RAPID COMMUNICATION
Activators of protein kinase C decrease
Ca2+ spark frequency in smooth
muscle cells from cerebral arteries
Adrian D.
Bonev1,
Jonathan H.
Jaggar1,
Michael
Rubart2, and
Mark T.
Nelson1
1 Department of Pharmacology,
College of Medicine, The University of Vermont, Colchester, Vermont
05446; and 2 Krannert Institute of
Cardiology, Indiana University Medical School, Indianapolis, Indiana
46202
 |
ABSTRACT |
Local
Ca2+ transients
("Ca2+ sparks") caused by
the opening of one or the coordinated opening of a number of tightly
clustered ryanodine-sensitive
Ca2+-release (RyR) channels in the
sarcoplasmic reticulum (SR) activate nearby
Ca2+-dependent
K+
(KCa) channels to cause an
outward current [referred to as a "spontaneous transient
outward current" (STOC)]. These
KCa currents cause membrane
potential hyperpolarization of arterial myocytes, which would lead to
vasodilation through decreasing
Ca2+ entry through
voltage-dependent Ca2+ channels.
Therefore, modulation of Ca2+
spark frequency should be a means to regulation of
KCa channel currents and hence
membrane potential. We examined the frequency modulation of
Ca2+ sparks and STOCs by
activation of protein kinase C (PKC). The PKC activators, phorbol
12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),
decreased Ca2+ spark frequency by
72% and 60%, respectively, and PMA reduced STOC frequency by 83%.
PMA also decreased STOC amplitude by 22%, which could be explained by
an observed reduction (29%) in
KCa channel open probability in
the absence of Ca2+ sparks. The
reduction in STOC frequency occurred in the presence of an inorganic
blocker (Cd2+) of
voltage-dependent Ca2+ channels.
The reduction in Ca2+ spark
frequency did not result from SR
Ca2+ depletion, since
caffeine-induced Ca2+ transients
did not decrease in the presence of PMA. These results suggest that
activators of PKC can modulate the frequency of
Ca2+ sparks, through an effect on
the RyR channel, which would decrease STOC frequency (i.e.,
KCa channel activity).
calcium-dependent potassium channels; caffeine; ryanodine; thapsigargin
 |
INTRODUCTION |
LOCAL RELEASE OF CALCIUM
("Ca2+ sparks") through
ryanodine-sensitive
Ca2+ release (RyR)
channels in the sarcoplasmic reticulum (SR) have recently been measured
in arterial smooth muscle cells, using a laser-scanning confocal
microscope and the fluorescent
Ca2+ indicator, fluo 3 (21).
Ca2+ sparks arise from the opening
of a single or a small number of tightly clustered RyR channels.
Ca2+ sparks activate nearby
Ca2+-sensitive
K+
(KCa) channels in smooth muscle
(13, 21), which causes outward currents (previously referred to as
"spontaneous transient outward currents" or STOCs) (2). An
increase in KCa channel current causes the membrane potential to hyperpolarize, which closes
voltage-dependent Ca2+ channels,
decreases Ca2+ entry, and lowers
average global intracellular Ca2+,
which exerts a vasorelaxing influence (22, 23). Thus activation of the
Ca2+ spark
KCa channel pathway appears to
oppose pressure-induced constrictions of myogenic cerebral arteries (5,
21). This work suggests that frequency modulation of
Ca2+ sparks would alter arterial
smooth muscle membrane potential and arterial tone (3, 4, 6, 21, 25).
Ca2+ spark frequency (i.e., the
open probability of RyR channels) increases with cytoplasmic
Ca2+ and SR
Ca2+ load (3, 4). In smooth
muscle, STOC frequency, which reflects Ca2+ spark frequency, has been
shown to increase with membrane depolarization (see, e.g., Refs. 2 and
34) and is associated with elevated cytoplasmic and SR
Ca2+ (2, 18, 29, 34). The
phosphorylation state of the RyR channel and certain drugs (caffeine
and ryanodine) may modulate Ca2+
spark frequency and thereby its consequences, independent of changes in
Ca2+. Recent evidence suggests
that protein kinase C (PKC) can phosphorylate RyR channels in cardiac
muscle (30), although the functional effect of this
phosphorylation on RyR channel properties is unknown.
