PKC regulation of cardiac CFTR
Cl
channel function in
guinea pig ventricular myocytes
Lisa M.
Middleton and
Robert D.
Harvey
Department of Physiology and Biophysics, Case Western Reserve
University, Cleveland, Ohio 44106-4970
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ABSTRACT |
The role of
protein kinase C (PKC) in regulating the protein kinase A
(PKA)-activated Cl
current
conducted by the cardiac isoform of the cystic fibrosis transmembrane
conductance regulator (cCFTR) was studied in guinea pig ventricular
myocytes using the whole cell patch-clamp technique. Although
stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu)
alone did not activate this
Cl
current, even when
intracellular dialysis was limited with the perforated patch-clamp
technique, activation of PKC did elicit a significant response in the
presence of PKA-dependent activation of the current by the
-adrenergic receptor agonist isoproterenol. PDBu
increased the magnitude of the
Cl
conductance activated by
a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% (n = 9) when added after
isoproterenol and by 36 ± 16% (n = 14) when introduced before isoproterenol. 4
-Phorbol
12,13-didecanoate, a phorbol ester that does not activate PKC, did not
mimic these effects. Preexposure to chelerythrine or
bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl
current by 79 ± 7.7% (n = 11) and 52 ± 10%
(n = 8), respectively. Our
results suggest that although acute activation of endogenous PKC alone
does not significantly regulate cCFTR
Cl
channel activity in
native myocytes, it does potentiate PKA-dependent responses, perhaps
most dramatically demonstrated by basal PKC activity, which may play a
pivotal role in modulating the function of these channels.
bisindolylmaleimide; chelerythrine; cross talk; phorbol
12,13-dibutyrate; protein kinase C; cardiac isoform of cystic fibrosis
transmembrane conductance regulator
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INTRODUCTION |
THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) gene encodes a Cl
channel found in epithelial tissue. An alternatively spliced isoform of
the protein has also been identified in cardiac muscle of a number of
different mammalian species (16, 35). This cardiac isoform of CFTR
(cCFTR) lacks exon 5, which encodes a cytoplasmic region located
between the second and third transmembrane segments. Aside from this
30-amino acid deletion, this isoform of the protein isolated from
rabbit ventricle is reported to retain >90% homology to human
epithelial CFTR (eCFTR) (13). Both isoforms of the channel protein have
been shown to contain a high concentration of phosphorylation sites in
a region referred to as the regulatory domain, which contains several
sites that can be phosphorylated by protein kinase A (PKA)
and/or protein kinase C (PKC). PKA phosphorylation sites
predominate in the regulatory domain, and PKA-dependent phosphorylation
is the well-known activation mechanism for both cCFTR and eCFTR. The
role of PKC phosphorylation is much less clear, especially for cCFTR.
Although the regulatory domain contains 9 of 10 consensus sites for PKA
phosphorylation, it contains only 7 of 29 consensus sites for PKC
phosphorylation, and 2 of the 22 PKC phosphorylation sites found
outside the regulatory domain are in exon 5 (26). The impact, if any,
of the PKC phosphorylation sites that are missing from the cCFTR
channel protein is not known but could underlie fundamental differences
in the way that cCFTR and eCFTR are regulated.
Studies involving eCFTR have clearly demonstrated that this protein is
phosphorylated by PKC (11). However, the functional consequence of
PKC-dependent phosphorylation is somewhat complex. In heterologous
expression systems, PKC alone stimulates eCFTR activity to a level that
is only 10-15% of that activated by PKA (3, 19, 30). Although the
response to PKC alone is relatively minor, this kinase also has been
shown to potentiate the effect of PKA-dependent stimulation such that
channel activity observed in the presence of both kinases has been
reported to be 50-200% greater than that activated by PKA alone
(19, 30). It has even been demonstrated that basal PKC activity,
although it does not sustain channel activity on its own, is actually
required for subsequent PKA-dependent stimulation (19). Activation of endogenous PKC and activation of endogenous PKA have also been shown to
produce qualitatively similar results in native epithelial cells, which
constitutively express eCFTR. In rat pancreatic duct cells, stimulation
of PKC alone activated no detectable
Cl
current, but activation
of PKC did increase the response to PKA-dependent stimulation by 31%
(37). Also, in T84 epithelial cells, there is indirect evidence that
basal PKC activity is necessary for PKA-dependent stimulation of eCFTR
(6).
