology, The University of Michigan, 7710 Medical
Science Building II, Ann Arbor, Ml 48109-0622.
of barium (Ba2+) on the channel were evaluated.
Additionally, contractile activity to Ba2+ was characterized in arterial segments from SHRSP and WKY
rats to evaluate functional properties related to
membrane-associated K+ channels.
Methods
Adult male and female SHRSP and WKY rats
(16-20 weeks old) were obtained from rat colonies
maintained in the Department of Anatomy and Cell
Biology (University of Michigan, Ann Arbor, Michigan). The systolic blood pressures were measured by
an indirect tail-cuff method using a pneumatic transducer (WKY, 118±4 mm Hg [n=12]; SHRSP, 176+4
mmHg [n = 12]\ p<0.05).
On the day of an experiment, the rats (one SHRSP
and one WKY rat) were anesthetized with sodium
pentobarbital (50 mg/kg), and tail arteries were
removed and placed in cold physiological salt solution (PSS). A 2-4 cm segment of each artery was cut
helically into a strip (0.7x10 mm). The endothelium
was removed from all segments by a rubbing procedure (confirmed by the absence of a relaxation
response to acetylcholine). The strips were then
mounted in an organ chamber containing PSS for
measurement of isometric development, as described
elsewhere.5 All preparations were allowed to equilibrate for 90 minutes before an experiment was
688
Hypertension
Vol 15, No 6, Part 2, June 1990
begun. The PSS was maintained at 37° C and was
aerated with a mixture of 95% O2 and 5% CO2. The
composition of the PSS (mmol/1) was as follows:
NaCl 130, KC14.7, KH2PO41.18, MgSO4 • 7H2O 1.17,
NaHCO3 14.9, CaCl2-H2O 1.6, dextrose 5.5, and
CaNa2-EDTA 0.03. The K+ and Ca2+ concentrations
of the buffer were altered without compensating for
changes in tonicity. All experiments were performed
in the presence of 10"6 M phentolamine to block the
actions of norepinephrine released from nerve endings by depolarizing conditions. Contractile responses to KC1 (12-120 mM) and Ba2+ (10' 5 to 10"3
M) were measured in all experiments.
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The remaining section of each tail artery was used in
studies of K+ channel activity by patch-clamp technique. To isolate cells, artery segments were incubated
in digestion medium (mg/ml) (collagenase 0.5, trypsin
inhibitor 0.3, papain 0.4, dithiothreitol 0.3, bovine
serum albumin 7.5, pH 7.4) for 90 minutes at 37° C.
Cells were then dispersed by trituration with firepolished Pasteur pipettes of decreasing tip diameter.
Single channel currents were measured with standard patch-clamp technique. A Dagan 8900 Patch
Clamp/Whole Cell Clamp (Dagan Corp., Minneapolis, Minnesota) was used to voltage-clamp membrane
patches in the inside-out configuration. Data were
digitized with a modified digital audio processor
(Sony PCM-701ES, Medical Systems Corp., Greenvale, New York) and recorded on videocassette tape
for analysis at a later time. Electrodes were prepared
from borosilicate glass (Kimble R-6, Richland Glass,
Richland, New Jersey).
The pipette-filling solution (PFS) always contained
(mM): KC1 145, HEPES (7V-[2-hydroxyethyl]
piperazine-7V'-[2-ethanesulfonic acid]) 10, glucose
10, and KOH to bring pH to 7.4. In the bath PFS or
a Ca2+-buffered solution was used that contained
(mM): KC1 113, EGTA 2.02, K2-PIPES (piperazine7\WV'-bis[2-ethanesulfonic acid]) 20.2, pH 7.0. Sufficient CaCl2 was added to yield calculated free Ca2+
concentrations of 100-1,000 nM as described by
Chang et al.« Ba2+ in the form BaCl2 (10~6 to 10"3 M)
was added to the bath PFS.
Unless otherwise stated, data are reported as the
mean±SEM. ED50 values (agonist dose producing a
half-maximal response) were determined after logit
transformation of normalized, dose-response curves.
Statistical comparisons were performed by Student's t test with the Bonferroni adjustment for
multiple procedures. A p value less than 0.05 was
considered significant.