In this study, we explored the possibility that activators of PKC
(phorbol ester and a diacylglycerol analog), which are potent vasoconstrictors, could affect
Ca2+ spark properties. Agents that
inhibit Ca2+ sparks
(ryanodine, thapsigargin, cyclopiazonic acid) have been shown to
depolarize and constrict myogenic cerebral arteries (21). We provide
the first evidence that activators of PKC can decrease Ca2+ spark frequency and,
consequently, STOC frequency. Activators of PKC also slightly reduced
STOC amplitude, which could be explained by a direct effect on the
KCa channels. Activators of PKC
reduced Ca2+ spark frequency, even
as they slightly elevated cytoplasmic
Ca2+ and SR
Ca2+ load. These results are
consistent with the idea that PKC acts directly on the RyR channel to
decrease its opening rate (i.e., Ca2+ spark frequency) and suggests
that frequency modulation of Ca2+
sparks may be important in the regulation of cell function.
 |
METHODS |
Cell isolation. Single smooth
muscle cells were enzymatically isolated from rat cerebral (basilar)
arteries. The cells were isolated with a papain and collagenase
digestion as described in Ref. 26. Only spindle-shaped cells with
intact membranes were used for measurements.
Ca2+ spark
measurements.
The procedure for the measurement of sparks is described in Ref. 21.
Briefly, the cells were loaded with the
Ca2+-sensitive indicator fluo 3 with a 20-min incubation in 5 µM of the acetoxymethyl ester (AM) of
fluo 3, 2.5 µg/ml Pluronic acid (Molecular Probes, Eugene, OR),
followed by a 20-min wash. All measurements were made 15-45 min
after the application of compounds. Control and treated cells from the
same cell isolation were examined randomly to minimize any bias or
time-dependent changes. The cells were scanned with a Bio-Rad MRC 1000 laser-scanning confocal microscope, housed in the University of Vermont
Cell Imaging Facility. 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. 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 is in the horizontal direction running from left to right, and position along
the scan line is given by the vertical displacement. Six consecutive
3-s line scan images were recorded from each cell along a single line.
Sparks were analyzed using custom-written analysis programs using
interactive data language (IDL) software (Research Systems, Boulder,
CO). Fractional fluorescence increases >1.3 with spreads (spatial
distribution determined as the width of the Gaussian distribution at
the half amplitude) of >1.2 µm were analyzed. Such events were not
observed in the presence of ryanodine or thapsigargin (21), indicating
that these events originated from the SR.
Electrophysiological recordings.
K+ currents were measured in the
whole cell, perforated-patch configuration (11) of the patch-clamp
technique (10), using 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 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4). The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (pH 7.2). Membrane currents were recorded while holding the cells
at a membrane potential of
40 mV. To determine the mean amplitude and frequency of the STOCs, analysis was performed off-line, using a custom analysis program. The threshold of STOCs was set at
three times the single channel amplitude at
40 mV. In the presence of ryanodine or thapsigargin, the simultaneous opening of
three single KCa channels was not
observed at
40 mV. The large amplitude and low open probability
of the KCa channel permitted the
measurement of single KCa channel
currents using the perforated-patch configuration of the whole cell
voltage clamp. To observe single KCa channel currents,
Ca2+ sparks and STOCs were
prevented by thapsigargin (31), which inhibits the SR
Ca2+-ATPase, and the cells were
clamped at 0 mV. Ca2+ sparks and
STOCs were measured in different cells.
Conventional
Ca2+ imaging.
Isolated smooth muscle cells 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 in the dark for 20 min before measurements
were made. Ca2+ was measured
ratiometrically (340:380 nm) using IMAGE-1/FL quantitative fluorescence
measurement software (Universal Imaging, West Chester, PA).
Fluorescence ratios were converted to
Ca2+ concentrations (as described
in Ref. 9), using an apparent dissociation constant for fura 2 of 282 nM (15).
Chemicals. Unless otherwise stated all
chemicals used in this study were obtained from Sigma Chemical (St.
Louis, MO) and Calbiochem-Novabiochem International (La Jolla, CA). All
experiments were conducted at room temperature (20-22°C).
Statistical analysis. Results are
expressed as means ± SE. Statistical significance was tested at the
95-99% confidence level using a paired or unpaired Student's
t-test, where applicable.
 |
RESULTS |
Activators of PKC decrease
Ca2+ spark
frequency.