In contrast to its effect on eCFTR channel function, the effect of PKC
on cCFTR channel activity is more controversial. We have previously
reported that phorbol ester stimulation of endogenous PKC does not
activate a macroscopic Cl
conductance in guinea pig ventricular myocytes (24). Although other
groups have described a phorbol ester-activated
Cl
current in these same
cells (5, 28, 33, 34), the relative magnitude of such PKC-activated
currents is unclear. Because the level of cCFTR expression varies
significantly from cell to cell (18), the most meaningful estimates
require comparison of PKC- and PKA-dependent responses obtained from
the same cell. Zhang et al. (39) described a PKC- and PKA-activated
Cl
current in cat
ventricular myocytes and found that PKC was as effective as PKA in
activating that current. They also found that maximal PKC- and
PKA-dependent responses were not additive. This suggests that
activation of PKC does not potentiate PKA-dependent stimulation of this
cardiac Cl
current.
However, the structure of the channel responsible for the
Cl
current in cat myocytes
has not been determined. Although similar findings have been reported
by Walsh and Long (34) in guinea pig ventricular myocytes, where the
PKA-activated Cl
current is
known to be conducted by the alternatively spliced cCFTR (16), the
interpretation of the results is complicated by the fact that their
experiments were carried out using myocytes dialyzed with exogenous PKC
isoforms. Furthermore, no one has yet examined whether basal PKC
activity has any effect on PKA-dependent regulation of cCFTR.
Therefore, to clarify the functional role of PKC in regulating cCFTR,
the purposes of the present study were
1) to reevaluate the effect of acute
activation of endogenous PKC alone on cCFTR, 2) to determine whether there is any
interaction between acute activation of endogenous PKC- and
PKA-dependent responses, and 3) to
examine the question of whether basal PKC activity affects PKA-dependent responses.
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MATERIALS AND METHODS |
Cell isolation.
Hearts excised from anesthetized adult guinea pigs of either sex were
perfused via the aorta with Krebs-Henseleit buffer (KHB) containing (in
mM) 120 NaCl, 4.8 KCl, 1.5 CaCl2,
2.2 MgSO4, 1.2 NaH2PO4,
25.0 NaHCO3, and 11.0 glucose. The
buffer pH was maintained at 7.35 by bubbling with 95%
O2-5%
CO2 at 36°C. Ventricular
myocytes were then isolated according to a previously described method (14). After dissection, the heart was first exposed to
Ca2+-containing KHB for 5 min.
Perfusion was then switched to
Ca2+-free KHB for another 5 min,
following which enough collagenase B (Boehringer Mannheim) was added to
the Ca2+-free buffer to obtain a
final concentration of ~0.5 mg/ml. After 30 min of digestion, the
ventricles were removed, minced, rinsed free of collagenase, and
reintroduced to Ca2+-containing
KHB. Gentle trituration freed individual cells for use in patch-clamp
experiments.
Data acquisition and analysis.
Except as noted, macroscopic membrane currents were recorded using the
conventional whole cell patch-clamp technique (12). Electrodes were
pulled from borosilicate glass capillary tubing (Corning 7052, Garner
Glass) and had resistances between 0.5 and 1.5 M
. The bath was
grounded with a 3 M KCl-agar bridge. Currents were recorded using an
Axopatch 200 voltage-clamp amplifier (Axon Instruments) and an
IBM-compatible computer with a TL-1-125 interface and pCLAMP software
(version 5.7, Axon Instruments). Recording the current elicited by
100-ms voltage-clamp steps to +50 mV once every 3 s provided a record
of the time course of change in
Cl
current in response to
application and removal of drug(s). Once a peak or steady-state
response to a particular drug was observed, a current-voltage
(I-V)
relationship was obtained by recording currents elicited during 100-ms
voltage steps to test potentials from
120 to +50 mV.