Results
Contractile Responses to Potassium
Chloride and Ba2+
Cumulative addition of KC1 (12-130 mM) to the
muscle bath caused contractions in tail arteries from
SHRSP and WKY rats. Maximal force generation in
tail arteries from SHRSP (525 ±36 mg, n=12) was
not different from that in WKY rat arteries (524±56
mg, n=l2). Arteries from SHRSP were more sensitive
to the contractile properties of potassium chloride
than arteries from WKY rats, as indicated by lower
EDJO values (SHRSP, -log ED*, value=1.368+0.016,
antilog=42.9 mM, n=12; WKY, -log EDJO value=
1.301±0.017, antilog=50.0 mM, «=12;p<0.05).
Addition of Ba2+ (10~5 to 10"3 M) to the muscle
bath had no effect on the resting force of tail artery
strips from SHRSP or WKY rats. In contrast, when
the arteries were first contracted with KC1 (18-50
mM), Ba2+ was then able to induce small contractions
in arteries from both rat groups (Figures 1 and 2).
Arteries from SHRSP developed significantly greater
force to Ba2+ chloride at 3xlO" 4 and 10~3 M in the
presence of 35 mM KC1 (Figure 2). When the K+
concentration of the bathing solution was varied from
18 to 50 mM, the magnitude of contractile responses
to 10~3 M Ba2+ increased in a direct manner (Figure
2, inset). Force development to Ba2+ (10~3 M) in
SHRSP arteries was statistically greater than that in
WKY rat arteries at K+ concentrations of 35 and 50
mM. Contractile responses to KC1 in these experiments were as follows: 1) 18 mM KC1: SHRSP, 27±6
mg; WKY, 13±7 mg; 2) 25 mM KC1: SHRSP, 67±18
mg; WKY, 53 ±16 mg; 3) 35 mM KC1: SHRSP,
195+28 mg; WKY, 124±29 mg; 4) 50 mM KC1:
SHRSP, 334±39 mg; WKY, 279±57 mg (n=6 in all
groups).
The Ca2+-dependence of contractile responses to
Ba2+ were examined in arteries incubated in Ca2+-free
depolarizing solution (35 mM KC1) containing 1.0 mM
EGTA (Figure 1) and in arteries treated with 10"6 M
nifedipine or 10~6 M verapamil. Further, the effect of
Ba2+ on contractile responses to caffeine were examined to determine if the cation had an effect on
subcellular stores of activator Ca2+. Contractile
responses to Ba2+ in Ca2+-free, depolarizing solution
in arteries from SHRSP (10±6 mg, n=6) were not
significantly different from WKY rat values (5 ±2 mg,
n=6). Nifedipine and verapamil completely blocked
contractions to 35 mM KQ and subsequent responses
to Ba2+ (10~3 M) in both normal PSS and in strips
incubated in Ca2+-free PSS. Ba2+(10"3 M) did not alter
contractile responses to caffeine (20 mM) in arteries
from SHRSP or WKY rats (Figure 1). Contractile
responses to caffeine in arteries from SHRSP (Ca2+free PSS, 200±17 mg; Ca2+- free depolarizing PSS,
196 ±20 mg; n=6) were greater than the responses in
WKY rat arteries (Ca2+-free PSS, 101 ±12 mg; Ca2+free depolarizing PSS, 102±13 mg; n=6).
K+ Channel Activity
We were able to identify and characterize a large
conductance K+ channel in inside-out patches of the
plasma membrane of tail artery vascular smooth
muscle cells from both strains of rat. The number of
channels per patch varied considerably (1-5) for cells
from both SHRSP and WKY rats. K+ currents were
recorded that had a slope conductance (psiemen) of
194.8+1.49 (n=5) and 201.3±1.44 (n=5) in SHRSP
and WKY rat tail artery cells, respectively (Figure 3).
