Frequency modulation of Ca2+
sparks is involved in regulation of arterial diameter by cyclic
nucleotides
Valerie A.
Porter1,
Adrian D.
Bonev1,
Harm J.
Knot1,
Thomas J.
Heppner1,
Andra S.
Stevenson1,
Thomas
Kleppisch1,
W. J.
Lederer2, and
Mark T.
Nelson1
1 Department of Pharmacology,
University of Vermont, Colchester, Vermont 05446; and
2 Department of Physiology and The
Medical Biotechnology Center, University of Maryland School of
Medicine, Baltimore, Maryland 21201
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ABSTRACT |
Forskolin, which elevates cAMP levels, and sodium nitroprusside
(SNP) and nicorandil, which elevate cGMP levels, increased, by two- to
threefold, the frequency of subcellular
Ca2+ release
("Ca2+ sparks") through
ryanodine-sensitive Ca2+ release
(RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated
from cerebral and coronary arteries of rats. Forskolin, SNP,
nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of
Ca2+-sensitive
K+
(KCa) currents
["spontaneous transient outward currents" (STOCs)] by
two- to threefold, consistent with
Ca2+ sparks activating STOCs.
These agents also increased the mean amplitude of STOCs by 1.3-fold, an
effect that could be explained by activation of
KCa channels, independent of
effects on Ca2+ sparks. To test
the hypothesis that cAMP could act to dilate arteries through
activation of the Ca2+
spark
KCa channel pathway,
the effects of blockers of KCa
channels (iberiotoxin) and of Ca2+
sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external
K+. Forskolin lowered average
Ca2+ in pressurized arteries while
increasing ryanodine-sensitive, caffeine-induced
Ca2+ transients. These experiments
suggest a new mechanism for cyclic nucleotide-mediated dilations
through an increase in Ca2+ spark
frequency, caused by effects on SR
Ca2+ load and possibly on the RyR
channel, which leads to increased STOC frequency, membrane potential
hyperpolarization, closure of voltage-dependent
Ca2+ channels, decrease in
arterial wall Ca2+, and,
ultimately, vasodilation.
cAMP; cGMP; vasodilation; sarcoplasmic reticulum; KCa channels
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INTRODUCTION |
LOCAL CALCIUM TRANSIENTS or
"Ca2+ sparks" have recently
been measured in smooth muscle cells from cerebral arteries (28). Ca2+ sparks in
arterial myocytes as well as in cardiac myocytes (6) appear to arise
from the opening of one or the coordinated opening of several tightly
clustered ryanodine-sensitive Ca2+
release (RyR) channels in the sarcoplasmic reticulum (SR). In arterial
myocytes, Ca2+ sparks activate
10-100 nearby sarcolemmal
Ca2+-sensitive
K+
(KCa) channels to cause an
outward K+ current (17, 28),
previously referred to as a "spontaneous transient outward
current" (STOC) (4). Ca2+
sparks, themselves, contribute little to overall cellular
Ca2+ in pressurized cerebral
arteries, since their activity is asynchronous (~1/s in a cerebral
artery myocyte) (28). Ca2+
spark-activated KCa channels can
continuously regulate, as a negative feedback element, the membrane
potential of smooth muscle cells in intact pressurized cerebral
arteries (7). Thus either blocking
Ca2+ sparks or decreasing their
frequency of occurrence (5) would lead to membrane potential
depolarization and vasoconstriction through a decrease in
KCa channel activity (Ref. 28; see
also Refs. 7 and 31). Therefore, these results predict that increasing Ca2+ spark frequency could lead to
vasodilation through activation of
KCa channels.
The cyclic nucleotides, cAMP and cGMP, mediate vasodilation of
important endogenous and therapeutic agents (e.g.,
-adrenergic agents, adenosine, calcitonin gene-related peptide, nitric oxide, atrial natriuretic factor, synthetic nitrovasodilators). Diverse mechanisms have been proposed to explain the vasodilator effect of
cyclic nucleotides, including the stimulation of
Ca2+ uptake by the SR (24), the
direct activation of KCa channels (39), activation of ATP-sensitive
K+ channels (18, 19, 36, 48),
activation of voltage-dependent K+
channels (1), and changes in the
Ca2+ sensitivity of the
contractile process (11, 15, 41). Relaxation of cerebral and coronary
arteries to agents that elevate cAMP or cGMP are inhibited by blockers
of KCa channels (29, 32, 33, 35,
42, 47). In fact, in a number of other types of smooth muscle (airway,
mesenteric, uterine, pulmonary), blockers of
KCa channels (iberiotoxin or
charybdotoxin) reduce significantly the relaxing effects of agents that
elevate cAMP and cGMP (2, 3, 8, 16, 23).
KCa channels can be activated by
cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase
(PKG) (39, 44). Recent evidence suggests that
KCa channels in pressurized arteries with tone are primarily activated by local
Ca2+ transients, i.e.,
Ca2+ sparks, and not directly by
average intracellular Ca2+ (28).
