(Hypertension. 1998;31:872-877.)
© 1998 American Heart Association, Inc.
Mechanisms Responsible for Forskolin-Induced Relaxation of Rat Tail Artery
Christopher M. Rembold;
; Xiao-Liang Chen
From the Cardiovascular Division, Departments of Internal Medicine and
Physiology, University of Virginia Health Sciences Center (Charlottesville).
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Abstract
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Abstract—The goal of the present
study was to determine the physiologically
relevant mechanisms for forskolin-induced relaxation of intact rat tail
artery. We stimulated deendothelialized rat
tail artery with phenylephrine and then relaxed the tissue
with the addition of forskolin, a specific activator of
adenylyl cyclase. We measured membrane potential with the use of
microelectrodes, estimated intracellular Ca2+ concentration
([Ca2+]i) with the use of fura 2, and
measured isometric force with a strain-gauge transducer. We found that
0.3 to 1.0 µmol/L forskolin relaxed 0.3 to 1.0 µmol/L
phenylephrine-stimulated rat tail artery by decreasing the
[Ca2+]i sensitivity of force as well as
through repolarization. There was no evidence for forskolin-induced
inhibition of Ca2+ influx beyond that associated with
repolarization. There also was no evidence for forskolin-induced
enhancement of Ca2+ efflux or sequestration. Inhibition of
ATP-activated K+ channels with 10 µmol/L
glibenclamide, Ca2+-activated K+
channels with 50 nmol/L iberiotoxin, Ca2+-activated
K+ channels with 3 or 10 mmol/L
tetraethylammonium ion, inwardly rectified
K+ channels with 20 µmol/L Ba2+, and
voltage-activated K+ channels with 0.5 mmol/L
4-aminopyridine did not significantly attenuate
forskolin-induced reductions in [Ca2+]i or
force. Forskolin-induced repolarization was not altered by 10
µmol/L glibenclamide or 0.5 mmol/L
4-aminopyridine. These data suggest that these
K+ channels were not individually involved in
forskolin-induced relaxation and that other channels and/or multiple
channels are involved in forskolin-induced repolarization of intact rat
tail artery. Our data also suggest that forskolin-induced relaxation of
intact rat tail artery occurred primarily through repolarization and
reductions in the [Ca2+]i sensitivity of
force.
Key Words: calcium ion concentration • repolarization • forskolin • membrane potential • potassium channels
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Introduction
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There are five
mechanisms responsible for agonist-induced arterial smooth
muscle contraction (for a review, see Reference 11 ): (1) depolarization,
(2) release of Ca2+ from the sarcoplasmic
reticulum, (3) direct activation of L-type Ca2+
channels to increase Ca2+ influx beyond that
expected for the level of depolarization, (4) activation of other
Ca2+-permeable channels, and (5) increasing the
[Ca2+]i sensitivity of
myosin light chain phosphorylation.
Increases in cAMP levels are hypothesized to induce smooth muscle
relaxation by decreasing
[Ca2+]i and/or the
sensitivity of the contractile apparatus to
[Ca2+]i. The action of
cAMP may not only be through activation of cAMP-dependent protein
kinase because large increases in [cAMP] can also activate
cGMP-dependent protein kinase.2 3
Most4 5 6 7 but not all8 9
studies in smooth muscle found that elevations in [cAMP] decreased
[Ca2+]i.
Adenosine decreased
45Ca2+ influx in
coronary artery,10 and forskolin reduced
Mn2+ influx (a surrogate for
Ca2+ influx) in swine carotid
artery.11 These results suggest that increases in
cAMP concentration may decrease
[Ca2+]i, at least
partially by decreasing Ca2+ influx.
Increases in cAMP levels are hypothesized to induce relaxation
through at least six specific mechanisms. Five of these six mechanisms
are the reverse of the above agonist-dependent contractile mechanisms:
(1) increases in [cAMP] can repolarize some smooth muscles,
potentially by activating KCa
channels.12 13 (2) Increases in [cAMP], via
activation of cGMP-dependent protein kinase, could inhibit release of
Ca2+ from the intracellular
Ca2+ store.14 15 (3) cAMP,
under certain conditions, may inactivate voltage-dependent
L-type Ca2+ channels.16 (4)
It is possible, but not reported, that cAMP could inhibit
Ca2+ influx through
Ca2+-permeable nonselective
Ca2+ channels. (5) cAMP can decrease the
[Ca2+]i sensitivity of
phosphorylation.7 17 This effect
was not caused by cAMP-dependent protein kinase
phosphorylation of myosin light chain
kinase.18 19 Finally, (6) cAMP may enhance
Ca2+ efflux or sequestration. This is the only
mechanism that is not the reverse of a mechanism responsible for
contraction. There are few data on cAMP, but there are several proposed
mechanisms for cGMP-dependent increases in Ca2+
extrusion and sequestration, such as cGMP-dependent protein kinase can
phosphorylate phospholamban, a process that
activates the sarcoplasmic reticulum
Ca2+-ATPase by removing the inhibition caused by
unphosphorylated
phospholamban.4 20 21 Second, cGMP-dependent
protein kinase also may activate a plasma membrane
Ca2+-ATPase.22 23 Third,
8-bromo-cGMP increased
Na+/Ca2+ exchange in
isolated rat aortic smooth muscle cells.24
The first goal of this study was to determine which of these mechanisms
were physiologically relevant during
cAMP-dependent rat tail artery relaxation. The second goal was to
characterize pharmacologically which membrane ion channels were
involved in cAMP-induced repolarization. Unlike prior studies, we
performed these experiments in sections of intact arteries rather than
isolated smooth muscle cells. This approach allowed determination of
changes in Em and isometric force in one set of
intact tissues and of
[Ca2+]i and isometric
force in a second set of intact tissues. We stimulated
deendothelialized rat tail artery with 0.3 to
1.0 µmol/L phenylephrine and then relaxed the tissue
by the addition of low concentrations of forskolin (0.3 to 1.0
µmol/L), a specific activator of adenylyl cyclase.