Data were also analyzed using pCLAMP software. The
Cl
current was defined as
the difference current obtained by subtracting currents recorded in the
absence of any drug from those recorded in the presence of drug(s). In
addition to the PKA-activated
Cl
current conducted by
cCFTR, guinea pig ventricular myocytes also possess a
swelling-activated Cl
current (32), and PKC has been reported to stimulate a similar current
found in dog atrial cells (7). However, DIDS inhibits the
swelling-activated Cl
current but not the cCFTR
Cl
current (32). Therefore,
we verified that the Cl
current enhanced by the activation of PKC was not inhibited by 250-500 µM DIDS (Sigma; see Fig. 5).
Current traces were measured over a 15-ms span at the end of each
100-ms voltage step. Slope conductances were calculated by linear
regression of
I-V
relationships over the range of membrane potentials positive to the
reversal potential. For dose-response relationships, data were fitted
to appropriate equations using a nonlinear, least squares curve-fitting
routine (SigmaPlot, Jandel Scientific). Results are reported as means ± SE. Statistical tests (SigmaStat, Jandel Scientific) used to
evaluate the potential significance of responses to different drug
treatments are indicated (see Figs. 3, 6, and 7).
Solutions.
For study of the
Cl
current, cells were
dialyzed with an internal solution composed of (in mM) 130 glutamic
acid, 5.0 HEPES, 5.0 EGTA, 20.0 tetraethylammonium (TEA) chloride, 5.0 MgATP, and 0.1 Tris-GTP; pH was brought to 7.2 with CsOH. Cells were
bathed in an external solution of (in mM) 140.0 NaCl, 5.4 CsCl, 2.5 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 11.0 glucose; pH was raised to 7.4 with NaOH. All
K+ currents were eliminated by
using these K+-free solutions
containing Cs+ and/or TEA.
L-type Ca2+ currents were
eliminated by adding 1 µM nisoldipine (Miles Laboratories) to all
external solutions, while Na+
channels were inactivated through the use of a
30 mV holding potential. For study of delayed rectifier
K+ currents, the internal solution
was modified by replacing glutamic acid with potassium glutamate, TEA
chloride with KCl, and CsOH with KOH; the external solution was
modified by replacing CsCl with KCl. For perforated patch experiments,
the technique used was as previously described (38). Amphotericin B
(Sigma; 240 µg/ml) was added to the internal solution described
above. All experiments were conducted at 36-37°C.
Drugs.
Cells were exposed to external solution after being placed in a 0.5-ml
chamber on the stage of an inverted microscope. Once the whole cell
configuration was obtained, each cell was positioned in front of a
rapid perfusion system, thereby making it possible to change the
solution bathing a cell in <1 s (38).
R(
)-isoproterenol-bitartrate (Research Biochemicals International) was dissolved in water. Chelerythrine chloride (Research Biochemicals International), phorbol
12,13-dibutyrate (PDBu; Sigma), 4
-phorbol 12,13-didecanoate (4
-PDD; Sigma), and bisindolylmaleimide (Calbiochem) were initially dissolved in DMSO (Sigma) and further diluted in water. All
drug-containing solutions were prepared by diluting stock solutions
1,000-fold. The final concentration of DMSO never exceeded 0.1%. In
experiments involving PDBu and 4
-PDD, 0.1% albumin (Sigma) was
added to all external solutions to prevent the hydrophobic phorbol
esters from adhering to the plastic tubing in our setup (2). Ascorbic
acid (Sigma; 50 µM) was present in all isoproterenol-containing
solutions to prevent oxidative degradation.
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RESULTS |
Effect of PDBu alone on cCFTR in guinea pig ventricular myocytes.
