Protein kinase C reduces the KCa current of rat
tail artery smooth muscle cells
Rudolf
Schubert1,
Thomas
Noack1, and
Vladimir N.
Serebryakov2
1 Institute of Physiology,
University of Rostock, D-18055 Rostock, Germany; and
2 Institute of Experimental
Cardiology, Cardiology Research Center, Academy of Medical
Sciences, 121552 Moscow, Russia
 |
ABSTRACT |
The hypothesis that protein kinase C (PKC) is
able to regulate the whole cell Ca-activated K
(KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog
1,2-dioctanoyl-sn-glycerol (DOG) at 10 µM attenuated the current-voltage
(I-V)
relationship of the KCa current significantly and reduced the KCa
current at +70 mV by 70 ± 4% (n = 14). In contrast, 10 µM DOG after pretreatment of the cells with 1 µM calphostin C or 1 µM PKC inhibitor peptide, selective PKC
inhibitors, and 10 µM
1,3-dioctanoyl-sn-glycerol, an
inactive diacylglycerol analog, did not significantly alter the
KCa current. Furthermore, the
catalytic subunit of PKC (PKCC)
at 0.1 U/ml attenuated the
I-V
relationship of the KCa current
significantly, reduced the KCa
current at +70 mV by 44 ± 3% (n = 17), and inhibited the activity of single
KCa channels at 0 mV by 79 ± 9% (n = 6). In contrast, 0.1 U/ml
heat-inactivated PKCC did not
significantly alter the KCa
current or the activity of single
KCa channels. Thus these results
suggest that PKC is able to considerably attenuate the
KCa current of freshly isolated
rat tail artery smooth muscle cells independently of effects of PKC on
local Ca release events, most likely by a direct effect on the
KCa channel.
catalytic subunit of protein kinase C; calcium-activated potassium
channel; local calcium release
 |
INTRODUCTION |
THE CALCIUM-ACTIVATED potassium
(KCa) channel has been
identified in all vascular smooth muscle cells investigated so far. The
K current carried by KCa channels
is one of the major outward currents of vascular smooth muscle cells.
Recently, it has been shown that in arteries exposed to a physiological
pressure level but not subjected to any vasoactive agonist this current
belongs to the ion currents that establish the membrane potential, one of the main factors determining the contractile state of these arteries
(5). Thus the contractile state of these arteries may be altered by
either an increase of the KCa
current, leading to membrane potential hyperpolarization and
vasodilation, or by a decrease of the
KCa current, leading to membrane
potential depolarization and vasoconstriction. Indeed, an increase of
the arterial KCa current has been
shown to be important, for example, for the protein kinase G
(PKG)-mediated vasodilation induced by NO (2) and estrogen (28) as well
as for the protein kinase A (PKA)-mediated vasodilation induced by
iloprost (23). Furthermore, a decrease of the arterial
KCa current has been shown to be
important for the vasoconstriction induced by
20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (30). However, in
contrast to numerous studies showing that vasodilations accompanied by
an increase of the KCa current are
mediated by intracellular messengers like PKA and PKG, the intracellular messengers mediating vasoconstrictions accompanied by a
decrease of the KCa current are
not well known. Yet, recent reports showing that another K current, the
ATP-sensitive K current, is involved in the vasoconstriction induced by
2D receptor agonists (26) and
that the decrease of this current produced by neuropeptide Y,
phenylephrine, serotonin, and histamine (4) is mediated by protein
kinase C (PKC) suggest that PKC may be an intracellular messenger
mediating vasoconstrictions accompanied by a decrease of the
KCa current. This hypothesis is
supported by recent findings demonstrating that spontaneous transient
outward currents, which represent
KCa currents induced by local Ca
release events, are inhibited by a PKC-mediated depletion of Ca stores
in rabbit portal vein (13) or by a PKC-mediated inhibition of ryanodine
receptor channel activity in rat cerebral arteries (3). In the latter study, it was additionally suggested that the PKC-mediated decrease of
the arterial smooth muscle KCa
current may be caused by some other mechanisms unrelated to local Ca
release events. Thus an inhibition of
KCa channels by PKC was proposed,
although this question had not been studied in detail (3). This
hypothesis is supported by the observation that activators of PKC can
inhibit the activity of single KCa
channels in cultured porcine coronary artery smooth muscle cells (18).
However, considerable alterations can occur in cultured smooth muscle
cells in comparison with native cells, including the appearance of
KCa channels with atypical properties (25) and the disappearance of
KCa channels (8). Thus the aim of
this study was to explore in more detail the possibility that a
PKC-mediated decrease of the arterial smooth muscle
KCa current may be produced, at
least partly, independently of effects of PKC on local Ca release
events. To accomplish this, experiments were performed to investigate
the effect of an activation of endogenous PKC by a diacylglycerol
analog, 1,2-dioctanoyl-sn-glycerol
(DOG), and of the application of PKC on the whole cell
KCa current of freshly isolated
arterial smooth muscle cells dialyzed with a strongly Ca-buffered
solution to eliminate local Ca release events as well as the effect of
the application of PKC on excised single KCa channels of these cells.
 |
METHODS |
Cell isolation and solutions.
Tail arteries were obtained from male Wistar-Kyoto rats, which were
killed by stunning and subsequent decapitation. A 2- to 3-cm-long piece
of an artery was placed into a microtube containing 1 ml of an enzyme
solution and stored there overnight at 4°C. The enzyme solution
contained (in mM) 110 NaCl, 5 KCl, 0.16 CaCl2, 2 MgCl2, 10 NaHEPES, 10 NaHCO3, 0.5 KH2PO4,
0.5 NaH2PO4,
10 glucose, 0.49 EDTA, and 10 taurine at pH 7.0, and also 1.5 mg/ml
papain (12,000 U/g), 1.6 mg/ml albumin, and 0.4 mg/ml
DL-dithiothreitol. The next day,
the microtube with the vessel was incubated for 5-10 min at
37°C. Single cells were released by trituration with a polyethylene
pipette into the experimental bath solution. The experimental bath
solution in the whole cell experiments contained (in mM) 135 NaCl, 6 KCl, 0.1 CaCl2, 1 MgCl2, 3 EGTA (purity 96%), and
10 HEPES at pH 7.4, with a calculated free Ca concentration of ~3 × 10
9 M. The
experimental bath solution in the inside-out experiments contained (in
mM) 140 KCl, 3 EGTA (purity 96%), 3 HEDTA (purity 98%), an
appropriate amount of MgCl2 to get
a free Mg concentration of 1 mM, an appropriate amount of
CaCl2 to get the desired free Ca
concentration, and 10 HEPES at pH 7.4. For a first estimate of the free
Ca concentrations, solution composition was calculated with the
following apparent reaction constants
(K) at pH 7.4: log
KCaEGTA = 7.17, log KMgEGTA = 1.93, log
KCaHEDTA = 5.67, and log KMgHEDTA = 4.37 (21). The exact free Ca concentration in the bath solution for
the inside-out experiments was measured using fura 2, and, after
calibration of the signal with K-based standard solutions (CALBUF-2;
World Precision Instruments), a concentration of 2.5 µM was
determined. The pipette solution contained (in mM) 102 KCl, 10 NaCl, 1 CaCl2, 1 MgCl2, 10 EGTA (purity 96%) or 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA; purity 98%), 10 HEPES, and 0.1 MgATP at pH 7.4, with a calculated free Ca concentration of ~8 × 10
9 M in the whole cell
experiments and 135 NaCl, 6 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES at pH 7.4 in
the inside-out experiments.