We therefore explored the possibility that cAMP and cGMP can increase
KCa channel activity by affecting
Ca2+ sparks. Specifically, our
results provide direct evidence that cAMP and cGMP increase the
frequency of local Ca2+ release
events (Ca2+ sparks) from the SR,
which activate KCa channels.
Furthermore, we provide evidence that cAMP can act, in part, to dilate
pressurized arteries through the
Ca2+ spark
KCa channel pathway.
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METHODS |
Cell isolation. Single smooth muscle
cells were enzymatically isolated from cerebral (basilar) and coronary
(septal) arteries of rats. The cell isolation procedure was slightly
modified from that previously described for cerebral (37) and
coronary (38) arteries. Briefly, an artery was dissected out and placed
in ice-cold Ca2+-free isolation
solution of the following composition (in mM): 60 NaCl, 85 sodium
glutamate, 5.6 KCl, 2 MgCl2, 10 glucose, and 10 HEPES (pH 7.4). After 10 min, the artery was
transferred to the first of two enzyme solutions, with the same ionic
composition as the Ca2+-free
isolation solution described above, containing 1 mg/ml albumin, 0.5 mg/ml papain (Worthington, Freehold, NJ), and 1 mg/ml dithioerythritol, and was digested for 25 min at 37°C. The tissue was then
transferred into isolation solution containing 0.1 mM
CaCl2 and 1 mg/ml of a collagenase
mixture of 30% type H + 70% type F (Sigma) and was digested for
another 10 min at 37°C. The tissue was then washed twice in
Ca2+-free fresh isolation solution
without enzymes for 10 min. Single smooth muscle cells were obtained
from the digested artery by gentle trituration with a polished
wide-bore pipette. After trituration, cells were stored in the same
solution at 4°C, to be used the same day. Cells were left to stick
to the coverslip in the experimental chamber for 15-20 min.
Ca2+ spark
measurements.
The procedure for the measurement of sparks is described in Ref. 28.
Briefly, the cells were loaded with the
Ca2+-sensitive indicator fluo 3 by
a 20-min incubation in 5 µM fluo 3-AM and 2.5 µg/ml pluronic
acid (Molecular Probes, Eugene, OR) followed by a 20-min
wash. All measurements were made within 30 min of the end of the wash.
The cells were scanned with a Bio-Rad 1000 laser scanning confocal
microscope, which was housed in the University of Vermont Cell Imaging
Facility. The resolution was 0.4 µm × 0.4 µm
(x and
y) × 0.8 µm
(z) (28). Images were
acquired using the line scan mode of the confocal microscope; this mode repeatedly scans a single line through a cell. A scan duration of 6 ms
was used. Cells were positioned so that the line would traverse the
long axis of the cell to detect sparks occurring in as much of the cell
volume as possible. The average length of the cell through which the
scan line passed was 36 µm. Scan lines are displayed vertically, and
each line is added to the right of the preceding line to form the line
scan image. In these images, time passes in the horizontal direction
running from left to right, and position along the scan line is given
by the vertical displacement. To minimize the possibility of laser
damage affecting the Ca2+ handling
of the cells, control and test measurements were made from different
cells, with each cell being scanned for the same duration, 18 s total
(6 consecutive line scan images of 512 lines, at 6 ms/line, were
recorded from each cell along a single line). Sparks were analyzed
using custom-written (IDL, Research Systems, Boulder, CO) analysis
programs. To test the significance in the changes of
Ca2+ spark frequency, a one-way
ANOVA on ranks (Student-Newman-Keuls) method was used.
Electrophysiological recordings.
K+ currents were measured in the
whole cell, perforated-patch configuration (14) of the patch-clamp
technique (13) with the use of an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA). The bathing solution (also used for
spark measurements) contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES
(pH 7.4). The pipette solution contained (in mM) 110 potassium
aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA.
All experiments were conducted at room temperature (23°C). Membrane
currents were recorded, while holding the cells at membrane potentials
of
40 mV, or as indicated. The STOCs were analyzed off-line,
with the use of custom analysis programs. Briefly, STOCs were
identified by setting a current threshold at three times the mean
KCa single-channel amplitude at
40 mV (~5 pA), and all events that were greater than this
threshold were included. The mean amplitude and frequency of the STOCs
were determined. The large amplitude and low open probability
(Po) of the
KCa channel permitted the
measurement of single KCa channel
currents with the use of the perforated-patch configuration of the
whole cell voltage clamp. To observe single KCa channel currents,
Ca2+ sparks and hence STOCs were
prevented by thapsigargin (28), which inhibits the SR
Ca2+-ATPase, and the cells were
clamped at 0 mV.
NPo was
calculated over 5-min intervals as
Nj=1 tj · j/T,
where tj is the
time spent with j = 1, 2, . . . N channels open,
N is the maximum number of channels
observed, and T is the duration of the
recording (5 min).