Activators of PKC, phorbol 12-myristate 13-acetate (PMA; 10 nM) and
1,2-dioctanoyl-sn-glycerol (1 µM),
decreased Ca2+ spark frequency
(determined as sparks per cell) from 2.85 ± 0.40 (n = 86 cells) to 0.80 ± 0.20 (n = 130) and 1.15 ± 0.38 (n = 20) or by 71.9% and 59.7%,
respectively (Fig. 1,
A and
B). The inactive phorbol ester
analog 4
-PMA (10 nM) had no effect on
Ca2+ spark frequency (Fig. 1,
A and
B). PMA caused a small
decrease in Ca2+ spark amplitude,
with no effect on spatial spread or rate of decay (Fig.
1C and Table
1).

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Fig. 1.
Protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA)
and 1,2-dioctanoyl-sn-glycerol (DOG)
decrease frequency of Ca2+ sparks
in cerebral artery myocytes. A: line
scan images from a control cell
(top) and from a cell incubated with
PMA (10 nM; middle) or nonactive
analog of PMA (4 -PMA; 10 nM
bottom). Scan lines are displayed
vertically in a continuous manner.
Inset: orientation of scanning line.
B: average data for number of sparks
per cell in control (2.85 ± 0.40;
n = 86 cells), with PMA (10 nM; 0.80 ± 0.20; n = 130), with 4 -PMA
(10 nM; 2.76 ± 0.52; n = 50), and with DOG (1 µM; 1.15 ± 0.38;
n = 20). PKC activators, PMA and DOG,
caused a statistically significant decrease in
Ca2+ spark frequency
(* P < 0.05;
** P < 0.01), whereas
nonactive analog 4 -PMA did not. Measurements were made after 15 min
of incubation with PMA, 4 -PMA, or DOG.
C:
Ca2+ spark image demonstrating
time course of fractional fluorescence
(F/Fo;
bottom) and spatial distribution of
Ca2+ spark
(left) fitted with a Gaussian
distribution (red line). Gray bar labeled "t" indicates region
over which fluorescence time course was averaged.
|
|
PMA decreases STOC frequency and
amplitude. Ca2+
sparks activate nearby KCa
channels to cause outward currents (STOCs). Therefore, if activators of
PKC decrease Ca2+ spark frequency,
then these activators should decrease STOC frequency. PMA (10 nM)
decreased STOC frequency from 1.23 ± 0.22 (n = 10) to 0.18 ± 0.03 Hz, or by
82.9 ± 3.6%, and decreased STOC amplitude by 22.0 ± 9.7% at
40 mV (Fig. 2,
A and
B). The nonactive analog 4
-PMA
(10 nM) had no effect on STOC frequency and amplitude (Fig. 2B). A decrease in STOC amplitude
could occur through direct inhibition of
KCa channels. To test this
possibility, currents through single KCa channels were measured in the
whole cell (perforated patch) configuration.
Ca2+ sparks, and hence STOCs, were
prevented by thapsigargin (100 nM), which depletes SR
Ca2+ by inhibiting the SR
Ca2+-ATPase. PMA significantly
decreased the activity [measured as the product of the number of
channels and open probability
(NPo)] of
KCa channels from 4.71 ± 0.97 × 10
3 to 3.43 ± 0.84 × 10
3
(n = 4 cells;
P < 0.01) or by 28.9 ± 4.1% (at
0 mV; Fig. 2C). This effect of PMA
on KCa channel
NPo should
contribute to the decrease in STOC amplitude.

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Fig. 2.
PMA decreases frequency and amplitude of spontaneous transient outward
currents (STOCs). A: original records
of STOCs recorded from single smooth muscle cells isolated from basilar
cerebral artery. Holding potential was 40 mV. PMA (10 nM)
decreased frequency and amplitude of STOCs (top
trace). Nonactive analog 4 -PMA (10 nM) was without
effect (bottom trace).
B: average changes in STOC frequency
and amplitude relative to pretreatment control levels with PMA (10 nM;
n = 11) and 4 -PMA (10 nM;
n = 5). PMA caused a statistically
significant decrease in frequency
(** P < 0.01), by 82.9 ± 3.6%, and in amplitude of STOCs
(* P < 0.05), by 22.0 ± 9.7%. Nonactive analog 4 -PMA did not have any significant effects.