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Methods
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Tissues
Rat tail arteries were obtained from 180- to 250-g male
Sprague-Dawley rats that were humanely killed with 0.15 mg/g
pentobarbital according to a protocol approved by the Animal Use
Committee of the University of Virginia. The adventitia was dissected,
and the arterial tissue was immobilized on a
dual-wire myograph (Mulvany-Halperin type; University of Vermont
[Burlington]) in a temperature-controlled tissue bath at 35°C (the
physiological temperature for this vessel). The
physiological salt solution contained (in
mmol/L) 140 NaCl, 4.7 KCl, 5 MOPS, 1.2
Na2HPO4, 1.6
CaCl2, 1.2 MgSO4, and 5.6
D-glucose, pH adjusted to 7.4 at 35°C. The
endothelium was removed through gently rubbing with a
wire, a procedure that eliminated acetylcholine-induced relaxation.
Removal of the endothelium was necessary to prevent
contamination of fura 2 signals by changes in
endothelial
[Ca2+]i. At the end of
each experiment, the tissue was depolarized with 90 mmol/L
[K+]o, and the fura 2 and
force responses were normalized to the 90 mmol/L
[K+]o response. When
tissues were depolarized with high
[K+]o, we added 3
µmol/L phentolamine to block the effect of
endogenously released norepinephrine.
The experimental design consisted of (1) the addition of the
K+ channel blockers (if included) for 5 minutes,
(2) stimulation with various concentrations of
phenylephrine for 5 minutes, and (3) the addition of
increasing concentrations of forskolin at 5-minute intervals. The only
exception to this was the iberiotoxin experiments, in which the high
cost of iberiotoxin required that iberiotoxin be added in only the
forskolin-containing solutions.
Measurement of Em
Em was measured in one set of tissues.
High-impedance microelectrodes (50 to 80 M
filled with 2 mol/L KCl)
were pulled with a Flaming Brown P87 puller (Sutter). Cells were
impaled with microelectrodes held in a Leitz micromanipulator, and
Em was measured with a Dagan model 8700
electrometer. Criteria for acceptance of recordings
were (1) an abrupt decrease in voltage on impalement, (2) stable
baseline for 3 minutes, and (3) unchanged electrode resistance and
potential after removal of the microelectrode from the tissue.
Isometric force was measured with a strain-gauge transducer (Kulite
BG-10; University of Vermont [Burlington]), and solutions were
perfused through the 6-mL chamber at 6 to 10 mL/min with a roller pump
(perfusion was necessary to prevent artifacts from changing solution
level). Measurements were made in tissues from four to six arteries and
presented as mean±1 SEM. The perfusion setup required large
solution volumes (120 mL) to maintain constant solution depth;
therefore, measurement of Em was not performed
when reagents were expensive (ie, iberiotoxin). In these cases, only
experiments with fura 2 were performed.
Measurement of Myoplasmic [Ca2+]i
[Ca2+]i was
estimated with fura 2 in a second set of tissue. Fura 2 was loaded
intracellularly via a 60-minute incubation in the AM ester of fura 2
(20 µmol/L). The fura 2 was washed out for 30 minutes before
recording to allow adequate hydrolysis and washout of fura 2-AM
(see Refs 11 and 25 for details of fura 2 loading and
fluorescence measurement equipment [University of
Pennsylvania, Philadelphia]). Isometric force was measured with a
capacitative force transducer (Harvard Apparatus).
Measurements were made in four to six tissue samples and
presented as mean±1 SEM. Fura 2 signals were calibrated
through incubation of the tissue sequentially in solutions containing
ionomycin with low Ca2+ (10 min) and high
Ca2+ (5 min); then, the fura 2 signal was
quenched with Mn2+ (5 min). The
low-Ca2+ calibration solution contained (in
mmol/L) 0.04 ionomycin, 2 EGTA, 145 KCl, 5 MOPS, 1.2
MgSO4, and 5.6 D-glucose, pH adjusted
to 7.4 at 35°C. The high-Ca2+ calibration
solution contained (in mmol/L) 0.04 ionomycin, 5
CaCl2, 145 KCl, 5 MOPS, 1.2
MgSO4, and 5.6 D-glucose, pH adjusted
to 7.4 at 35°C. The Mn2+ quench solution
contained (in mmol/L) 5 MnCl2, 145 KCl, 5
MOPS, 1.2 MgSO4, and 5.6 D-glucose.