Intact pressurized artery.
Resistance-sized posterior-cerebral arteries from rats were isolated,
cannulated, and pressurized to 60 mmHg as described in Refs. 7, 31, and
20. The maximal diameter of the pressurized arteries was estimated from
the arterial diameter in 30 µM diltiazem. Arterial diameter was
measured with a video dimension analyzer (Living Systems
Instrumentations, Burlington, VT). Results are expressed as means (in
µm) ± SE for n vessels in
diameter experiments. Student's
t-test was used to compare values. A
value of P < 0.05 was considered
significant.
Conventional
Ca2+ imaging.
Isolated smooth muscle cells and intact arteries were loaded with the
Ca2+ indicator dye fura 2. Cells
were incubated with 0.25 µM fura 2-AM for 15 min. Cells were then
washed and allowed to sit for 20 min before measurements were made.
Arteries were loaded by incubation with 2 µM fura 2-AM for 45 min.
Arteries were then cannulated and continuously superfused with
physiological salt solution at 37°C.
Ca2+ was measured ratiometrically
(340:380 nm) using the IMAGE-1/FL quantitative fluorescence measurement
program (Universal Imaging, West Chester, PA).
Chemicals. Unless otherwise stated,
all chemicals used in this study were obtained from Sigma Chemical (St.
Louis, MO), including the following drugs: forskolin, adenosine,
dibutyryl-cAMP (DBcAMP), sodium nitroprusside (SNP), thapsigargin, and
diltiazem. H-89 and ryanodine were obtained from Calbiochem (La Jolla,
CA), and iberiotoxin was from Peptides International (Louisville, KY). Nicorandil was a gift from Chugai Pharmaceuticals (Tokyo, Japan).
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RESULTS |
Forskolin, SNP, and nicorandil increase
Ca2+ spark
frequency.
To test the hypothesis that increases in cAMP and cGMP can increase the
frequency of Ca2+ sparks, the
effects of forskolin (an activator of adenylyl cyclase that elevates
cAMP), SNP, and nicorandil (nitric oxide donors, which elevate cGMP) on
Ca2+ sparks were examined in
single smooth muscle cells isolated from rat cerebral and coronary
arteries. Forskolin significantly increased (P < 0.05) the
Ca2+ spark frequency 2.3-fold
(Fig. 1, A
and B), as measured with a laser
scanning confocal microscope and the
Ca2+ fluorescence indicator fluo
3. This increase was prevented by an inhibitor of PKA, H-89 (1 µM),
which binds to the active center of the catalytic subunit and
suppresses the kinase activity (Fig. 1,
A and
B). The nitrovasodilators, SNP and
nicorandil, also increased Ca2+
spark frequency 2.2- and 2.6-fold, respectively (Fig.
1C; P < 0.05). Forskolin, H-89, or SNP did not cause statistically
significant changes in Ca2+ spark
amplitude or rate of decay. Nicorandil did, however, cause a small but
significant increase in Ca2+ spark
amplitude (8% increase). If, indeed,
Ca2+ sparks can activate
sarcolemmal KCa channels, then
forskolin, SNP, and nicorandil should increase the frequency of STOCs
in these preparations, as had been suggested to occur in other
preparations (9, 22).

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Fig. 1.
Forskolin, sodium nitroprusside, and nicorandil increase
Ca2+ spark frequency in smooth
muscle cells from cerebral and coronary arteries.
A: forskolin (10 µM) increases
Ca2+ spark frequency, and an
inhibitor of protein kinase A (PKA), H-89 (1 µM), prevents the
effects of forskolin on single smooth muscle cells from rat cerebral
arteries. First column shows a series of 6 consecutive line scan images
each of 3 s duration from a control cerebral artery myocyte in which
sparks were observed in scans 1 and
2. Second column shows 6 line scan
images from a different cerebral artery myocyte bathed in forskolin for
20 min, in which sparks were observed in all scans. Third column shows
6 line scans from a 3rd cerebral artery myocyte bathed for 20 min in 10 µM forskolin and the PKA inhibitor H-89, in which sparks were
observed in scans 1, 2, 3, and
5. In each of the line scan images,
position across scan line is given by vertical displacement, and time
progresses from left to
right.
Inset (left of the line scan images):
orientation of scan line through a smooth muscle cell.
B: bar graph summarizing number of
sparks per cerebral artery myocyte during 18 s of scanning in control
(n = 40 cells), forskolin
(n = 40 cells), and forskolin + H-89
cells (n = 40 cells). Spark frequency
was significantly (* P < 0.05)
increased by forskolin. This effect was blocked by presence of H-89.
C: bar graph of effect of
nitrovasodilators sodium nitroprusside (SNP; 10 µM) and nicorandil
(100 µM) on Ca2+ spark
frequency in coronary artery myocytes.