Patch-clamp recordings.
All patch-clamp experiments were performed at room temperature
(22-24°C). Patch pipettes were prepared from borosilicate
glass (WP Instruments) and had a resistance in the range of 1-3
M
when filled with the pipette solution. Recordings were made with
an Axopatch 200 amplifier (Axon Instruments) with the conventional whole cell and inside-out configuration.
Whole cell experiments.
In the whole cell experiments, stimulation of currents with pulse and
ramp protocols, data sampling at a rate of 1 kHz for ion currents and
of 50 kHz for cell capacitance and series resistance determination, and
data analysis were all done with the software package ISO2 (MFK).
Experiments were performed only on cells with negligible leaks. Series
resistance compensation was not employed because often cells were lost
even at slight overcompensation. Instead, series resistance was
measured every 2 min during the course of the experiment. Data were
used for analysis if the command voltage error due to series resistance
changed <2 mV. In a few cases, a voltage error change of 5 mV was
accepted, but these data were corrected using the current-voltage
(I-V)
relationship obtained during ramp pulses. Whole cell currents were
elicited every 5 s by either 500-ms step pulses from a holding
potential of
40 mV to a test potential of +70 mV or by 1,000-ms
ramp pulses from
70 to +100 mV. Current amplitudes were either
determined from step pulse stimulation and measured as the mean current
of the last 100 ms of the average of three consecutive current traces or determined from ramp pulse stimulation at the desired potentials. Preliminary tests showed that the current amplitudes at a certain potential obtained from consecutive step pulse and ramp pulse stimulations do not differ significantly. The difference in command potential to obtain the same current amplitude with ramp pulse stimulation compared with step pulse stimulation at +70 mV was only 1.8 ± 0.3 mV (n = 34).
Isolation of the KCa current.
The KCa current was defined as the
difference of the net outward current amplitude under the desired
condition, e.g., control or PKC application, and the outward current
amplitude after application of 100 nM iberiotoxin, the specific
inhibitor of KCa channels (9, 10),
which was given at the end of each experiment. The correctness of this
procedure was proven by the observation that the iberiotoxin-resistant
currents after the application of DOG or PKC were not significantly
different from the currents without application of any substance (data
not shown). The threshold potential of the
KCa current was determined as the
potential of the intercept of the
I-V
relationships of the KCa current
before and after iberiotoxin application. To be able to test the
hypothesis that PKC may have an effect on the
KCa current independently of
effects of PKC on local Ca release events, freshly isolated arterial
smooth muscle cells were dialyzed with a strongly Ca-buffered solution
to eliminate local Ca release events. Two ligands were employed to
buffer the intracellular Ca concentration, EGTA, a widely used Ca
buffer, and BAPTA, a Ca buffer with fast binding kinetics. Under these conditions, it was observed that the control
KCa current at +70 mV after 6 min
was 87 ± 11% (n = 5) of the
current at the beginning of the registration period when 10 mM EGTA was
used in the intracellular solution and 93 ± 8%
(n = 4) of the current at the
beginning of the registration period when 10 mM BAPTA was used in the
intracellular solution, which are not significantly different
(P = 0.69). The active diacylglycerol
analog DOG at 10 µM decreased the
KCa current 6 min after the
beginning of the application at +70 mV by 70 ± 4%
(n = 8) compared with the current at
the beginning of the application period when 10 mM EGTA was used in the
intracellular solution and by 69 ± 8%
(n = 6) compared with the current at
the beginning of the application period when 10 mM BAPTA was used in
the intracellular solution, which are not significantly different
(P = 0.84). The catalytic subunit of
PKC (PKCC) at 0.1 U/ml decreased
the KCa current 6 min after the
beginning of the application at +70 mV by 47 ± 5%
(n = 10) compared with the current at
the beginning of the application period when 10 mM EGTA was used in the
intracellular solution and by 40 ± 3%
(n = 7) compared with the current at
the beginning of the application period when 10 mM BAPTA was used in
the intracellular solution, which are not significantly different (P = 0.28). Because there were no
significant differences of the effect of DOG and
PKCC on the
KCa current in the experiments
using EGTA or BAPTA, respectively, in the intracellular solution, these data were pooled. In addition to the independence of the observed results from the Ca buffer used in the intracellular solution, further
evidence for a successful elimination of local Ca release events under
our experimental conditions are the inability to observe spontaneous
transient outward currents and the absence of an effect of ryanodine, a
well-known blocker of local Ca release events, and of cyclopiazonic
acid, an inhibitor of the Ca pump in the sarcoplasmic reticulum, on the
KCa current. Thus 10 µM ryanodine decreased the KCa
current 6 min after the beginning of the application at +70 mV by 3 ± 6% (n = 7) compared with the current at the beginning of the application period. This is not significantly different from the 10 ± 7%
(n = 9) decrease of the time-matched
control current at +70 mV (P = 0.43).
Furthermore, 10 µM cyclopiazonic acid increased the
KCa current 6 min after the
beginning of the application at +70 mV by 2 ± 7%
(n = 9) compared with the
current at the beginning of the application period. This is not
significantly different from the 10 ± 7%
(n = 9) decrease of the time-matched
control current at +70 mV (P = 0.22).
Inside-out experiments.
In the inside-out experiments, single-channel data were stored on a
DTR-1800 data recorder (Biologic, France) and later replayed for
analysis. They were filtered at 1 kHz with use of an eight-pole Bessel
filter (model 902, Frequency Devices, USA) and digitized at 5 kHz.