There are many uncertainties in fura 2
calibration26 27 ; for example, the presence of
various proteins (0.3 to 1 mmol/L) in a calibration solution
increased the measured Kd value of fura 2
by as much as 200%.27 For these reasons, we
primarily present data as background-subtracted 340 nm/380 nm
ratios normalized to the response observed 5 minutes after stimulation
with 90 mmol/L
[K+]o. The force reported
in the figures was that measured in the experiments with fura 2. The
force measured in the Em experiments was similar
to that observed in the experiments with fura 2.
Statistical Analysis
Measurements were compared with the use of Student's
t test if there were two groups or with an ANOVA and the
Student-Newman-Keuls test if there were more than two groups.
Regression analysis was performed with the least-squares
method, and slopes were compared by calculating residual variances and
a t test.28 When possible (see Fig 2A
and 2B), regression was performed on measured values from individual
experiments. However, this was not possible in the unpaired comparison
of Em and fura 2–estimated
[Ca2+]i (see Fig 2C). A
value of P<.05 was considered significant.
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Figure 2. The relation between forskolin-induced changes in
Em, fura 2–estimated [Ca2+]i,
and contraction. A, Effect of forskolin on the relation between
Em and force; data from individual rat tail
arterial tissues unstimulated or stimulated with 0.1, 0.3,
or 1.0 µmol/L phenylephrine ( ) or relaxed with
0.3 or 1.0 µmol/L forskolin after stimulation with 0.3 or 1
µmol/L phenylephrine ( ). Force was analyzed as
log10 force to correct for the nonlinear the relation
between Em and force; this accounts for the curvature of
the regression lines. Statistical analysis revealed that the
regression line for tissues treated with phenylephrine and
forskolin (log10 force=0.048 · Em+2.64,
r2=.52, dashed line) was significantly
(P<.001) different than the regression line for tissues
treated with phenylephrine alone (log10
force=0.075 · Em+3.82,
r2=.88, solid line). B, Effect of forskolin
on the relation between fura 2–estimated
[Ca2+]i and force; data from individual rat
tail arterial tissues unstimulated or stimulated with 0.1,
0.3, or 1.0 µmol/L phenylephrine () or relaxed
with 0.3 or 1 µmol/L forskolin after stimulation with 0.3 or
1 µmol/L phenylephrine (). Statistical
analysis revealed that the regression line for tissues treated
with phenylephrine and forskolin (force=0.94 ·
fura+5.15, r2=.60, dashed line) was
significantly different (P<.001) than the regression
line for tissues treated with phenylephrine alone
(force=1.96 · fura+20.74, r2=.75,
solid line). C, Effect of forskolin on the relation between
Em and fura 2–estimated [Ca2+]i;
mean±1 SEM data from rat tail arterial tissues
unstimulated or stimulated with 0.1, 0.3, or 1.0 µmol/L
phenylephrine () or relaxed with 0.3 or 1.0
µmol/L forskolin after stimulation with 0.3 or 1 µmol/L
phenylephrine (). Data from the individual tissues could
not be analyzed because Em and fura 2–estimated
[Ca2+]i were not measured in the same set of
tissues and therefore are unpaired. Statistical analysis
revealed that the regression line of the mean values for tissues
treated with phenylephrine and forskolin (fura=2.95
· Em+140, r2=.97, dashed line)
was not statistically different (0.4>P>.2) than the
regression line for tissues treated with phenylephrine
alone (fura=7.26 · Em+289,
r2=.90, solid line). Because the number of
mean values was low, the lack of statistical significance could result
from a type II error.
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Results
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Stimulation with phenylephrine induced a
dose-dependent depolarization, increase in fura 2–estimated
[Ca2+]i, and contraction
(Fig 1). The addition of 0.3 and 1.0
µmol/L forskolin to 0.3 and 1.0 µmol/L
phenylephrine–stimulated tissue induced repolarization,
reductions in fura 2–estimated
[Ca2+]i, and reduction in
force. These data show that forskolin induced repolarization and
suggest that the repolarization may be at least partially responsible
for relaxation.