Furspan and Webb K+ Channels and Vascular Reactivity
689
WKY
rinse
SHRSP
4 mln
33 mM
1.0 mU
I
- fre«,
1.0 mM EQTA
I
35 mM 1.0 mM
KCI
BaCfc
I
20 mM
CafMne
I
C«2+. frae,
20 mM
1.0 raM EQTA CaffalM
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FIGURE 1. Recordings showing contractile responses to Ba1+. After contraction induced by 35 mM KCI, Ba2+
(10~3 M) caused small contractions in tail arteries from both stroke-prone spontaneously hypertensive rats
(SHRSP) and Wistar-Kyoto (WKY) rats. Incubation in Ca2+-freesolution (l.OmMEGTA) abolished contractions
to elevated KCI and markedly attenuated contractions to Ba2+. Treatment with Ba2+ under depolarizing conditions
in Ca2+-free solution did not alter contractile responses to caffeine (20 mM; compare with second caffeine-induced
contraction in Ca2+-free solution alone).
The channel did not exhibit rectification and was
highly selective for K+ (K + >Na + , Rb+). Channel
activity did not respond to varying Ca2+ concentrations in the bath (1(T9 to 10~6 M). This channel,
however, exhibited pronounced voltage dependence.
Channel activity increased as holding potential
60-
imMBaO
120-
(bath) was made more positive. There was little or no
activity at the equivalent of resting membrane potential in whole cells. There were no apparent differences in the voltage dependence of channels from
WKY rat and SHRSP tail artery cells (Figure 3). The
channel was blocked in a concentration-dependent
-
1
*
•S 8 0 -
50"
I 60 "
/
/
S. 4 0 TO
40-
§
20-
y\s^L
/
WKY
30
[KO] (mM)
10
ha
O
FIGURE 2.
m
100-
30"
/
100 ^
.
/
/
SHRSP
(n=6)
i
/
2035 mM KCI
T
/
/I
10
WKY
]
^
L (n=6)
]
"
0^
•
10-5
i
•
i
10-4
[BaCI2] (M)
•
10-3
Plottings of dose
response to Ba2+. Mean contractile responses to Ba2+ (10'! to
10~3 M) in stroke-prone spontaneously
hypertensive
rat
(SHRSP) arteries that are significantly greater than in tail arteries from Wistar-Kyoto (WKY)
rats are denoted by asterisks
(p<0.05). All dose-response
effects ofBa2+ were evaluated in
arteries that werefirstcontracted
with 35 mM KCL Inset shows
contractile responses to 1.0 mM
Ba2* when arteries were first
treated with four different concentrations of potassium chloride (KCI) (18-50 mM). *In the
inset, indicate
significantly
greater responses to Ba2+ in tail
arteries from SHRSP compared
with WKY rat values (p<0.05).
Values are mean±SEM for six
rats in each group.
690
Hypertension
Vol 15, No 6, Part 2, June 1990
-20
-100
100
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FIGURE 3. Plottings showing current-voltage relation for a K* channel from tail artery cells of stroke-prone spontaneously
hypertensive rats (SHRSP) (right panel) and Wistar-Kyoto (WKY) rats (left panel). Lines represent the average of five
current-voltage plots; five cells per five rats. SEMs for each point are smaller than the symbol before reduction.
manner by BaCl2 (10~6 to 10"4 M) (Figure 4). The
effect of Ba2+ did not differ in its effect on channels
from WKY rat and SHRSP cells (Figure 4).
smooth muscle cells from both SHRSP and WKY
rats.
The cellular mechanism that explains augmented
responsiveness to Ba2+ in SHRSP arteries is not
clear. Ba2+ is rather nonselective in that it blocks
most K+ channels.2 In many of these channels, it
blocks at the K+-selectivity filter, which is approximately halfway through the channel.7 Based on
patch-clamp studies, it has been noted that Ba2+ is
more potent (10,000-fold) at the inner surface of the
membrane compared with the outer surface.1 Presumably, this indicates that Ba2+ ions can first enter
the cell through Ca2+ channels before blocking K+
Discussion
This study demonstrates that the contractile properties of Ba2+ in tail arteries from SHRSP are augmented compared with those in tail arteries from
WKY rats. This augmented responsiveness does not
appear to be related to a difference in the biophysical
properties of a voltage-gated, Ca2+-insensitive, K+selective channel of large conductance. This channel
was observed in membrane patches isolated from
BaCI2(liM)
WKY
SHRSP
0
MM 1nil r 'I I f f I'M
*m U | l MWJ!'