Thereafter, they were analyzed offline with the software package ASCD
(G. Droogmans, Lab. Fysiologie, KU Leuven, Louvain, Belgium). The
single-channel amplitudes were determined by fitting Gaussian
distributions to the amplitude histograms of the closed and the open
state, respectively. The activity of the channel in a patch was
determined as
NPo, where
Po is the open
probability of one channel and N is
the number of channels in the patch, which could not be determined in
most cases because the patch was lost before application of a high-Ca
solution. NPo was
calculated as the sum of the times finding 1, 2, 3, · · · , N simultaneously open channels divided
by the registration time. The registration time was 2-3 min in the
control and 3-5 min during PKC and PKC inhibitor peptide
application. All potentials are expressed as membrane potentials.
Drugs and chemicals.
Albumin, DL-dithiothreitol, and
the salts for the solutions were obtained from Sigma, except for BAPTA,
which was purchased from Molecular Probes. Papain was from Ferak
(Berlin, Germany). Iberiotoxin and ryanodine were from Research
Biochemicals International. PKCC,
the PKC inhibitor peptide (19
31), DOG, and calphostin C were obtained
from Calbiochem, and
1,3-dioctanoyl-sn-glycerol was from Biomol.
Statistics.
All data are presented as means ± SE;
n is the number of cells. Statistical
analysis was performed with use of paired and unpaired
t-tests, one-way ANOVA followed by a
Bonferroni test, or repeated measures ANOVA, as appropriate.
 |
RESULTS |
In a recent publication, we characterized the properties of the outward
current of rat tail artery smooth muscle cells in detail (22). Briefly,
this current showed a marked outward rectification, a strong dependence
on the intracellular Ca ion concentration, and a high sensitivity to
iberiotoxin, the selective blocker of KCa channels (9, 10), which
produced a considerable inhibition of this current. Neither
glibenclamide, the selective blocker of ATP-sensitive K channels, nor
4-aminopyridine, a blocker of voltage-dependent K channels, affected
this current. Additionally, it was shown in the cited publication that
in excised patches from these cells a K channel with a high
conductance, a steep voltage dependence of activation, and a prominent
Ca sensitivity is the main channel, which was blocked by low
concentrations of tetraethylammonium and by iberiotoxin. Thus, under
our experimental conditions, the outward current of these cells
consists largely of a KCa
current, which is carried by
KCa channels. The
KCa current in the present study
was defined as the difference of the net outward current amplitude
under the desired experimental condition and the outward current
amplitude after application of 100 nM iberiotoxin and was independent
from local Ca release events (for details, see
Isolation of the
KCa current, above).
Effect of an activation of PKC on the KCa
current.
For the investigation of the effect of PKC on the
KCa current of rat tail artery
smooth muscle cells, DOG (7), a cell-permeable active analog of
diacylglycerol, was used to activate PKC. DOG at a concentration of 10 µM was applied to the bath solution 2 min after the whole cell
configuration was obtained. Control experiments had shown that this
time was necessary but sufficient to get stable currents (data not
shown). Therefore, the beginning of any application period was marked
time
0. A typical example of the time
course of the KCa current elicited
with use of a voltage step from a holding potential of
40 mV to
a test potential of +70 mV under control conditions and after DOG
application is shown in Fig. 1A.
A transient increase of the KCa
current was observed ~1-2 min after the beginning
of DOG application. This increase did not reach significance in
comparison with the time-matched control current and was, therefore,
not studied in detail. Subsequently, the
KCa current started to decline,
and 6-8 min after the beginning of DOG application, the
KCa current reached a steady state
at a considerably lower level compared with the time-matched control current. For example, in the control series, the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV was 131 pA at the beginning of the control
application period and 99 pA 6 min after the beginning of the control
application period (Fig. 1B,
top).
I-V
relationships of the control KCa
current were obtained with use of a voltage ramp from
70 mV to
+100 mV based on a holding potential of
40 mV and showed that
the changes of the control current with time were similar at all
potentials tested (see Fig. 1B,
bottom, for a typical example). In
contrast, in the DOG series, the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV was, for example, 214 pA at the beginning of the
DOG application period and only 33 pA 6 min after the beginning of the
DOG application period (Fig. 1C,
top).
I-V
relationships of the KCa current
obtained with use of a voltage ramp from
70 mV to +100 mV based
on a holding potential of
40 mV showed that DOG produced a
marked decrease of the KCa current
in the potential range tested. This is demonstrated in Fig.
1C,
bottom, with a typical example and, in
addition to the original traces, the
I-V
relationship of the pure KCa
current, i.e., the difference of the net outward current and the
iberiotoxin-insensitive current, is shown before and after DOG
application (Fig. 1C,
bottom right). In summary, the
I-V
relationship of the KCa current
was altered significantly, seen as a reduction of this current (Fig. 2), and the threshold potential of the
current changed significantly, from 23.6 ± 3.4 to 40.0 ± 2.9 mV
or by 16.4 ± 2.8 mV (n = 7;
P < 0.01). On average, 10 µM DOG
decreased the KCa current 6 min after the beginning of the application at +70 mV by 70 ± 4%
(n = 14) compared with the current at
the beginning of the application period. This is a significant decrease
in comparison with the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (see Fig.
4; P < 0.001).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of 1,2-dioctanoyl-sn-glycerol
(DOG) on Ca-activated K (KCa)
current of rat tail artery smooth muscle cells.
A: examples of time course of control
(con) KCa current and of time
course of effect of DOG and of inactive (inact) DOG on
KCa current.
KCa current
(IKCa) was
elicited with use of a voltage step from a holding potential of
40 mV to a test potential of +70 mV and was normalized to
current at beginning of application period
(time
0).
B: examples of traces of
KCa current under control
conditions. Top: from
left, stimulation protocol, control
current at time
0 together with current after
iberiotoxin (IBTX) application, and control current after 6 min
together with current after IBTX application.
Bottom: from
left, stimulation protocol and control
current-voltage
(I-V)
relationship at time
0 together with control
I-V
relationship after 6 min as well as
I-V
relationship after IBTX application.
C: examples of traces of
KCa current in DOG series.
Top: from
left, stimulation protocol, current at
beginning (0) of DOG application together with current after IBTX
application, and current after 6 min of DOG application together with
current after IBTX application.