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Figure 1. Forskolin-induced relaxation of
phenylephrine-stimulated rat tail artery. Em
(mV; top), fura 2 340 nm/380 nm ratio (an estimate of
[Ca2+]i; as a percent of the response after 5
minutes to 90 mmol/L [K+]o [middle]),
and force (as a percentage of the response after 5 minutes to 90
mmol/L [K+]o; bottom) in rat tail artery
tissues either (1) unstimulated, (2) stimulated with 1.0 µmol/L
phenylephrine alone, (3) stimulated with 1.0 µmol/L
phenylephrine and then relaxed with 0.3 µmol/L
forskolin, (4) stimulated with 1.0 µmol/L
phenylephrine and then relaxed with 1.0 µmol/L
forskolin (after 0.3 µmol/L forskolin), (5) stimulated with
0.3 µmol/L phenylephrine alone, (6) stimulated with
0.3 µmol/L phenylephrine and then relaxed with
0.3 µmol/L forskolin, (7) stimulated with 0.3 µmol/L
phenylephrine for 5 minutes and then relaxed with 1.0
µmol/L forskolin (after 0.3 µmol/L forskolin), and (8)
stimulated with 0.1 µmol/L phenylephrine alone (n=4
to 7). Each treatment was 5 minutes in duration, and measurements were
made 5 minutes after each treatment. Data are presented as
mean±1 SEM. The dashed lines 1 and 2 refer to the discussion in the
text.
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Further evaluation of the data in Fig 1
revealed that repolarization
was not responsible for the entire relaxation. First, we compared the
combination of 1 µmol/L phenylephrine and 0.3
µmol/L forskolin with 0.3 µmol/L phenylephrine
alone (Fig 1, dashed line 1). The measured fura 2 signals did not
statistically differ (t test, P=.71), but the
contraction was statistically larger than that with
phenylephrine alone (t test, P=.002).
Second, we compared the combination of 1 µmol/L
phenylephrine and 1.0 µmol/L forskolin, the
combination of 0.3 µmol/L phenylephrine and 0.3
µmol/L forskolin, and 0.1 µmol/L phenylephrine
alone (Fig 1, dashed line 2). The measured fura 2 signals did not
statistically differ (ANOVA, P=.39), but the contraction was
statistically larger with phenylephrine alone than the
contraction observed in the presence of 0.3 or 1.0 µmol/L
forskolin (ANOVA, P<.001). These data suggest that some of
the forskolin-induced relaxation was caused by reductions in the
[Ca2+]i sensitivity of
force.
These conclusions were supported with a comparison of the relationships
among Em, fura 2 signals, and contraction. First,
we compared Em and force. The addition of 0.3 or
1.0 µmol/L forskolin to 0.3 or 1.0 µmol/L
phenylephrine–stimulated rat tail artery decreased
contractile force at intermediate and high Em
levels compared with phenylephrine stimulation alone (Fig 2A, and , respectively; the
regression lines are based on a log10 transform
of force data, and they differed with a value of P<.001).
This suggests that factors beyond repolarization were responsible for
at least part of the relaxation.
Second, we compared fura 2–estimated
[Ca2+]i and force. The
addition of 0.3 or 1.0 µmol/L forskolin to 0.3 or 1.0
µmol/L phenylephrine–stimulated rat tail artery
decreased contractile force at intermediate and high levels of fura
2–estimated [Ca2+]i
compared with phenylephrine stimulation alone (Fig 2B,
and , respectively; the linear regression lines differed with a
value of P<.001). These data suggest that forskolin
decreased the [Ca2+]i
sensitivity of force.
Third, we compared the relation between Em and
fura 2–estimated
[Ca2+]i. The plot in Fig 2C suggests that the addition of forskolin was associated with a
modestly higher fura 2–estimated
[Ca2+]i at lower
Em values compared with the use of
phenylephrine alone. Because the Em
and fura 2 data are not paired, statistical comparison regarding
individual data points is not possible. Evaluation of the regression
line through the mean values revealed no significant difference. These
data suggest that the combination of phenylephrine and
forskolin either had no effect or, possibly, increased
[Ca2+]i above that
expected on the basis of the Em. More
importantly, this finding demonstrates that forskolin is not inhibiting
potential independent Ca2+ influx, inhibiting
nonselective cation channels, or activating Ca2+
efflux/sequestration (mechanisms 3, 4, and 6 listed in the
introduction, respectively). These three mechanisms should reduce
[Ca2+]i below that
expected on the basis of measured Em.
We examined the relative importance of forskolin-induced repolarization
compared with other mechanisms by reversing the repolarization with
increasing [K+]o (Fig 3). Stimulation with 0.3 µmol/L
phenylephrine depolarized and contracted the tissues. The
addition of 1 µmol/L forskolin repolarized and relaxed the
tissues. The further addition of 30, 35, and 40 mmol/L
[K+]o depolarized the
tissues and increased force. The addition of 35 mmol/L
[K+]o depolarized the
tissues to a level that did not statistically differ from that observed
with 0.3 µmol/L phenylephrine alone. The force
observed with 0.3 µmol/L phenylephrine plus 1
µmol/L forskolin plus 35 mmol/L
[K+]o was 49% of the
value observed with 0.3 µmol/L phenylephrine alone.
These data suggest that approximately half of the forskolin-induced
relaxation was caused by the repolarization and the other half by
decreases in the [Ca2+]i
sensitivity of force.