Ca2+ spark frequency
(n = 164 cells) was significantly
increased (* P < 0.05) with
both SNP (n = 132 cells) and
nicorandil (n = 95 cells). Error bars
are SE.
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Forskolin, SNP, and nicorandil increase STOC frequency
and amplitude. KCa
channel currents were measured in arterial myocytes clamped at
40 mV, a potential similar to that of intact pressurized cerebral arteries (7, 20, 31). Whole cell currents were measured using
the perforated-patch mode of the whole cell configuration, so as not to
disturb the intracellular contents of the cells. Forskolin increased
STOC frequency by 2.8 ± 0.5-fold (from 0.9 ± 0.2 Hz;
n = 9; Figs.
2 and
3A). The
PKA inhibitor H-89 (1 µM) completely reversed the effect of forskolin
(Figs. 2 and 3A). Forskolin caused a
small but significant (P < 0.05)
increase in the average STOC amplitude by 1.3 ± 0.1-fold, which was
also reversed by H-89 (Figs. 2 and
3B). Neither forskolin nor H-89
affected the decay of the STOCs. Forskolin had no effect on STOC
frequency or amplitude in the continued presence of H-89. The
nitrovasodilators, SNP (10 µM) and nicorandil (100 µM), also
increased STOC frequencies (SNP, 4.0 ± 1.5-fold,
n = 4; nicorandil, 3.3 ± 0.8-fold,
n = 8) and amplitudes (SNP, 1.2 ± 0.1-fold, n = 4; nicorandil, 1.3 ± 0.1-fold, n = 8). These results are
consistent with the idea that forskolin, through cAMP, and SNP and
nicorandil, through cGMP, can increase
Ca2+ spark and STOC frequency by
200-300% and STOC amplitude by ~30%.

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Fig. 2.
Forskolin increases Ca2+-sensitive
K+
(KCa) channel currents in single
smooth muscle cells from cerebral arteries.
A: forskolin (10 µM) increases
spontaneous transient outward current (STOC) frequency in a single
smooth muscle cell isolated from a cerebral artery. PKA inhibitor H-89
(1 µM) reversed effects of forskolin. Whole cell currents were
recorded with perforated-patch technique at holding potential
(Vh) of
40 mV. Also shown on expanded time scale is average current
signal of 20 STOCs averaged under control, forskolin-treated, and
forskolin + H-89 conditions. B:
expanded portion of experiment in A
before and after forskolin. C: time
course of STOC frequency from experiment in
A, plotted in 20-s bins. Mean STOC
amplitude and frequencies in this experiment were 25.4 pA and 2.08 Hz
(control), 36.5 pA and 4.8 Hz (forskolin), and 28.8 pA and 1.97 Hz
(H-89), respectively.
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Fig. 3.
Adenosine, forskolin, and dibutyryl-cAMP (db-cAMP) increase STOC
frequency (A) and amplitude
(B), and H-89 reversed the effects
of forskolin. Changes in STOC frequency and amplitude relative to
pretreatment control levels with application of adenosine (10 µM,
n = 7), forskolin (10 µM,
n = 9), dibutyryl-cAMP (500 µM,
n = 5), and forskolin (10 µM) in the
presence of H-89 (1 µM, n = 9) are
illustrated. Error bars are means ± SE. Statistical significance
was tested with a paired Student's
t-test
(* P < 0.05, ** P < 0.01).
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To provide additional evidence that cAMP can increase STOC
frequency, the effects of the membrane-permeable cAMP analog DBcAMP (500 µM) on STOC frequency and amplitude were examined. Externally applied DBcAMP increased STOC frequency 2.0 ± 0.2-fold
(n = 5; Fig.
3A). In further support of cAMP/PKA
increasing STOC frequency, the vasodilator adenosine (10 µM), which
activates adenylyl cyclase, significantly
(P < 0.05) increased STOC frequency
2.6 ± 0.7-fold (n = 11) and
amplitude 1.3 ± 0.1-fold (Fig. 3,
A and
B).
Forskolin increases the
NPo of
KCa channels in the absence of
Ca2+ sparks.
The forskolin-induced increase in
Ca2+ spark frequency would explain
the observed elevation of STOC frequency. However, the effect of
forskolin on STOC amplitude cannot be explained by its action on
Ca2+ sparks alone, because the
amplitude of the spark was unaffected. A possible explanation for the
1.3-fold increase in STOC amplitude to forskolin is through an
elevation of open state probability (Po) of
KCa channels caused by PKA
phosphorylation of the channel, as has been demonstrated in excised
patches by others (23). An elevated
Po of
KCa channels would lead to higher
Po of the KCa channels during a spark and
hence increase the STOC amplitude, if it were not already maximal at
that voltage. To test the effects of forskolin on
KCa channels, single-channel
currents through KCa channels were
recorded from isolated arterial myocytes in the whole cell mode, using
the perforated-patch technique. Single KCa channels were identified by
their characteristic large single-channel conductance, voltage
dependence, and sensitivity to block by tetraethylamonium and
iberiotoxin (7, 23, 28, 31).