Bottom: from
left, stimulation protocol,
I-V
relationship at beginning of DOG application together with
I-V
relationship after 6 min of DOG application as well as
I-V
relationship after IBTX application, and
I-V
relationship of pure KCa current,
i.e., difference of net outward
I-V
relationship and
I-V
relationship after IBTX application, at beginning of DOG application
and after 6 min of DOG application. In
A, data points of different
experimental series do not coincide in time because of variable length
of period for series resistance control. Also, capacitive artifacts
shown in B and
C are distorted due to filtering of
current traces at low frequency and limited
x-axis resolution of graphic
presentation.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Dependence of KCa current density
on membrane potential (MP). con,
KCa current at beginning of
application of 10 µM DOG. DOG,
KCa current after 6 min of
application of 10 µM DOG. A repeated measures ANOVA showed a
significant difference between control and DOG
(n = 7;
P < 0.01).
|
|
To obtain evidence that the effect of DOG on the
KCa current is mediated by PKC,
the influence of calphostin C, a selective PKC inhibitor (14), on the
DOG effect was tested. Calphostin C at a concentration of 1 µM was
added to the pipette solution. Control experiments showed that
calphostin C itself did not affect the
KCa current (see Fig. 4). A
typical example of the time course of the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV after calphostin C application is shown in Fig.
3A,
together with an example of the time course of the
KCa current after DOG application with calphostin C pretreatment and after DOG application without PKC
blocker pretreatment. During the application of 10 µM DOG to cells
pretreated with 1 µM calphostin C, the
KCa current was measured with the
same protocol as in the DOG series. An increase of the
KCa current was observed ~2 min
after the beginning of DOG application, but subsequently there was no
decline of the KCa current;
rather, the KCa current stayed at
the increased level. For example, in the experiments with calphostin C
pretreatment, the KCa current
elicited with use of a voltage step from a holding potential of
40 mV to a test potential of +70 mV was 682 pA at the beginning
of the DOG application period and 779 pA 6 min after the beginning of
the DOG application period (Fig. 3B,
top).
I-V relationships of the KCa current
obtained with use of a voltage ramp from
70 mV to +100 mV based
on a holding potential of
40 mV showed that DOG after calphostin
C pretreatment produced a small increase of the
KCa current at all potentials
tested (see Fig. 3B,
bottom, for a typical example). In
summary, 10 µM DOG after calphostin C pretreatment increased the
KCa current 6 min after the
beginning of the application at +70 mV by 5 ± 8%
(n = 6) compared with the current at
the beginning of the application period. This is significantly
different from the 70 ± 4% (n = 14; P < 0.001) decrease of the
KCa current after DOG application without PKC blocker pretreatment at +70 mV (Fig.
4).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of protein kinase C (PKC) inhibitor calphostin C (cal) on
DOG-induced decrease of KCa
current of rat tail artery smooth muscle cells.
A: examples of time course of effect
of calphostin C on KCa current, of
time course of effect of DOG on
KCa current after calphostin C
pretreatment, and of time course of effect of DOG on
KCa current without any
pretreatment (taken from Fig. 1 for comparison).
KCa current was elicited with use
of a voltage step from a holding potential of 40 mV to a test
potential of +70 mV and was normalized to current at beginning of
application period (time
0).
B: examples of traces of
KCa current in DOG series with
calphostin C pretreatment. Top: from
left, stimulation protocol, current at
beginning of DOG application with calphostin C pretreatment together
with current after IBTX application, and current after 6 min of DOG
application with calphostin C pretreatment together with current after
IBTX application. Bottom: from
left, stimulation protocol and
I-V
relationship at beginning of DOG application with calphostin C
pretreatment together with
I-V
relationship after 6 min of DOG application with calphostin C
pretreatment as well as
I-V
relationship after IBTX application. In
A, data points of different
experimental series do not coincide in time because of variable length
of period for series resistance control. Also, capacitive artifacts
shown in B are distorted due to
filtering of current traces at low frequency and limited
x-axis resolution of graphic
presentation.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Summary of effect of DOG (10 µM) on
KCa current of rat tail artery
smooth muscle cells 6 min after beginning of application. Data are
expressed as ratio of KCa current
6 min after beginning of application under specified experimental
conditions (exp) to KCa current at
beginning of application period under same conditions (con). cal, 1 µM calphostin C; PKC-I, 1 µM PKC inhibitor peptide; inact DOG, 10 µM inactive DOG. * Significant difference compared with control
determined by 1-way ANOVA (P < 0.001); no. of cells investigated appears on bar for each condition.
|
|
To obtain further evidence that the effect of DOG on the
KCa current is mediated by PKC,
the influence of PKC inhibitor peptide (19
31), another selective PKC
inhibitor, on the DOG effect was tested. PKC inhibitor peptide at a
concentration of 1 µM was added to the pipette solution. Control
experiments showed that PKC inhibitor peptide itself did not affect the
KCa current (Fig. 4). In summary, 10 µM DOG after PKC inhibitor peptide pretreatment increased the KCa current 6 min after the
beginning of the application at +70 mV by 3 ± 16%
(n = 5) compared with the current at
the beginning of the application period. This is significantly
different from the 70 ± 4% (n = 14; P < 0.001) decrease of the
KCa current after DOG application
without PKC blocker pretreatment at +70 mV (Fig. 4).
To test for a possible nonspecific effect of DOG on the
KCa current, the effect of
1,3-dioctanoyl-sn-glycerol (inactive
DOG), a cell-permeable inactive analog of diacylglycerol, was tested. A
typical example of the time course of the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV after application of inactive DOG is shown in Fig.
1A, together with an example of the
time course of the KCa current
after DOG application and of the control
KCa current. During the
application of 10 µM inactive DOG, the
KCa current was measured with the
same protocol as in the DOG series. An increase of the
KCa current was observed 2 min
after the beginning of application of inactive DOG, and subsequently
there was no decline of the KCa
current; rather, the KCa current
stayed at the increased level. In summary, 10 µM inactive DOG
increased the KCa current 6 min
after the beginning of the application at +70 mV by 18 ± 10%
(n = 4) compared with the current at
the beginning of the application period. This is significantly
different from the 70 ± 4% (n = 14; P < 0.001) decrease of the
KCa current after DOG application
at +70 mV (Fig. 4).
Effect of PKC on the KCa current.
For the further investigation of the effect of PKC on the
KCa current of rat tail artery
smooth muscle cells, PKCC (27), which is active in the absence of phospholipids and Ca, was employed. PKCC was added to the pipette
solution at 0.1 U/ml. A higher activity of
PKCC was not used so as to avoid
possible artifacts caused by components of the
PKCC storage buffer. A typical
example of the time course of the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV under control conditions and after
PKCC application is shown in Fig.
5A. The
KCa current started to decline
~3-4 min after the beginning of
PKCC application, and 6-8 min
after the beginning of PKCC
application the KCa current
reached a steady state at a considerably lower level compared with the
time-matched control current. For example, the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test potential of +70 mV was 209 pA at the beginning of the
PKCC application period and only
79 pA 6 min after the beginning of the
PKCC application period (Fig.