Phorbol ester-dependent activation of endogenous PKC has been reported
to elicit a measurable macroscopic
Cl
current in guinea pig
ventricular myocytes (28, 33, 34). However, in a previous study, we saw
no evidence for such a response (24). One possible explanation for the
discrepancy is that intracellular dialysis associated with the whole
cell patch-clamp technique caused the loss of a key component necessary
for PKC-dependent activation of such a current. Therefore, we conducted
a similar study using the perforated patch-clamp technique to limit
intracellular dialysis. Cells were exposed to 100 nM PDBu, which is
approximately 10 times the concentration necessary for half-maximal
binding to and activation of PKC (23) and twice the concentration
reported to effectively stimulate
Cl
channel
activity in guinea pig ventricular myocytes (5, 33). Figure 1 demonstrates that we were still
unable to detect any evidence for PKC-dependent stimulation of cCFTR
Cl
channel activity under
these conditions. Subsequent activation of the
Cl
current after exposure
to isoproterenol was used to confirm that these channels were present
in each cell tested. Identical results were obtained in three different
cells. The lack of response to PDBu cannot be due to its inability to
activate PKC because the same concentration of PDBu enhanced the
delayed rectifier K+ current and
this effect was blocked by the PKC inhibitor bisindolylmaleimide (30 nM; n = 3; data not shown). Therefore,
these results indicate that phorbol ester activation of endogenous PKC
alone does not elicit a measurable macroscopic
Cl
current conducted by
cCFTR in our cells, a conclusion consistent with our previous findings
(24). This observation is also consistent with the lack of effect that
activation of endogenous PKC alone is reported to have on epithelial
CFTR channel activity in native cells (37).

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Fig. 1.
Phorbol ester activation of endogenous protein kinase C (PKC) alone
does not produce a Cl
current in guinea pig ventricular myocytes.
A: time course of changes in membrane
current recorded during 100-ms voltage-clamp steps to +50 mV applied
once every 3 s. Exposure to 100 nM phorbol 12,13-dibutyrate (PDBu), a
phorbol ester that activates PKC, does not stimulate cardiac cystic
fibrosis transmembrane conductance regulator (cCFTR)
Cl channels, which are
clearly present, as indicated by positive response to 1 µM
isoproterenol (ISO), a specific -adrenergic receptor agonist. Dashes
indicate data that were observed but not saved.
B: membrane potential
(Vm) dependence
of difference current ( I)
obtained by subtracting currents recorded under control conditions
(a) from currents recorded in
presence of 100 nM PDBu alone (b),
in presence of 1 µM isoproterenol alone
(c), and after isoproterenol washout
(d). This experiment was conducted
using dialysis-limiting perforated patch-clamp technique.
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Effect of PDBu after PKA-dependent activation of cCFTR.
Despite the fact that activation of PKC alone has been reported to have
little or no effect on basal activity of eCFTR, it has been clearly
demonstrated that activation of PKC does have a significant effect
after the channel has been first activated by PKA (4, 19, 30). To test
the hypothesis that PKC may have a similar effect on cCFTR, we first
exposed guinea pig myocytes to 100 nM isoproterenol to stimulate cCFTR
Cl
channel activity through
the PKA-dependent pathway. Cells were then exposed to 100 nM PDBu in
the continued presence of isoproterenol. Figure
2 illustrates a representative experiment
demonstrating that, under these conditions, PDBu does exert an effect
on channel activity. The addition of PDBu did not affect the time
independence, voltage dependence, or reversal potential of the current,
consistent with the idea that PDBu had simply enhanced the magnitude of
the current already activated by isoproterenol. Furthermore, in similar experiments, the current activated by PDBu in the presence of isoproterenol was not inhibited by DIDS
(n = 3). This suggests that the
PKC-activated current was conducted by cCFTR.

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Fig. 2.
Phorbol ester activation of endogenous PKC further stimulates
Cl current after it has
been first activated by protein kinase A. A: time course of changes in membrane
current recorded during 100-ms voltage-clamp steps to +50 mV applied
once every 3 s. A supramaximally stimulating concentration of
isoproterenol (100 nM) elicits a
Cl current that is further
enhanced by subsequent addition of 100 nM PDBu.
B: membrane currents recorded at time
points in protocol illustrated in A.
Currents were elicited by 100-ms voltage-clamp steps from holding
potential of 30 mV to
Vm values between
120 and +50 mV in 10-mV increments.
C:
Vm dependence of
I obtained by subtracting currents
recorded under control conditions
(a) from currents recorded in
presence of 100 nM isoproterenol alone
(b), in presence of 100 nM
isoproterenol + 100 nM PDBu (c), and
after washout of isoproterenol and PDBu
(d).
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This stimulatory effect of PDBu was not mimicked by 4
-PDD, a phorbol
ester that does not activate PKC (Fig.