Forskolin increased single KCa
channel activity (measured as
NPo) from 0.041 ± 0.015 to 0.055 ± 0.02 (n = 7) measured over 5 min at 0 mV (Fig. 4).
The single-channel current amplitude was not affected by forskolin (at
0 mV: control, 5.0 ± 0.2 pA; forskolin, 5.0 ± 0.2 pA,
n = 7). The forskolin-induced increase
in NPo did not
appear to be the result of an elevation in global
Ca2+ in these voltage-clamped
cells, since Ca2+ measured with
fura 2 did not increase (106.0 ± 5.4% of the control, n = 4). Forskolin would not be
expected to decrease average Ca2+
under these conditions, since the voltage of the cells was controlled. These results are consistent with the idea that forskolin increased NPo by 1.3-fold
through an effect of cAMP/PKA directly on the KCa channel and thus provide an
explanation for the increase in STOC amplitude to forskolin.

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Fig. 4.
Forskolin increases open probability
(NPo) of single
KCa channels, in absence of
Ca2+ sparks, in a single smooth
muscle cell isolated from a cerebral artery. Single-channel currents
were recorded with the whole cell, perforated-patch technique from a
single isolated smooth muscle cell at 0 mV. STOCs were indirectly
inhibited by blocking sarcoplasmic reticulum (SR)
Ca2+ uptake with thapsigargin (100 nM). Shown is a series of channel openings from a single cell recorded
before (NPo of
this series = 0.043) and during
(NPo of this
series = 0.054) application of 10 µM forskolin at 0 mV.
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Role of Ca2+
sparks and KCa channels in forskolin-induced
dilations.
We tested the hypothesis that part of the forskolin dilation of
pressurized cerebral arteries is through increasing
Ca2+ spark frequency, activating
KCa channels. Consistent with this hypothesis, an inhibitor of Ca2+
sparks, ryanodine, inhibited 79.8 ± 8.1% of the forskolin-induced dilation of pressurized cerebral arteries with tone (Fig.
5D). Furthermore, ryanodine constricted pressurized cerebral arteries to a
similar diameter in the absence (to 81 ± 7 µm) (28) or presence
of forskolin (100 nM; to 85 ± 20 µm,
n = 5), consistent with forskolin
acting on a ryanodine-sensitive process. The blocker of
KCa channels, iberiotoxin, also
inhibited 82.3 ± 7.0% of the forskolin-induced dilation (Fig.
5D) (as shown
previously, see Ref. 42). The effects of ryanodine and iberiotoxin were
not additive (Fig. 5, B and
D; see also Ref. 28), consistent with the idea that Ca2+ sparks act to
stimulate KCa channels (28). Part
of the dilation to forskolin was insensitive to iberiotoxin or
ryanodine, particularly at higher forskolin concentrations, suggesting
that forskolin also acts through other mechanisms (1, 11, 15, 18, 19, 36, 41, 48). In this preparation, however, forskolin (10 µM or less)
was ineffective on pressurized arteries that were constricted with high
(60 mM) K+ (Fig. 5,
A and
D), consistent with forskolin acting
in large part through activation of
K+ channels (1, 31). Ryanodine and
iberiotoxin were ineffective on pressurized arteries previously dilated
with the Ca2+ channel blocker
diltiazem (1 µM; Fig. 5C), a
condition that would lower Ca2+
spark frequency and hence KCa
channel activity (7, 20, 28, 31) through reducing average intracellular
Ca2+ and SR
Ca2+ content. These results
suggest that forskolin dilates pressurized cerebral arteries through
activation of K+ channels, with
part of the dilation due to activation of the Ca2+
spark
KCa channel pathway.

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Fig. 5.
Forskolin dilations of pressurized (60 mmHg) cerebral arteries were
reversed by ryanodine, but diltiazem dilations were not. Arteries were
partially constricted (~35%) by pressure (60 mmHg), as indicated by
horizontal dotted line. A: forskolin
did not dilate pressurized cerebral arteries constricted by 60 mM
K+. Transient dilation to
K+ was due to activation of inward
rectifier K+ channel present in
these arteries (31). B: ryanodine (10 µM) constricted pressurized arteries dilated by forskolin.
KCa channel blocker iberiotoxin
(IbTx; 100 nM) was without effect in presence of ryanodine.
C: ryanodine and iberiotoxin were
without effect on pressurized arteries dilated by
Ca2+ channel blocker, diltiazem (1 µM). Ryanodine constricted arteries when diltiazem was removed.
D: dilations to 30 nM forskolin were
inhibited by presence of either ryanodine (10 µM,
n = 5) or iberiotoxin (100 nM,
n = 4). Ryanodine and iberiotoxin
together (n = 5) were no more
effective than either agent alone. Forskolin is ineffective in the
presence of 60 mM external K+
(n = 4).