5B,
top).
I-V
relationships of the KCa current
obtained with use of a voltage ramp from
70 mV to +100 mV based
on a holding potential of
40 mV showed that
PKCC produced a marked decrease of
the KCa current in the potential
range tested. This is demonstrated in Fig.
5B,
bottom, with a typical example and, in
addition to the original traces, the
I-V
relationship of the pure KCa
current, i.e., the difference of the net outward current and the
iberiotoxin-insensitive current, is shown before and after
PKCC application (Fig.
5B, bottom
right). In summary, the
I-V
relationship of the KCa current was altered significantly, seen as a reduction of this current (Fig.
6), and the threshold potential of the
current changed significantly, from 30.0 ± 2.2 to 41.1 ± 2.2 mV
or by 11.1 ± 2.0 mV (n = 9;
P < 0.01). On average, 0.1 U/ml
PKCC decreased the
KCa current 6 min after the
beginning of the application at +70 mV by 44 ± 3% (n = 17) compared with the current at
the beginning of the application period. This is a significant decrease
in comparison with the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (see Fig.
8; P < 0.01).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of catalytic subunit of PKC
(PKCC) on
KCa current of rat tail artery
smooth muscle cells. A:
examples of time course of control
KCa current (taken from Fig. 1 for
comparison) and of time course of effect of
PKCC on
KCa current.
KCa current was elicited with use
of a voltage step from a holding potential of 40 mV to a test
potential of +70 mV and was normalized to current at beginning of
application period (time
0).
B: examples of traces of
KCa current in PKC series.
Top: from
left, stimulation protocol, current at
beginning (0) of PKCC application
together with current after IBTX application, and current after 6 min
of PKC application together with current after IBTX application.
Bottom: from
left, stimulation protocol,
I-V
relationship at beginning of PKCC
application together with
I-V
relationship after 6 min of PKCC
application as well as
I-V
relationship after IBTX application, and
I-V
relationship of pure KCa current,
i.e., difference of net outward
I-V
relationship and
I-V
relationship after IBTX application, at beginning of
PKCC application and after 6 min
of PKCC application. In
A, data points of different
experimental series do not coincide in time because of variable length
of period for series resistance control. Also, capacitive artifacts
shown in B are distorted due to
filtering of current traces at low frequency and limited
x-axis resolution of graphic
presentation.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Dependence of KCa current density
on membrane potential. con, KCa
current at beginning of application of 0.1 U/ml
PKCC. PKCC,
KCa current after 6 min of
application of 0.1 U/ml PKCC. A
repeated measures ANOVA showed a significant difference between control
and PKCC
(n = 9;
P < 0.01).
|
|
To obtain evidence that the effect of
PKCC on the
KCa current is not due to an
artifact, the effect of PKCC
inactivated by heating at 60°C for 30 min was tested.
Heat-inactivated PKCC at 0.1 U/ml
was added to the pipette solution. A typical example of the time course
of the KCa current elicited with
use of a voltage step from a holding potential of
40 mV to a
test potential of +70 mV after application of heat-inactivated
PKCC is shown in Fig.
7A,
together with an example of the time course of the
KCa current after application of
active PKCC. However, there was no change of the KCa current during
the whole application period. For example, the
KCa current elicited with use of a
voltage step from a holding potential of
40 mV to a test
potential of +70 mV was 184 pA at the beginning of the application
period of heat-inactivated PKCC
and 152 pA 6 min after the beginning of the application period (Fig.
7B,
top).
I-V
relationships of the KCa current
obtained with use of a voltage ramp from
70 mV to +100 mV based
on a holding potential of
40 mV showed that the changes of the
control current with time were similar at all potentials tested (see
Fig. 7B, bottom, for a typical example). In
summary, 0.1 U/ml heat-inactivated PKCC decreased the
KCa current 6 min after the
beginning of the application at +70 mV by 13 ± 9%
(n = 5) compared with the current at
the beginning of the application period. This is significantly different from the 44 ± 3% (n = 17; P < 0.01) decrease of the KCa current after application of
active PKCC at +70 mV (Fig.
8).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of heat-inactivated PKCC on
KCa current of rat tail artery
smooth muscle cells. A: examples of
time course of effect of heat-inactivated
PKCC on
KCa current and of time course of
effect of active PKCC on
KCa current (taken from Fig. 5 for
comparison). KCa current was
elicited with use of a voltage step from a holding potential of
40 mV to a test potential of +70 mV and was normalized to
current at beginning of application period
(time
0).
B: examples of traces of
KCa current in series with
heat-inactivated PKCC.
Top: from
left, stimulation protocol, current at
beginning of application of heat-inactivated
PKCC together with current after
IBTX application, and current after 6 min of application of
heat-inactivated PKCC together
with current after IBTX application.
Bottom: from
left, stimulation protocol and
I-V
relationship at beginning of application of heat-inactivated
PKCC together with
I-V
relationship after 6 min of application of heat-inactivated
PKCC as well as
I-V
relationship after IBTX application. In
A, data points of different
experimental series do not coincide in time because of variable length
of period for series resistance control. Also, capacitive artifacts
shown in B are distorted due to
filtering of current traces at low frequency and limited
x-axis resolution of graphic
presentation.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Summary of effect of PKCC on
KCa current of rat tail artery
smooth muscle cells 6 min after beginning of application. Data are
expressed as ratio of KCa current
6 min after beginning of application under specified experimental
conditions (0.1 U/ml active or heat-inactivated
PKCC) and
KCa current at beginning of
application period under same conditions. * Significant
difference compared with control determined by 1-way ANOVA
(P < 0.01); no. of cells
investigated appears on bar for each condition.
|
|
Effect of PKC on single KCa channels.
For the investigation of the effect of PKC on the
KCa current of rat tail artery
smooth muscle cells, the direct effect of PKCC on single
KCa channels was tested.
PKCC was added to the bath
solution at 0.1 U/ml together with 100 µM MgATP after the registration of channel control activity. In these single-channel experiments, a spontaneous decrease of the activity of the
KCa channel (rundown) was often
observed, the nature of which was not established in the present study.
Therefore, all measurements showing a rundown in the registration of
channel control activity were excluded from further analysis.
Furthermore, to be able to differentiate the expected PKC-induced
decrease of KCa channel activity
from rundown, the experimental protocol included a test of the action
of PKC inhibitor peptide after a stable level of channel activity was
obtained after PKC application. Only experiments in which PKC inhibitor
peptide was able to reverse the effect of PKC on the
KCa current were included in the
analysis. A typical example of the time course of the activity of
single KCa channels at 0 mV under
control conditions and after PKCC
and subsequent PKC inhibitor peptide application is shown in Fig.