3A).
Whereas 100 nM PDBu increased the magnitude of the
Cl
current by 21 ± 3.3% (n = 9) when added subsequent to
channel activation by 100 nM isoproterenol, the magnitude of the
Cl
current actually
decreased by 6.0 ± 1.2% (n = 5)
in the presence of 100 nM 4
-PDD (Fig.
3B). This decrease is most likely
due to current rundown, a common phenomenon in which the magnitude of the current activated by an agonist diminishes with time in dialyzed cells (38). This suggests that the magnitude of the PDBu-dependent response is slightly underestimated. These data are also consistent with the idea that the PDBu-induced increase in
Cl
current magnitude is due
to activation of PKC.

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Fig. 3.
PDBu-induced increase in isoproterenol-activated
Cl current is due to
phorbol ester activation of PKC. A:
time course of changes in membrane current recorded during 100-ms
voltage-clamp steps to +50 mV applied once every 3 s; 100 nM
isoproterenol elicits a Cl
current that is not further enhanced by concurrent exposure to 100 nM
4 -phorbol 12,13-didecanoate (4 -PDD), a phorbol ester that does
not activate PKC. B: scatter plot
depicting slope conductance
(GCl) of CFTR
Cl current recorded in
presence of phorbol ester + isoproterenol normalized to that recorded
in presence of isoproterenol alone, before addition of phorbol ester.
Dashed line represents no change in
GCl on addition
of phorbol ester. Solid lines in each column indicate averages for each
group. * Statistically significant difference between PDBu and
4 -PDD responses (P < 0.0001;
unpaired t-test).
n, No. of cells.
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Having seen a response to activation of PKC in the presence of a
supramaximally stimulating concentration (100 nM) of isoproterenol but
not in its absence, we next wanted to determine whether the magnitude
of the PKC-dependent response was affected by the level of
-adrenergic receptor stimulation. Therefore, we compared the response to activation of PKC in the presence of concentrations of
isoproterenol between 1 and 30 nM. All responses were normalized to the
magnitude of current elicited by that concentration of isoproterenol
alone, in the absence of PDBu. We found that activation of PKC had no
effect in the presence of a subthreshold concentration of isoproterenol
(1 nM; n = 6), even though subsequent
exposure to 1 µM isoproterenol clearly demonstrated that cCFTR
channels were present. However, after exposure to 3, 10, and 30 nM
isoproterenol, subsequent addition of 100 nM PDBu increased the
magnitude of the Cl
current
by 17 ± 4.5 (n = 4), 31 ± 5.4 (n = 5), and 28 ± 5.6% (n = 6), respectively. The magnitudes
of these PKC-dependent responses were statistically independent of the
concentration of isoproterenol that was present. Figure
4 illustrates the fact that PDBu activation of endogenous PKC increases the magnitude of the current without affecting the threshold sensitivity of channel activation.

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Fig. 4.
Effect of activating PKC after exposure to various concentrations of
isoproterenol. Cells were first exposed to isoproterenol alone,
followed by subsequent addition of 100 nM PDBu. Magnitude of
GCl of
I activated by PDBu in presence of
isoproterenol was normalized to magnitude of
GCl activated by
that concentration of isoproterenol alone in same cell. Each cell could
be used to determine effect of PDBu in presence of only a single
concentration of isoproterenol. To illustrate effect of PDBu on
concentration-response relationship for activation of
Cl current by isoproterenol
alone, these results are plotted relative to concentration dependence
for activation of Cl
current by isoproterenol alone, which was determined in a previous
study (38). Each data point represents average of 4-9 experiments.
Solid line represents nonlinear least squares fit of data points to
equation GCl = Gmax/{1 + (EC50/[ISO])n};
maximum value of
GCl
(Gmax) is 1.2, concentration of isoproterenol at which activation of
GCl is
half-maximal (EC50) is 7.7 nM,
and slope factor (n) is 2.2. Dashed
line represents previously determined relationship between
concentration of isoproterenol ([ISO]) and relative
magnitude of GCl
activated in absence of PDBu;
Gmax is 1, EC50 is 8.3 nM, and
n is 1.9. Note that PDBu increased
relative magnitude of response to isoproterenol consistently at all
concentrations, without significantly affecting either
EC50 or minimal concentration of
isoproterenol necessary to activate current.