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Figure 3. Reversal of forskolin-induced repolarization
increases force to approximately half of that observed with
phenylephrine alone. Em (mV; top) and force (as
a percentage of the response after 5 minutes to 90 mmol/L
[K+]o; bottom) in rat tail artery tissues
either (1) unstimulated or stimulated with (2) 0.3 µmol/L
phenylephrine, (3) 0.3 µmol/L
phenylephrine plus 1.0 µmol/L forskolin, and (4 to
6) 0.3 µmol/L phenylephrine, 1.0 µmol/L
forskolin, plus either 30, 35, or 40 mmol/L
[K+]o, respectively (n=5). Measurements were
made 5 minutes after treatment. Data are presented as mean±1
SEM. *Significant difference from unstimulated. #Significant difference
from 0.3 µmol/L phenylephrine. &Significant
difference from 0.3 µmol/L phenylephrine plus
1.0 µmol/L forskolin by Student-Newman-Keuls test.
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We next evaluated the role of K+ channels in
forskolin-induced relaxation. We inhibited KATP
channels with 10 µmol/L glibenclamide, KCa
channels with 50 nmol/L iberiotoxin, both KATP
and KCa channels with 10 µmol/L
glibenclamide and 50 nmol/L iberiotoxin, KCa
channels with 3 or 10 mmol/L TEA+,
KIR channels with 20 µmol/L
Ba2+, and KV channels with
0.5 mmol/L 4-aminopyridine. Preliminary studies
found that 10 µmol/L glibenclamide, a specific
inhibitor of KATP channels,
completely inhibited 10 µmol/L pinacidil–induced relaxation of
rat tail artery precontracted with 1 µmol/L
phenylephrine (data not shown). There is no good agonist
for KCa channels; therefore, we used a standard
dose (50 nmol/L29 ) of iberiotoxin.
TEA+ is a less-specific K+
channel antagonist, acting at low concentrations on
KCa channels (EC50
0.2 µmol/L) and at higher concentrations on
KATP channels (EC50
7 mmol/L) and KV channels
(EC50 >5 mmol/L)
channels.29 The KIR channel
is inhibited by Ba2+ with an
IC50 value of 2
µmol/L.29 The KV channel
is inhibited by 4-aminopyridine with an
IC50 value of 0.3 to 1
mmol/L.29
We incubated rat tail artery tissue with the above
K+ channel blockers, stimulated the tissue with
0.3 µmol/L phenylephrine, and then evaluated the
response to 0.3 µmol/L forskolin. None of tested
K+ channel blockers significantly attenuated the
forskolin-induced reduction in fura 2–estimated
[Ca2+]i or force (Fig 4). Em was measured
with only some of the K+ inhibitors.
In the presence of TEA+, 0.3 µmol/L
forskolin induced APs and transient contractions in five of seven
preparations. The reported mean Em value is from
the two preparations that did not exhibit APs (Fig 4, AP). The
forskolin-induced repolarization was not significantly altered by
10 µmol/L glibenclamide or 0.5 mmol/L
4-aminopyridine. Similar results were observed with
1.0 µmol/L forskolin–induced relaxation of 0.3 µmol/L
phenylephrine–stimulated rat tail artery (data not shown).
This higher forskolin concentration abolished
TEA+-induced APs, and the mean repolarization did
not differ from that observed with 1.0 µmol/L forskolin without
TEA+. These data suggest that these
K+ channels were not individually responsible for
forskolin-induced relaxation of phenylephrine-induced
contraction in the deendothelialized rat tail
artery.
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Figure 4. Effect of K+ channel blockers on
0.3 µmol/L forskolin–induced relaxation of 0.3 µmol/L
phenylephrine–stimulated rat tail artery. Em
(mV; top), fura 2 340 nm/380 nm ratio (as a percentage of the response
after 5 minutes to 90 mmol/L [K+]o;
middle), and force (as a percentage of the response after 5 minutes to
90 mmol/L [K+]o; bottom) in rat tail
artery tissues either (1) unstimulated or (2) stimulated with 0.3
µmol/L phenylephrine, (3) stimulated with 0.3
µmol/L phenylephrine and then relaxed with 0.3
µmol/L forskolin, or (4 to 10) stimulated with 0.3 µmol/L
phenylephrine and then relaxed with 0.3 µmol/L
forskolin in the presence of the K+ channel blockers
glibenclamide (10 µmol/L; column 4), iberiotoxin (50 nmol/L;
column 5), both glibenclamide and iberiotoxin (10 µmol/L and 50
nmol/L, respectively; column 6), TEA+ (3 mmol/L;
column 7), TEA+ (10 mmol/L; column 8),
Ba2+ (20 µmol/L; column 9), and
4-aminopyridine (0.5 mmol/L; column 10). Each
treatment was 5 minutes in duration, and measurements were made 5
minutes after each treatment. Data are presented as mean±1 SEM
(n=4 to 7). ND indicates not done; APs were present in some
tissues; the data are from tissues without APs.