9A.
KCa channel activity started to
decline ~2 min after the beginning of the application of
PKCC together with 100 µM MgATP,
and KCa channel activity reached a
steady state ~4 min after the beginning of PKCC application. Subsequently,
PKC inhibitor peptide application restored
KCa channel activity, and, ~3
min after the beginning of PKC inhibitor peptide application,
KCa channel activity reached a
steady state. In this example (Fig.
9B),
KCa channel activity (NPo) at 0 mV
was 0.56 in the control period, 0.07 during the steady state after
PKCC application, and 0.51 during
the steady state after the subsequent addition of PKC inhibitor
peptide. On average, 0.1 U/ml PKCC
together with 100 µM MgATP decreased KCa channel activity in the steady
state at +0 mV significantly by 79 ± 9%
(n = 6) compared with the activity in
the control period (Fig. 9C;
P < 0.001). PKC inhibitor peptide
reversed the PKCC-induced decrease
of KCa channel activity, and in
the steady state a nonsignificant decrease of channel activity by 5 ± 22% (n = 6) compared with the
activity in the control period was observed (Fig.
9C; P = 0.82). The amplitude of single
KCa channel openings was not
changed after the addition of PKCC
or PKC inhibitor peptide (data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of PKCC on single
KCa channel activity of rat tail
artery smooth muscle cells. A:
example of a diary plot of channel number times open probability
(NPo) of
KCa channel at 0 mV with
application of PKCC and PKC-I.
B: examples of traces of
KCa channel activity at 0 mV taken
from same patch as in A during control
(Ctrl), PKCC application and
subsequent addition of PKC-I. C:
summary of effect of PKCC on
single KCa channel activity. Data
are expressed as ratio of
NPo of channel
after reaching a steady state during application of 0.1 U/ml native
PKCC, 0.1 U/ml heat-inactivated
PKCC, or additional application of
1 µM PKC-I, respectively
[NPo(exp)],
and NPo of
channel in control period
[NPo(ctrl)].
* Significant difference compared with control
(P < 0.001); no. of cells
investigated appears on bar for each condition.
|
|
To obtain evidence that the effect of
PKCC on single
KCa channel activity is not due to
an artifact, the effect of PKCC
inactivated by heating at 60°C for 30 min was tested using the same
experimental protocol as for native
PKCC. Heat-inactivated
PKCC at 0.1 U/ml together with 100 µM MgATP did not significantly alter
KCa channel activity (Fig.
9C). The subsequent addition of PKC
inhibitor peptide also had no significant effect on
KCa channel activity. The
amplitude of single KCa channel
openings was not changed after the addition of heat-inactivated
PKCC or PKC inhibitor peptide
(data not shown).
 |
DISCUSSION |
Effect of an activation of PKC on the KCa
current of rat tail artery smooth muscle cells.
The aim of the first series of experiments was to investigate the
effect of an activation of the endogenous PKC on the
KCa current. To activate PKC, an
active analog of the natural PKC activator diacylglycerol, DOG, was
used (7). In the present experiments, the PKC activator DOG entered the
cells from the extracellular space, in contrast to the natural
conditions, in which the PKC activator is released from membrane
phospholipids. Therefore, it was necessary to obtain evidence that the
effect of DOG on the KCa current
is mediated by PKC. In the present study the DOG-induced decrease of
the KCa current was completely
blocked by the specific PKC inhibitors calphostin C and PKC inhibitor peptide. Calphostin C acts by an interaction with the diacylglycerol binding site of the regulatory domain of PKC (14), and PKC inhibitor peptide acts as pseudo-substrate by binding to the active site of PKC.
An unspecific action of DOG on the
KCa current seems unlikely also
because the inactive diacylglycerol analog
1,3-dioctanoyl-sn-glycerol was unable
to reduce the KCa current.
Therefore, the decrease of the KCa
current induced by DOG is probably mediated by an activation of the PKC
present in these cells.
Interestingly, in recent publications it has been shown that DOG
decreases the ATP-sensitive K current in rabbit mesenteric artery
smooth muscle cells (4) and the voltage-dependent K current in rabbit
portal vein smooth muscle cells (1) and that the latter effect was
blocked by calphostin C. Additionally, phorbol ester decreased
spontaneous transient outward K currents, which are generated by the
action of spontaneously released Ca from intracellular stores on
KCa channels, in rabbit portal
vein smooth muscle cells, and this effect was also blocked by
inhibition of PKC (13). Furthermore, a phorbol ester-mediated decrease
of KCa currents was also observed
in cultured endothelial cells (29) and in rat pituitary tumor cells
(24). In summary, our data show that the
KCa current of rat tail artery
smooth muscle cells can be inhibited by an activation of the endogenous
PKC of these cells.
Effect of PKC on the KCa current and
single KCa channels of rat tail artery
smooth muscle cells.
The aim of the second series of experiments was to investigate the
effect of exogenous PKC on the KCa
current and on single KCa
channels. To ensure a straightforward interpretation of these experiments, PKCC was used, which
does not require Ca or phosphatidylserine to be active (27). Thus the
number of factors that could affect the
KCa current or
KCa channel on their own was
limited. In the present study, it was shown for the first time that the
application of PKCC together with
MgATP, which was permanently present in the intracellular solution in
all experiments, considerably decreased the
KCa current in freshly isolated
arterial smooth muscle cells. Furthermore, the data on the direct
inhibition of single-KCa channel activity by PKCC together with
MgATP in freshly isolated smooth muscle cells extend earlier findings
showing an inhibition of single
KCa channel activity in cultured
smooth muscle cells from porcine coronary arteries after a direct
application of PKC to this channel (18).
It should be noted that the PKC storage buffer contained a variety of
components, for example, 100 mM NaCl, 15 mM dithiothreitol, and 10%
glycerol, some of which are known to affect
KCa channel function. Therefore,
the PKC stock solution was diluted 100 times to avoid artifacts caused
by the components of the PKC storage buffer. This, however, limited the
maximal employable PKC activity to 0.1 U/ml and, consequently, it was
decided not to use higher activities of PKC to test whether or not PKC
is able to inhibit the KCa current
completely. To obtain evidence for a specific action of PKC on the
KCa current and the
KCa channel, the PKC stock
solution was heated for 30 min at 60°C. Application of solution containing heat-treated PKC did not produce any effect on the KCa current or the
KCa channel, demonstrating that a
heat-sensitive component in the PKC stock solution, probably the
protein PKC, is responsible for the decrease of the
KCa current and
KCa channel activity. In summary,
the present data show that the KCa
current and the activity of KCa
channels of freshly isolated rat tail artery smooth muscle cells can be
inhibited by exogenous PKC, supporting earlier conclusions that this
current can be inhibited by an activation of the endogenous PKC.