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Activation of KCa channels leads
to membrane potential hyperpolarization of the arterial myocytes, which
closes voltage-dependent Ca2+
channels, decreasing Ca2+ entry,
which should cause vasodilation through a reduction of arterial wall
Ca2+. As predicted, forskolin
caused average Ca2+ in the wall of
a pressurized artery (60 mmHg) to fall from 184 ± 5 nM
(n = 20) to 123 ± 22 nM
(n = 7) (Fig. 6,
A and
B). As expected from the previous
diameter measurements (Fig. 5) (28), ryanodine increased arterial wall
Ca2+ (to 202 ± 12 nM) through
membrane potential depolarization. Caffeine, which opens RyR channels
(34), caused a larger release of SR Ca2+ in the presence of forskolin
than in its absence (P < 0.05; Fig. 6). In contrast, caffeine released less
Ca2+ from arteries dilated by
diltiazem (Fig. 6), presumably because SR
Ca2+ content had been reduced.
Caffeine was relatively ineffective in the presence of ryanodine (Fig.
6). These results taken together support the idea that forskolin (cAMP)
can lower arterial wall Ca2+ by
increasing Ca2+ spark frequency.
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DISCUSSION |
The present study proposes a new mechanism of action for vasodilators
that work through cAMP and cGMP (Fig.
7). Our data
suggest that cAMP and cGMP can increase
KCa channel activity in two ways: 1) by increasing
Ca2+ spark frequency and
2) by directly increasing the open
probability of the KCa channel.
Together these actions lead to increased frequency and amplitude of
STOCs, which, when summed across the vessel wall, should cause membrane
potential hyperpolarization and, ultimately, relaxation of the artery.

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Fig. 6.
Forskolin decreases and ryanodine increases arterial wall
Ca2+ in an intact pressurized (60 mmHg) cerebral artery (A).
Ca2+ signal is from an intact
artery loaded with fura 2. Elevating pressure from 10 to 60 mmHg
increased Ca2+ from ~100 to 200 nM and constricted the artery by ~35%. Rapid application of caffeine
(10 mM) to the bath produced a transient increase in cytosolic
Ca2+ (arrows).
B: ratio images of cerebral artery
loaded with fura 2 (same artery as in
A) pressurized to 10 mmHg (No tone;
a), to 60 mmHg physiological salt
solution (PSS tone; b), at 60 mmHg
during a caffeine pulse (PSS/Caf pulse;
c), at 60 mmHg dilated with
forskolin (FSK tone; d), at 60 mmHg
with forskolin present during a caffeine pulse (FSK/Caf pulse;
e), and at 60 mmHg in presence of
forskolin after ryanodine-induced constriction (FSK + RYA tone;
f). Diameters of the artery are
indicated below images. C: forskolin
increases and diltiazem and ryanodine decrease caffeine-induced
Ca2+ transients. Peak change in
Ca2+ to caffeine is shown for
control (PSS) and in presence of 100 nM forskolin, 3 µM diltiazem,
and 10 µM ryanodine. * Caffeine transient was significantly
increased (P < 0.05) in presence of
forskolin and decreased in presence of diltiazem (which reduced
arterial wall Ca2+ to 126 ± 4 nM, n = 8) or ryanodine
(P > 0.05).
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Fig. 7.
Proposed mechanisms of action of forskolin and nitrovasodilators on
Ca2+ sparks and
KCa channels in arterial smooth
muscle cells. Adenylyl cyclase (AC) produces cAMP when activated by
externally applied forskolin or adenosine, which in turn stimulates
PKA. Activation of PKA has a direct effect on the
KCa channel to increase its open
probability, as has been previously demonstrated in inside-out excised
patches. However, the predominant effect of PKA is on the SR to
increase Ca2+ spark frequency.
Possible targets of PKA and PKG are the KCa channel, RyR
receptors, and Ca2+-ATPase
(phospholamban). Combined effects of increased spark activity and
direct effects on the KCa channel
lead to significantly increased activity of the
KCa channel. Resulting
hyperpolarization closes voltage-dependent
Ca2+ channels, leading to reduced
global intracellular Ca2+ and
vasodilation. GC, guanylate cyclase; G, Gs protein.
|
|
Comparison of
Ca2+ spark and
STOC frequency.
The mean Ca2+ spark
frequency (0.14 Hz) was ~15% of the mean STOC frequency (0.96 Hz).
We estimate that we could detect
Ca2+ sparks in no more than 23%
of cell volume, based on the average spread of a
Ca2+ spark (4.8 µm) and maximum
width of a cell (10 µm) (28) (assuming a circular cross section of a
cell). This detection volume would be less when a scan line is close to
the side of the cell and when the length of the scan line is less than
the length of the cell, which is the case when the cells are not
straight. These results are consistent with the idea that most
Ca2+ sparks cause STOCs (17).