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Discussion
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The first goal of the present study was to determine the
physiologically relevant mechanism or
mechanisms responsible for forskolin-induced rat tail artery
relaxation. Our approach was to measure Em, fura
2–estimated [Ca2+]i, and
force in intact phenylephrine-stimulated
deendothelialized rat tail artery. This
approach allows delineation of some but not all of the known
mechanisms. Specifically, we can determine whether relaxation is caused
by repolarization (mechanism 1, as given), by decreases in the
[Ca2+]i sensitivity of
force (mechanism 5), or by an alteration in the relation between
Em and fura 2–estimated
[Ca2+]i that would
indicate mechanism 3 (direct inactivation of Ca2+
channels), mechanism 4 (inactivation of nonselective cation channels),
or mechanism 6 (enhancement of Ca2+ efflux or
sequestration) was involved in the relaxation. In this study, we did
not investigate the role of Ca2+ release
inhibition (mechanism 2).
We found that forskolin-induced both a repolarization (Figs 1 and 2A)
and a decrease in the
[Ca2+]i sensitivity of
force (Fig 2B). Both were observed in maximally as well as submaximally
phenylephrine-stimulated tissues (Fig 1). Approximately
half of the overall relaxation of 0.3 µmol/L
phenylephrine–stimulated rat tail artery was associated
with repolarization (Fig 3). When the forskolin-induced repolarization
was reversed by the addition of 35 mmol/L
[K+]o, force increased to
49% of the value observed with phenylephrine alone. The
other half of the overall relaxation was associated with a decrease in
the [Ca2+]i sensitivity
of force.
Forskolin was associated with the similar or slightly higher
[Ca2+]i for a certain
Em compared with that observed with
phenylephrine alone (Fig 2C). This suggests that there may
be a mechanism keeping
[Ca2+]i higher than that
expected given the measured Em. It is possible
that (1) forskolin is activating L channels and enhancing
Ca2+ influx,16 (2)
forskolin is inhibiting Ca2+ efflux, or 3)
forskolin-induced repolarization is uncovering
phenylephrine-dependent activation of L channels
(contractile mechanism 330 31 ). However,
statistical analysis of our data did not clearly demonstrate
this effect. More importantly, our data suggest that forskolin did not
relax phenylephrine-stimulated rat tail artery by relaxing
mechanism 3 (voltage-independent inactivation of L channels), relaxing
mechanism 4 (inactivation of nonselective cation channels), or
mechanism 6 (enhanced Ca2+ efflux or
sequestration). These three mechanisms would reduce
[Ca2+]i below that
expected given the relative depolarization; this was not observed (Fig 2C).
In the histamine-stimulated swine carotid artery, we found that
forskolin reduced Mn2+ influx (a surrogate for
Ca2+ influx), fura 2–estimated
[Ca2+]i, and
force.11 We suggested that the reduction in
Mn2+ influx resulted from repolarization or
voltage-independent inactivation of Ca2+
channels. The results of the present study suggest that the former
is more important in the rat tail artery. It appears that
forskolin-induced repolarization and the resulting decrease in
Ca2+ influx occur in both the swine carotid and
rat tail artery (although we cannot measure Em in
the former for technical reasons). It should be noted that high
concentrations of forskolin (50 µmol/L) have been reported to
alter Na+ and K+ currents
independent of cAMP concentration.32 33 Our
experiments were performed with low forskolin concentrations (
1
µmol/L), at which forskolin is a specific activator of
adenylyl cyclase rather than a channel blocker.
The second goal of this study was to determine whether regulation of
K+ channels was
physiologically relevant in forskolin-induced
repolarization. The results of several studies suggested that increases
in cAMP concentration activate KCa
channels in bilayer12 and whole-cell
studies.13 Calcitonin gene–related peptide, a
stimulus that increases cAMP concentration, also activates
KATP channels.34 The
activation of these or other K+ channels could
induce repolarization, thereby decreasing Ca2+
influx and [Ca2+]i. We
found that inhibition of KATP channels with
glibenclamide, KCa channels with
TEA+, and KV channels with
4-aminopyridine did not alter forskolin-induced
repolarization, reduction in fura 2–estimated
[Ca2+]i, or the
relaxation (Fig 4). In addition, the blockage of
KCa channels with iberiotoxin and
KIR channels with Ba2+ did
not alter forskolin-induced reductions in fura 2–estimated
[Ca2+]i or relaxation.
These data suggest that other mechanisms, perhaps inactivation of
Na+ channel(s), activation of other
K+ channel(s), and/or inactivation of multiple
channels, are responsible for forskolin-induced repolarization in
intact rat tail artery. It is possible that blockage of one
K+ channel may not significantly alter
repolarization if the other channel or channels compensate for the
block. These data do not rule out a role for individual
K+ channels in forskolin-induced repolarization
of other arteries.