Mechanism of the PKC effect on the KCa
current of rat tail artery smooth muscle cells.
When discussing the mechanism of the PKC effect on the
KCa current, it is important to
note that in whole cell experiments the
KCa current has been shown to be
influenced markedly by changes of the intracellular Ca concentration
induced by the inositol trisphosphate-mediated Ca release (13), the
ryanodine-sensitive Ca release (19), or the Na/Ca exchange (6). The
activity of these mechanisms regulating the intracellular Ca ion
concentration is also affected by PKC (3, 15). Therefore, specific
experimental conditions with a strongly buffered low intracellular Ca
concentration and a low extracellular Ca concentration were selected,
which have been shown to greatly reduce the influence of mechanisms regulating the intracellular Ca concentration on the
KCa current (6). The absence of an
effect of ryanodine, which blocks local Ca release events spontaneously
occurring even in resting cells, on the
KCa current demonstrated that
under the selected experimental conditions it was possible to
investigate the effect of PKC on the
KCa current under conditions of a
minimized influence of alterations of the intracellular Ca concentration.
Because the whole cell KCa current
equals the product of N,
Po, and
single-channel amplitude, the observed decrease of the KCa current may be caused by a
decrease of any one of these factors. The whole cell data of the
present study show that DOG as well as PKC produce a larger reduction
of the KCa current at lower compared with higher membrane potentials and a significant shift of the
current threshold. This indicates that PKC affects mainly the
Po of the
KCa channel. Indeed, the
experiments on the effect of PKC on single
KCa channels demonstrated that PKC
does not affect the single-channel amplitude or
N but considerably alters the Po. Thus, under
the conditions of the present study, PKC inhibits the
KCa current by a direct
interaction with the channel or a closely related protein leading to a
decrease of single-channel activity.
As mentioned above, under more physiological conditions of
extracellular and intracellular Ca concentrations, PKC could affect the
KCa current also by an alteration
of the activity of mechanisms regulating the intracellular Ca
concentration. Therefore, as in the recent study on the regulation of
the KCa current by ryanodine receptor channel-dependent Ca release events (3), it should be
investigated whether the KCa
current is additionally affected by a PKC-mediated effect on, for
example, the Ca pump in the sarcoplasmic reticulum, the Ca pump in the
plasmalemma, the Ca channel in the plasmalemma, or the Na/Ca exchange.
Because of this multitude of possibilities, this question, however,
should be addressed in a separate study.
Possible functional role of the PKC effect on the
KCa current of rat tail artery smooth muscle
cells.
Because the KCa current in a
variety of arterial smooth muscle cells in vessels exposed to a
physiological pressure level but not subjected to any vasoactive
agonist belongs to the ion currents establishing the membrane potential
(5), a PKC-mediated decrease of this current may have important
functional consequences. Thus a decrease of the
KCa current, leading to membrane
potential depolarization and vasoconstriction, may mediate the effect
of transmural pressure or of different vasoconstrictors, because these
stimuli have been shown to activate PKC (12, 15). However, the
hypothesis of a functional importance of the PKC-mediated decrease of
the vascular smooth muscle KCa
current is highly speculative at the moment. Thus a phospholipase
C-induced activation of PKC under physiological conditions is
accompanied by an inositol trisphosphate-mediated release of Ca from
intracellular stores. This Ca may increase the
KCa current and, therefore,
counteract its decrease by PKC. Additionally, the regulation of the
KCa current by PKC-dependent local
Ca release events has to be taken into account (3). The data available
do not allow estimation of the net effect of the simultaneous action of
Ca and PKC on the KCa current.
Furthermore, PKC alters the activity of several other mechanisms, which
also can produce vessel constriction. Limiting this consideration only to mechanisms regulating the membrane potential, PKC, for example, decreases the ATP-sensitive K current (4) and the voltage-dependent K
current (1) and increases the voltage-dependent Ca current (16). Again,
the data available do not allow estimation of whether the PKC-related
response to the above-mentioned physiological stimuli is mediated
mainly by the PKC-induced decrease of the KCa current or mainly by one of
the other mechanisms. Additionally, although the membrane potential and
the intracellular Ca concentration can be adjusted in patch-clamp
experiments to simulate physiological conditions for the
KCa channel, the functional state
of a variety of other factors affecting its function is quite different
in an intact artery compared with an isolated cell, e.g., PKA is activated by transmural pressure (11), PKG is activated by flow-induced release of NO (20), or G proteins are activated by epoxyeicosatrienoic acids (17). Moreover, their functional state in the intact artery is
not known and, therefore, cannot be simulated in an isolated cell. Thus
the functional role of the PKC effect on the
KCa current can be established
only in studies on intact vessel preparations. In conclusion, the
complete mechanism and the functional role of the PKC-induced
regulation of the KCa current have
to be established in future studies.
In summary, this study presents the novel observation that the
KCa current of freshly isolated
rat tail artery smooth muscle cells was decreased by
PKCC and by an active analog of
diacylglycerol, an activator of PKC, independently of their effects on
local Ca release events. The effect of the active analog of
diacylglycerol was inhibited by calphostin C and PKC inhibitor peptide,
selective PKC inhibitors, and was not mimicked by an inactive analog of diacylglycerol, providing evidence for a PKC-selective action of the
diacylglycerol analog. Furthermore, the finding of the present study
that the activity of single KCa
channels was decreased by PKCC in
freshly isolated vascular smooth muscle cells suggests that the effect
of PKC on KCa currents is mediated
by its direct action on the channel.
 |
ACKNOWLEDGEMENTS |
This research was supported by Deutsche Forschungsgemeinschaft
Grant 436 RUS 113/11 (to R. Schubert), a University of Rostock Research Grant (to R. Schubert), and Russian Foundation
for Basic Research Grant 98-04-04106 (to V. Serebryakov).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. Schubert, University of Rostock,
Institute of Physiology, PSF 100888, D-18055 Rostock, Germany.
Received 9 March 1998; accepted in final form 2 December 1998.
 |
REFERENCES |
1.
Aiello, E. A.,
O. Clement-Chomienne,
D. P. Sontag,
M. P. Walsh,
and
W. C. Cole.
Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H109-H119,
1996[Abstract/Free Full Text].
2.