C: consecutive records of single
Ca2+-dependent
K+
(KCa) channel openings before
(control) and 15 min after application of PMA (10 nM). Thapsigargin
(100 nM) was added 10 min before starting the experiment to block
Ca2+ sparks and hence STOCs.
Currents were recorded with perforated-patch configuration of whole
cell voltage-clamp technique. Holding potential was 0 mV. Number of
channels times open probability
(NPo) of
KCa channels was determined by
analyzing 5-min sections of data in control and in presence of PMA. PMA
caused a small but statistically significant reduction in
NPo from
4.71 ± 0.97 × 10 3 to 3.43 ± 0.84 × 10 3
(P < 0.01) or by 28.9 ± 4.1%
(n = 4). Amplitude of single
KCa channel openings was not
affected by PMA (control, 4.9 ± 0.4 pA; with PMA, 4.9 ± 0.3 pA). D: average relative changes in
STOC frequency and amplitude (n = 4)
with CdCl2 (250 mM) and after
application of PMA (10 nM) in continued presence of
CdCl2.
Cd2+ reduced STOC frequency by
43.6 ± 10.9% (* P < 0.05)
but did not affect STOC amplitude. PMA reduced STOC frequency by 81.2 ± 5.8% and amplitude by 36.4 ± 1.5%
(* P < 0.05), comparable with
that observed in absence of
Cd2+.
|
|
PMA could conceivably decrease
Ca2+ spark and STOC frequency
indirectly by reducing Ca2+ entry
through voltage-dependent Ca2+
channels (17, 28). To exclude this possibility, we examined the effects
of PMA on STOC frequency and amplitude in the presence of the inorganic
Ca2+ channel blocker,
Cd2+ (250 µM) (12).
Cd2+ reduced STOC frequency by
43.5 ± 10.8% (n = 4 cells), but
it did not affect STOC amplitude (Fig.
2D). STOC frequency, in the presence
of Cd2+, remained constant for a
relatively long period of time (40-50 min). Nevertheless, PMA, in
the presence of Cd2+, reduced STOC
frequency by 81.1 ± 5.8% and amplitude by 36.4 ± 1.5% (Fig.
2D), suggesting that the effects of
PMA are independent of changes in
Ca2+ entry through
voltage-dependent Ca2+ channels.
PMA does not decrease caffeine-induced
Ca2+ transients.
PMA could decrease Ca2+ spark
frequency by decreasing cytoplasmic or SR
Ca2+, which would decrease the
opening rate of RyR channels (8, 19, 33). To test this possibility,
global intracellular Ca2+ was
measured in isolated myocytes, with the use of fura 2. PMA (10 nM)
caused a slight elevation of global
Ca2+ from 105.6 ± 3.8 to 153.1 ± 3.3 nM (n = 7 cells; Fig.
3A).
Caffeine (10 mM), which opens RyR channels, caused
Ca2+ transients of 425.0 ± 56.5 and 419.4 ± 32.4 nM (Fig. 3,
A and B). After 30 and 60 min of
application of PMA, caffeine-induced Ca2+ transients were 507.2 ± 43.2 and 626.3 ± 42.1 nM, respectively. These results argue against
changes in cytoplasmic or SR Ca2+
load leading to a decrease in Ca2+
spark or STOC frequency. The remaining likely possibility is that PKC
directly decreases the open rate of RyR channels through channel
phosphorylation, a possibility that remains to be explored.

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Fig. 3.
PMA does not inhibit intracellular
Ca2+ transients induced by
caffeine (Caff). A: transient
increases in cytosolic Ca2+
([Ca2+]cyt
) in response to application of 10 mM caffeine before and after
application of PMA (10 nM). Trace is average of
[Ca2+]cyt
signals from 7 cells. Cells were loaded with
Ca2+ indicator fura
2-acetoxymethyl ester, and Ca2+
was measured ratiometrically (340:380 nm) using IMAGE-1/FL quantitative
fluorescence equipment. B: average
changes in cytosolic Ca2+
( [Ca2+]cyt)
induced by caffeine (10 mM) in control
(1 and
2) and 30 min
(3) and 60 min
(4) after application of PMA (10 nM). C: proposed mechanism for action
of PKC on Ca2+ sparks and
KCa channels.
|
|
 |
DISCUSSION |
Our results are consistent with activation of PKC decreasing
Ca2+ spark frequency through a
direct action on the RyR receptor channel (Fig.