In conclusion, we demonstrated that repolarization and decreases in the
[Ca2+]i sensitivity of
force appear to equally participate in forskolin-induced relaxation of
phenylephrine-stimulated rat tail artery. The activation of
glibenclamide-, iberiotoxin-, TEA+-,
Ba2+-, or
4-aminopyridine–sensitive K+
channels did not appear to be individually responsible for
forskolin-induced repolarization. Potentially, other channels or
multiple channels are involved in forskolin-induced repolarization in
deendothelialized rat tail artery.
|
Selected Abbreviations and Acronyms
|
|---|
| AM |
= |
acetylmethoxy |
| AP |
= |
action potential |
| [Ca2+]i |
= |
intracellular Ca2+ concentration |
| [K+]o |
= |
extracellular K+ concentration |
| KATP |
= |
ATP-activated K+ channel |
| KCa |
= |
Ca2+-activated K+ channel |
| KIR |
= |
inwardly rectified K+ channel |
| KV |
= |
voltage-activated K+ channel |
| MOPS |
= |
3-(N-morpholino)propanesulfonic acid |
| TEA+ |
= |
tetraethylammonium ion |
|
|
Acknowledgments
|
|---|
This work was supported by grant J-360 from the Jeffress Trust.
The authors thank Barbara Weaver for technical support and Dr Terrence
Smith for helpful discussions.
|
Footnotes
|
|---|
Reprint requests to Christopher M. Rembold, MD, Box 146, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
Received September 10, 1997;
first decision October 10, 1997;
accepted October 29, 1997.
|
References
|
|---|
1.
Rembold CM. Electromechanical and
pharmacomechanical coupling. In: Barany M, ed. Biochemistry of
Smooth Muscle Contraction. Chicago, Ill: Academic Press;
1996:227–239.
2.
Lincoln TM, Cornwell TL, Taylor AE. cGMP-dependent
protein kinase mediates the reduction of Ca2+ by
cAMP in vascular smooth muscle cells. Am J Physiol. 1990;258:C399–C407.[Abstract/Free Full Text]
3.
Francis SH, Noblett BD, Todd BW, Wells JN, Corbin JD.
Relaxation of vascular and tracheal smooth muscle by cyclic
nucleotide analogs that preferentially activate
purified cGMP-dependent protein kinase. Mol Pharmacol. 1988;34:506–517.[Abstract]
4.
Kimura M, Kimura I, Kobayashi S. Relationship Between
cyclic AMP-dependent protein kinase activation and calcium uptake
increase of sarcoplasmic reticulum fraction of hog biliary muscles
relaxed by cholecystokinin-C-terminal peptides. Biochem
Pharmacol. 1982;31:3077–3083.[Medline]
[Order article via Infotrieve]
5.
Parker I, Ito Y, Kuriyama H, Miledi R.
ß-Adrenergic agonists and cyclic AMP decrease intracellular
resting free-calcium concentration in ileum smooth muscle. Proc R
Soc Lond. 1987;230:207–214.[Medline]
[Order article via Infotrieve]
6.
Gunst SJ, Bandyopadhyay S. Contractile force and
intracellular Ca2+ during relaxation of canine
tracheal smooth muscle. Am J Physiol. 1989;257:C355–C364.[Abstract/Free Full Text]
7.
McDaniel NL, Rembold CM, Richard HL, Murphy RA. cAMP
relaxes arterial smooth muscle predominantly by decreasing
cell [Ca2+]. J Physiol (Lond). 1991;439:147–160.[Abstract/Free Full Text]
8.
Takuwa Y, Takuwa N, Rasmussen H. The effects of
isoproterenol on intracellular calcium concentration. J Biol
Chem. 1988;263:762–768.[Abstract/Free Full Text]
9.
Morgan JP, Morgan KG. Alteration of cytoplasmic
ionized calcium levels in smooth muscle by vasodilators in the ferret.
J Physiol (Lond). 1984;357:539–551.[Abstract/Free Full Text]
10.
Motulsky HJ, Michel MC. Neuropeptide Y mobilizes
Ca2+ and inhibits adenylate cyclase
in human erythroleukemia cells. Am J Physiol. 1988;255:E880–E885.[Abstract/Free Full Text]
11.
Chen X-L, Rembold CM. Cyclic nucleotide
dependent regulation of Mn2+ influx,
[Ca2+]i, and
arterial smooth muscle relaxation. Am J
Physiol. 1992;263:C468–C473.[Abstract/Free Full Text]
12.
Savaria D, Lanoue C, Cadieux A, Rousseau E. Large
conducting potassium channel reconstituted from airway smooth muscle.
Am J Physiol. 1992;262:L327–L336.[Abstract/Free Full Text]
13.
Anwer K, Toro L, Oberti C, Stefani E, Sanborn BM.
Ca2+-activated K+
channels in pregnant rat myometrium: modulation by a beta-adrenergic
agent. Am J Physiol. 1992;263:C1049–C1056.[Abstract/Free Full Text]
14.
Rapoport RM. Cyclic guanosine monophosphate inhibition
of contraction may be mediated through inhibition of
phosphatidylinositol hydrolysis in rat aorta. Circ Res. 1986;58:407–410.[Abstract/Free Full Text]
15.