Archer, S. L.,
J. M. C. Huang,
V. Hampl,
D. P. Nelson,
P. J. Shultz,
and
E. K. Weir.
Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
91:
7583-7587,
1994[Abstract].
3.
Bonev, A.,
and
M. T. Nelson.
Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C.
J. Gen. Physiol.
108:
315-323,
1996[Abstract].
4.
Bonev, A. D.,
J. H. Jaggar,
M. Rubart,
and
M. T. Nelson.
Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries.
Am. J. Physiol.
273 (Cell Physiol. 42):
C2090-C2095,
1997[Abstract/Free Full Text].
5.
Brayden, J. E.,
and
M. T. Nelson.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992[Medline].
6.
Bychkov, R.,
M. Gollasch,
C. Ried,
F. C. Luft,
and
H. Haller.
Regulation of spontaneous transient outward potassium currents in human coronary arteries.
Circulation
95:
503-510,
1997[Abstract/Free Full Text].
7.
Davis, R. J.,
B. R. Ganong,
R. M. Bell,
and
M. P. Czech.
sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitagenesis.
J. Biol. Chem.
260:
1562-1566,
1985[Abstract].
8.
Fan, S. F.,
and
C. Y. Kao.
On the apparent absence of maxi-K+ channel in rat aortic myocyte.
Proc. Soc. Exp. Biol. Med.
202:
465-469,
1993[Abstract].
9.
Galvez, A.,
G. Gimenez Gallego,
J. P. Reuben,
L. Roy Contancin,
P. Feigenbaum,
G. J. Kaczorowski,
and
M. L. Garcia.
Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.
J. Biol. Chem
265:
11083-11090,
1990[Abstract/Free Full Text].
10.
Giangiacomo, K. M.,
M. L. Garcia,
and
O. B. McManus.
Mechanism of iberiotoxin block of the large-conductance calcium-activated potassium channel from bovine aortic smooth muscle.
Biochemistry
31:
6719-6727,
1992[Medline].
11.
Hopp, H.-H.,
R. Schubert,
V. N. Serebryakov,
and
H. Mewes.
The level of the spontaneous myogenic tone of rat tail resistance arteries is determined by protein kinase A activation of Ca-activated K-channels (Abstract).
Pflügers Arch.
429:
R72,
1995.
12.
Karibe, A.,
J. Watanabe,
S. Horiguchi,
M. Takeuchi,
S. Suzuki,
M. Funakoshi,
H. Katoh,
M. Keitoku,
S. Satoh,
and
K. Shirato.
Role of cytosolic Ca and protein kinase C in developing myogenic contraction in isolated rat small arteries.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H1165-H1172,
1997[Abstract/Free Full Text].
13.
Kitamura, K.,
Z. L. Xiong,
N. Teramoto,
and
H. Kuriyama.
Roles of inositol trisphosphate and protein kinase-C in the spontaneous outward current modulated by calcium release in rabbit portal vein.
Pflügers Arch.
421:
539-551,
1992[Medline].
14.
Kobayashi, E.,
H. Nakano,
M. Morimoto,
and
T. Tamaoki.
Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
159:
548-553,
1989[Medline].
15.
Lee, M. W.,
and
D. L. Severson.
Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action.
Am. J. Physiol.
267 (Cell Physiol. 36):
C659-C678,
1994[Abstract/Free Full Text].
16.
Lepretre, N.,
and
J. Mironneau.
Alpha(2)-adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes.
Pflügers Arch.
429:
253-261,
1994[Medline].
17.
Li, P. L.,
and
W. B. Campbell.
Epoxyeicosatrienoic acids activate K+ channels in coronary smooth muscle through a guanine nucleotide binding protein.
Circ. Res.
80:
877-884,
1997[Abstract/Free Full Text].
18.
Minami, K.,
K. Fukuzawa,
and
Y. Nakaya.
Protein kinase-C inhibits the Ca2+-activated K+ channel of cultured porcine coronary artery smooth muscle cells.
Biochem. Biophys. Res. Commun.
190:
263-269,
1993[Medline].
19.
Nelson, M. T.,
H. Cheng,
M. Rubart,
L. F. Santana,
A. D. Bonev,
H. J. Knot,
and
W. J. Lederer.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
20.
Robertson, B. E.,
R. Schubert,
J. Hescheler,
and
M. T. Nelson.
cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C299-C303,
1993[Abstract/Free Full Text].
21.
Schubert, R.
Multiple ligand-ion solutions: a guide for solution preparation and computer program understanding.
J. Vasc. Res.
33:
86-98,
1996[Medline].
22.
Schubert, R.,
V. N. Serebryakov,
H. Engel,
and
H.-H. Hopp.
Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1203-C1211,
1996[Abstract/Free Full Text].
23.
Schubert, R.,
V. N. Serebryakov,
H. Mewes,
and
H.-H. Hopp.
Iloprost dilates rat small arteries: role of KATP- and KCa-channel activation by cAMP-dependent protein kinase.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H1147-H1156,
1997[Abstract/Free Full Text].
24.
Shipston, M. J.,
and
D. L. Armstrong.
Activation of protein kinase C inhibits calcium-activated potassium channels in rat pituitary tumor cells.
J. Physiol. (Lond.)
493:
665-672,
1996[Abstract].
25.
Shoemaker, R. L.,
and
R. T. Worrell.
Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats.
Proc. Soc. Exp. Biol. Med.
196:
325-332,
1991[Abstract].
26.
Tateishi, J.,
and
J. E. Faber.
ATP-sensitive K+ channels mediate alpha(2D)-adrenergic receptor contraction of arteriolar smooth muscle and reversal of contraction by hypoxia.
Circ. Res.
76:
53-63,
1995[Abstract/Free Full Text].
27.
VanRenterghem, B.,
M. D. Browning,
and
J. L. Maller.
Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system.
J. Biol. Chem.
269:
24666-24672,
1994[Abstract/Free Full Text].
28.
White, R. E.,
D. J. Darkow,
and
J. L. F. Lang.
Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism.
Circ. Res.
77:
936-942,
1995[Abstract/Free Full Text].
29.
Zhang, H.,
B. Weir,
and
E. E. Daniel.
Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.
Pharmacology
50:
247-256,
1995[Medline].
30.
Zou, A. P.,
J. T. Fleming,
J. R. Falck,
E. R. Jacobs,
D. Gebremedhin,
D. R. Harder,
and
R. J. Roman.
20-HETE is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles.
Am. J. Physiol.
270 (Regulatory Integrative Comp. Physiol. 39):
R228-R237,
1996[Abstract/Free Full Text].
Am J Physiol Cell Physiol 276(3):C648-C658
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society