Effects on
Ca2+ sparks.
Forskolin, SNP, and nicorandil increased
Ca2+ spark frequency. Forskolin,
through cAMP/PKA, could act directly on the RyR channel (43, 46)
and/or indirectly through an elevation of SR
Ca2+ load, which has been shown to
increase the open state probability of RyR channels in skeletal and
cardiac muscle (12, 26, 45, 46). cAMP/PKA and cGMP/PKG could increase
SR Ca2+ load by enhancing SR
Ca2+-ATPase activity by
phosphorylating phospholamban (25). The observed increase in
caffeine-induced Ca2+ transients
in the presence of forskolin (Fig. 6) is consistent with cAMP/PKA
increasing SR Ca2+ load. However,
forskolin did not cause a detectable increase in
Ca2+ spark amplitude, which could
be indicative of SR Ca2+ load.
Nicorandil did increase Ca2+ spark
amplitude by ~10%. Our data do not distinguish between a direct
effect on ryanodine receptors and an indirect effect of SR
Ca2+ load on
Ca2+ spark frequency. cAMP (PKA)
and cGMP (PKG) could conceivably act on both the
Ca2+-ATPase and the RyR channel to
affect SR function and STOC frequency (see Fig. 7) (49).
Contribution of STOCs to overall KCa
channel activity.
KCa channels do not appear to
contribute significantly to regulation of smooth muscle membrane
potential in intact pressurized cerebral arteries when
Ca2+ sparks are blocked (28). This
suggestion is based on the observation that blockers of
KCa channels (iberiotoxin)
were without effect on pressurized cerebral arteries when inhibitors of
Ca2+ sparks (ryanodine,
thapsigargin, or cyclopiazonic acid) were present. Furthermore,
iberiotoxin was without effect in the presence of ryanodine and
forskolin (Fig. 5B), suggesting
that, even in the presence of forskolin,
KCa channels do not contribute
significantly to the regulation of arterial diameter without
Ca2+ sparks.
The relative activity
(NPo) of
KCa channels with and without
Ca2+ sparks can be estimated from
the data presented here and from Ref. 28. At
40 to
30 mV,
NPo contributed
by Ca2+ sparks is ~0.4 (assuming
peak NPo = 13 lasting 30 ms, frequency 1/s). Baseline
NPo is estimated
to be in the range of 0.01 at physiological membrane potentials and
Ca2+ in pressurized arteries
(
40 mV, 200 nM Ca2+),
based on extrapolation from direct measurements (Fig. 4). Assuming a
slope conductance of KCa channels
of 79 pS (estimate at
30 to
40 mV from current-voltage
relationship), this would correspond to an average
KCa channel conductance of ~32
pS with sparks, consistent with a significant contribution to membrane
conductance of smooth muscle cells with a 10 G
input resistance (see
Ref. 31). However, <5% of the
KCa channel conductance would be
contributed by channels not activated by
Ca2+ sparks. Forskolin would
increase NPo
caused by sparks two- to threefold or from 0.4 to
0.8-1.2. However, forskolin would directly increase
NPo in the
absence of sparks by 1.3 or to ~0.013-0.026. Regardless of the
uncertainties, these estimates support the idea that
Ca2+ sparks should have a profound
impact on the overall activity of
KCa channels and that forskolin
acts largely (99%) on KCa
channels through increasing Ca2+
spark frequency.
Proposed mechanism for regulation of arterial diameter by cAMP/PKA
or by cGMP/PKG via
Ca2+ sparks and
KCa channels.
Elevation of intravascular pressure causes a graded membrane potential
depolarization and vasoconstriction of small myogenic arteries (Fig. 7)
(7, 20, 27, 31). The membrane potential depolarization increases the
steady-state open probability of voltage-dependent
Ca2+ channels (30, 40), which
elevates Ca2+ entry and global
intracellular Ca2+ (21, 27).
Ca2+ channel inhibitors (e.g.,
nimodipine, diltiazem) prevent pressure-induced constrictions of
myogenic cerebral arteries (7, 20, 30). Intracellular
Ca2+ could increase
Ca2+ spark frequency through
cytoplasmic and SR luminal activation of RyR channels.
Ca2+ sparks, by themselves, would
have little direct effect on intracellular Ca2+, even at the highest observed
rate (4/s) (28). Ca2+ sparks
would, however, have a profound effect on the activity of
KCa channels, which would oppose
the pressure-induced membrane potential depolarization. In support of
this proposed mechanism, blockers of
KCa channels (e.g., iberiotoxin)
and Ca2+ sparks (ryanodine,
cyclopiazonic acid, thapsigargin) depolarize and constrict pressurized
(60 mmHg) myogenic cerebral arteries by ~8 mV and 30% (7, 20, 28).