Chen X-L, Rembold CM. Nitroglycerin
relaxes rat tail artery primarily by decreasing
[Ca2+]i sensitivity and
partially by repolarization and inhibiting Ca2+
release. Am J Physiol. 1996;271:H962–H968.[Abstract/Free Full Text]
16.
Ishikawa T, Hume JR, Keef KD. Regulation of
Ca2+ channels by cAMP and cGMP in vascular smooth
muscle cells. Circ Res. 1993;73:1128–1137.[Abstract/Free Full Text]
17.
Nishimura J, Van Breemen C. Direct regulation of smooth
muscle contractile elements by second messengers. Biochem Biophys
Res Commun. 1989;163:929–935.[Medline]
[Order article via Infotrieve]
18.
Stull JT, Hsu L-C, Tansey MG, Kamm KE. Myosin light
chain kinase phosphorylation in tracheal smooth muscle.
J Biol Chem. 1990;265:16683–16690.[Abstract/Free Full Text]
19.
Van Riper DA, Weaver BA, Stull JT, Rembold CM. Myosin
light chain kinase phosphorylation in swine carotid
artery contraction and relaxation. Am J Physiol. 1995;268:H2466–H2475.[Abstract/Free Full Text]
20.
Lincoln TM, Cornwell TL. Towards an understanding of
the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle
relaxation. Blood Vessels. 1991;28:129–137.[Medline]
[Order article via Infotrieve]
21.
Twort CH, Van Breemen C. Cyclic guanosine
monophosphate-enhanced sequestration of Ca2+ by
sarcoplasmic reticulum in vascular smooth muscle. Circ Res. 1988;62:961–964.[Abstract/Free Full Text]
22.
Furakawa K, Tawada Y, Shigekawa M. Regulation of the
plasma membrane Ca2+ pump by cyclic
nucleotides in cultured vascular smooth muscle cells.
J Biol Chem. 1988;263:8058–8065.[Abstract/Free Full Text]
23.
Popescu LM, Foril CP, Hinescu M, Panoiu C, Cinteza M,
Gherasim L. Nitroglycerin stimulates the sarcolemmal
Ca++-extrusion ATPase of coronary smooth
muscle cells. Biochem Pharmacol. 1985;34:1857–1860.[Medline]
[Order article via Infotrieve]
24.
Furukawa K-I, Ohshima N, Tawada-Iwata Y, Shigekawa M.
Cyclic GMP stimulates
Na+/Ca2+ exchange in
vascular smooth muscle cells in primary culture. J Biol
Chem. 1991;266:12337–12341.[Abstract/Free Full Text]
25.
Gilbert EK, Weaver BA, Rembold CM. Depolarization
decreases the
[Ca2+]i-sensitivity of
myosin light chain kinase in arterial smooth muscle: a
comparison of aequorin and fura 2 [Ca2+]
estimates. FASEB J. 1991;5:2593–2599.[Abstract]
26.
Shuttleworth TJ, Thompson JL. Effect of temperature on
receptor-activated changes in
[Ca2+]i and their
determination using fluorescent probes. J Biol
Chem. 1991;266:1410–1414.[Abstract/Free Full Text]
27.
Uto A, Arai H, Ogawa Y. Reassessment of fura-2 and the
ratio method for determination of intracellular
Ca2+ concentrations. Cell Calcium. 1991;12:29–37.[Medline]
[Order article via Infotrieve]
28.
Armitage P, Berry G. Statistical Methods in
Medical Research, 3rd ed. Oxford, UK: Blackwell Scientific
Publications; 1994:291–311.
29.
Nelson MT, Quayle JM. Physiological
roles and properties of potassium channels in arterial
smooth muscle. Am J Physiol. 1995;268:C799–C822.[Abstract/Free Full Text]
30.
Nelson MT, Standen NB, Brayden JE, Worley JF III.
Noradrenaline contracts arteries by activating
voltage-dependent calcium channels. Nature. 1988;336:382–385.[Medline]
[Order article via Infotrieve]
31.
Chen X-L, Rembold CM. Phenylephrine
contracts rat tail artery by one electromechanical and three
pharmacomechanical mechanisms. Am J Physiol. 1995;268:H74–H81.[Abstract/Free Full Text]
32.
Wagoner PK, Pallotta BS. Modulation of acetylcholine
receptor desensitization by forskolin is independent of cAMP.
Science. 1988;240:1655–1658.[Abstract/Free Full Text]
33.
Hoshi T, Garber SS, Aldrich RW. Effect of forskolin
on voltage-gated K channels is independent of adenylate
cyclase activation. Science. 1988;240:1652–1654.[Abstract/Free Full Text]
34.
Quayle JM, Bonev AD, Brayden JE, Nelson MT. Calcitonin
gene-related peptide activated ATP-sensitive
K+ currents in rabbit arterial smooth
muscle via protein kinase A. J Physiol (Lond). 1994;475:9–13.[Abstract/Free Full Text]
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Abstract
Introduction