10
1
m
I ,.
IK
•1 1
•
II
•1
W
in
V
r
TnttTTT
1 ll
mi T I I
nn
III I
I
nopA
2min
FIGURE 4. Recordings showing representative response ofK* channel to Ba2+ (10~6 to 10'4 M). Effect ofBa2+ on channel
activity was similar for channels from both strains of rat. BaCl2 was added to the bath solution additively. In this experiment,
the patch from the stroke-prone spontaneously hypertensive rat (SHRSP) cell contained one channel, and the Wistar-Kyoto
(WKY) rat cell contained at least three channels.
Furspan and Webb K+ Channels and Vascular Reactivity
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channels from the inside. This might explain the
differences in Ba2+ sensitivity observed in the present
study with respect to K+ channels in membrane
patches (inside-out configuration) and contractile
activity in intact arterial segments.
We speculate that the augmented responsiveness
to Ba2+ in SHRSP arteries might be related to an
alteration in Ca2+ permeability. Ba2+ did not cause
contraction in the resting state, indicating that it was
not able to enter the cell and sufficiently block K+
channels to cause membrane depolarization. If the
arteries were first treated with depolarizing solution,
however, the arteries were able to subsequently
contract to Ba2+. Because Ca2+ permeability is
increased in arteries from hypertensive rats,4'8 it
might be that in the depolarized state, more Ba2+
enters the cell, resulting in a greater block of K+
channels in the hypertensive rat arteries. Further
support for this hypothesis is that the contractile
properties of Ba2+ were markedly attenuated in
Ca2+-free depolarizing solution, and nifedipine and
verapamil completely blocked the contractions. Furthermore, Lamb and Webb9 reported that 10"4 M
Ba2+ caused added depolarization of tail artery that
had been depolarized with norepinephrine.
This study characterized augmented responsiveness to Ba2+ in tail arteries from SHRSP compared
with normotensive WKY rat values. This augmented
contractile activity is not due to a specific change in
the biophysical properties of a K+ channel characterized in isolated membrane patches. We speculate
691
that the augmented responsiveness might be due to
an increased entry of Ba2+ into the smooth muscle
cells through Ca2+ channels, with subsequent block of
the K+ channels from the inside.
References
1. Benham CD, Bolton TB, Lang RJ, Takewaki T: The mechanism of action of Ba2+ and TEA on single Ca2+-activated
K+-channels in arterial and intestinal smooth muscle cell
membranes. Pflugers Arch 1985;403:120-127
2. Cook NS: The pharmacology of potassium channels and their
therapeutic potential. Trends Pharm Sci 1988;9:21-28
3. Jones AW: Arterial tissue cations, in Genest J, Kuchel O,
Hamet P, Cantin M (eds): Hypertension. New York, McGrawHill Book Co, 1983, pp 488-497
4. Bohr DF, Webb RC: Vascular smooth muscle membrane in
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5. Lamb FS, Webb RC: Regenerative electrical activity and
arterial contraction in hypertensive rats. Hypertension 1989;
13:70-76
6. Chang D, Hsieh PS, Dawson DC: CALCIUM: A program in BASIC
for calculating the composition of solutions with specified free
concentrations of calcium, magnesium and other divalent cations. Comput Biol Med 1988;18:351-366
7. Latorre R, Miller C: Conductance and selectivity in potassium
channels. J Membr Biol 1983;71:ll-3O
8. vanBreemen C, Cauvin C, Johns A, Leijten P, Yamamoto H:
Ca2+ regulation of vascular smooth muscle. Fed Proc 1986;
45:2746-2751
9. Lamb FS, Webb RC: Potassium conductance and oscillatory
contractions in tail arteries from genetically hypertensive rats. /
Hypertens 1989;7:457-463
KEY WORDS • barium • potassium • vascular smooth muscle
• stroke-prone spontaneously hypertensive rats
Potassium channels and vascular reactivity in genetically hypertensive rats.
P B Furspan and R C Webb
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Hypertension. 1990;15:687-691
doi: 10.1161/01.HYP.15.6.687
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1990 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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