Lowering intravascular pressure (7) or blocking
Ca2+ channels (7, 20, 28), which
would decrease intracellular Ca2+
and thus KCa channel activity,
abolished the effects of iberiotoxin and ryanodine on membrane
potential and arterial diameter (also see Fig.
6B with diltiazem). Furthermore,
iberiotoxin was without effect on membrane potential and diameter in
intact pressurized arteries, when
Ca2+ sparks were blocked by
ryanodine or thapsigargin (28) (see also Fig.
6A), suggesting that global
intracellular Ca2+ does not cause
sufficient activation of KCa
channels to modulate membrane potential (see above).
Iberiotoxin blocks a significant fraction of the dilation of rat
cerebral arteries to forskolin (see Fig. 5 and Ref. 42). However, high
extracellular K+ blocked the
entire dilation to forskolin. cAMP/PKA has also been shown to activate
ATP-sensitive K+
(KATP) channels (18, 19, 36, 48)
and voltage-dependent K+ channels
(1) in smooth muscle. Forskolin dilations of cerebral arteries were not
affected by glibenclamide, an inhibitor of
KATP channels (data not shown;
also observed in Ref. 42), suggesting that
KATP channels do not contribute to
the observed dilations. Voltage-dependent
K+ channels contribute
significantly to the regulation of the membrane potential and diameter
of pressurized cerebral arteries (20), suggesting the possibility that
forskolin acts in part on this channel (cf. Ref. 1). These results
indicate that the forskolin response in our preparation is entirely
mediated by effects on the K+
conductance of the cell and that part of this change in
K+ conductance is due to effects
on the KCa channel.
Here we demonstrate that agents that elevate cGMP (SNP and nicorandil)
increase Ca2+ spark and STOC
frequency, as well as increase STOC amplitude in coronary arteries from
rat. In the same intact coronary artery preparation, SNP (10 µM) has
been shown to cause a 13-mV hyperpolarization and almost maximal
dilation, which was substantially blocked by iberiotoxin (47). PKG has
also been shown to activate KCa
channels in excised patches from the same coronary artery myocytes
(Ref. 47; see also Refs. 39 and 44). cGMP/PKG can increase SR Ca2+ uptake, presumably through an
action on phospholamban (10). Furthermore, smooth muscle relaxations to
nitrovasodilators in mesenteric (8, 16), pulmonary (3), and cerebral
arteries (33) can be partially inhibited by iberiotoxin, suggesting a role for KCa channels in
relaxations of a number of types of vascular smooth muscle to cGMP.
These results support the general mechanism presented in Fig. 7 (see
also Fig. 4 in Ref. 28).
In conclusion, the present study proposes a new mechanism of action for
vasodilators that work through cAMP and cGMP in cerebral and coronary
arteries (Fig. 7). Our data indicate that cAMP and cGMP can increase
Ca2+ spark frequency two- to
threefold as well as have a small, direct effect on
KCa channel open probability (23,
39, 44). Together these actions lead to increased frequency and
amplitude of STOCs, which, when summed across the vessel wall, should
cause membrane potential hyperpolarization and, ultimately, relaxation
of the artery through decreasing
Ca2+ entry through
voltage-dependent Ca2+ channels
(7, 28, 31), thereby lowering arterial wall
Ca2+ (Fig. 6). The frequency of
Ca2+ sparks, even with elevated
cAMP or cGMP (2-3
sparks · s
1 · cell
1),
would have little direct effect on average cytoplasmic
Ca2+. Our results would then
explain with one integrated mechanism (Fig. 7) the apparently disparate
observations that cAMP and cGMP can increase SR
Ca2+ uptake and lower arterial
Ca2+, and, yet, relaxations to
these cyclic nucleotides are often inhibited by blockers of
KCa channels (16, 33, 35, 42). This mechanism (frequency modulation of
Ca2+ sparks), along with
activation of KATP channels and
voltage-dependent K+ channels,
inhibition of Ca2+ channels, and
changes in Ca2+ sensitivity, would
contribute to the actions of cAMP and cGMP. We suggest that frequency
modulation of Ca2+ sparks by
cAMP/cGMP may be a general mechanism for the SR to regulate
plasmalemmal Ca2+ entry through
alterations in a cell's membrane potential.
 |
ACKNOWLEDGEMENTS |
We thank the late Dr. Fred Fay and Dr. John Walsh for insights and
comments on this study.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-44455, HL-51728, and HL-58231, National Science Foundation
Grant IBN-9631416, a grant from the Alexander Von Humboldt Foundation
(to T. Kleppisch), and a fellowship (to V. A. Porter) from the American
Heart Association, New Hampshire and Vermont Affiliates.
Address for reprint requests: M. T. Nelson, Dept. of Pharmacology, 55A
South Park Dr., Univ. of Vermont, Colchester, VT 05446.
Received 9 September 1997; accepted in final form 13 January 1998.
 |
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