3C). This would, therefore, be the
first functional evidence that PKC can affect RyR channels and is
consistent with the observation that PKC can phosphorylate RyR channels
(30). It seems very unlikely that PKC activation decreases
Ca2+ spark frequency through a
reduction in cytoplasmic or SR
Ca2+, since neither cytoplasmic
Ca2+ nor caffeine-induced
Ca2+ transients declined over 60 min of exposure to PMA (Fig. 3, A and
B). Activators of PKC have been
reported to inhibit STOCs in rabbit portal vein (14). This inhibitory
effect was ascribed to a depletion of the SR, since caffeine failed to
produce an outward current in the presence of PKC activators (14). In
our experiments, PMA clearly did not decrease caffeine-induced
Ca2+ transients. Because PKC
activation appears to inhibit KCa
channels (Fig. 2C), it is
conceivable that the inhibitory effects of PKC activators on STOCs and
caffeine-induced current transients observed in portal vein (14) were
due to inhibition of KCa channels
and not of Ca2+ sparks.
Alternatively, PKC activators depleted SR
Ca2+ in this preparation, which
led to a loss of caffeine-induced current transients and STOCs. The
mechanism by which PKC activation inhibits
KCa channels is unclear.
Activators of PKC have been shown to inhibit
KCa channels in cultured and
freshly isolated smooth muscle cells (20, 27).
Receptor-mediated vasoconstrictors may have complicated effects on
Ca2+ sparks. Most
receptor-mediated vasoconstrictors can cause membrane depolarization,
which increases Ca2+ entry through
voltage-dependent Ca2+ channels.
Vasoconstrictors can also directly activate voltage-dependent Ca2+ channels (24), which could
increase Ca2+ spark frequency (1).
These effects would increase cytoplasmic Ca2+ and SR
Ca2+ and thus elevate
Ca2+ spark frequency.
Vasoconstrictors also cause a transient increase in inositol
trisphosphate (IP3)
production, which would release SR
Ca2+ through
IP3-sensitive channels.
IP3-induced
Ca2+ release could increase or
decrease Ca2+ spark activity (3,
7, 14, 16), depending on the extent of the elevation of cytoplasmic
Ca2+ near the RyR receptors, which
would tend to increase Ca2+ spark
frequency, and of the depletion of SR
Ca2+, which should decrease
Ca2+ spark frequency and
amplitude. Vasoconstrictors also activate PKC through diacylglycerol,
which, as shown here, could cause a steady-state decrease in
Ca2+ spark frequency. Furthermore,
PKC activation could inhibit IP3 formation (32). The steady-state effect of vasoconstrictors on
Ca2+ spark properties would
therefore be a function of all these factors.
In conclusion, our results support the concept of frequency modulation
(3, 4, 6, 21, 25) of Ca2+ sparks
regulating KCa channels.
Vasodilators that elevate adenosine 3',5'-cyclic
monophosphate and guanosine 3',5'-cyclic monophosphate (35)
have been shown to increase Ca2+
spark and STOC frequency (25). In contrast, we demonstrate that
activators of PKC decrease Ca2+
spark frequency and hence STOC frequency. This effect would tend to
depolarize smooth muscle, which would open voltage-dependent Ca2+ channels, increase
Ca2+ entry, and constrict. Our
results therefore suggest a new mechanism of control of
Ca2+ spark frequency, which could
contribute to the action of vasoconstrictors.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Gary Mawe, Joseph E. Brayden, Valerie A. Porter, and
Karen M. Lounsbury for discussion and comments on the manuscript.
 |
FOOTNOTES |
This study was supported by National Institutes of Health Grants
HL-44455, HL-51728, and NS-26995, National Science Foundation Grant
IBN-9631416, and American Heart Association, Indiana Affiliate, Grant
INN-97-700-GIAR.
Address for reprint requests: M. T. Nelson, Department of Pharmacology,
College of Medicine, The University of Vermont, 55A South Park Dr.,
Colchester, VT 05446.
Received 24 June 1997; accepted in final form 12 September 1997.
 |
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