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Effect of PDBu on subsequent PKA-dependent activation of cCFTR.
Metabolic labeling studies have demonstrated that the order in which
eCFTR is exposed to PKA and PKC significantly affects the relative
degree of protein phosphorylation (4). It was found that more labeled
phosphate was incorporated when exposure to PKC preceded exposure to
PKA. Assuming that phosphorylation of cCFTR is also subject to this
kind of hierarchical effect, we performed experiments to determine
whether it might be reflected in a difference in the functional
response. For these experiments, cells were first exposed to PDBu alone
for 5 min, after which time the response to 100 nM isoproterenol was
measured in the continued presence of the phorbol. However, to
determine whether the response to isoproterenol had been altered by
preexposure to PDBu, it was necessary to precede this protocol with a
brief exposure to the same concentration of isoproterenol alone. In this way, the effect of PDBu on isoproterenol-activated current could
be compared with the current activated by isoproterenol alone in the
same cell. This protocol is depicted in Fig.
5A. We
found that the response to isoproterenol observed following exposure to
PDBu was significantly greater than the magnitude of the response to
isoproterenol alone. On average, the magnitude of the current
activated by isoproterenol following exposure to PDBu was 36 ± 16%
(n = 14) greater than the magnitude of
the current activated by isoproterenol alone in the same cell.
Consistent with the previous set of experiments, the current activated
by isoproterenol in the presence of PDBu exhibited the same time independence, voltage dependence, and reversal potential as the current
activated by isoproterenol alone. In addition, as expected for the
cCFTR Cl
current, this
conductance was not blocked by DIDS (n = 4).

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Fig. 5.
Isoproterenol-induced cCFTR
Cl current is enhanced when
preceded by activation of PKC. A: time
course of changes in membrane current recorded during 100-ms
voltage-clamp steps to +50 mV applied once every 3 s. Although 100 nM
PDBu alone stimulates no measurable
Cl conductance on its own,
exposure to a supramaximally stimulating concentration of isoproterenol
(100 nM) in continued presence of PDBu (100 nM) elicits more
Cl current than does 100 nM
isoproterenol alone in same cell. Increase in current is insensitive to
DIDS (500 µM). B: time course of
changes in membrane current recorded during 100-ms voltage-clamp steps
to +50 mV applied once every 3 s. Inactive phorbol ester 4 -PDD (100 nM) does not stimulate any
Cl current on its own. When
4 -PDD is followed by concurrent exposure to 100 nM isoproterenol,
resulting current is actually smaller than that elicited by 100 nM
isoproterenol alone in same cell. Dashed lines in both
A and
B indicate magnitude of current
elicited during initial exposure to isoproterenol alone.
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To verify that the increase in the magnitude of the response to
isoproterenol observed in the presence of PDBu was due to activation of
PKC, the same protocol was repeated using 4
-PDD (Fig.
5B). We found that the magnitude of
the Cl
current activated by
isoproterenol in the presence of 4
-PDD was actually 22 ± 9.7%
(n = 9) smaller than that activated by isoproterenol alone. This decrease is most likely due to current rundown. The greater degree of rundown in these experiments is probably
due to the longer duration of the protocol (38). To confirm this
conclusion, we repeated the same protocol in the absence of any phorbol
ester and found that the magnitude of the Cl
current activated by the
second exposure to isoproterenol was 11 ± 6.9%
(n = 11) smaller than that activated
by the same concentration of isoproterenol 5 min earlier. The magnitude
of this decrease is not statistically different from that observed in
the presence of 4
-PDD, consistent with the idea that the decrease
observed in the presence of this inactive phorbol ester is due to
current rundown and not a direct effect of 4
-PDD. However, the
magnitude of the response to isoproterenol following exposure to
4
-PDD (as well as that in the absence of phorbol ester) is
significantly different from the magnitude of the response to
isoproterenol observed following exposure to PDBu. These results with
4
-PDD are consistent with the idea that the effect of PDBu is due to activation of PKC. The fact that there was some rundown of the isoproterenol response in the presence of 4
-PDD indicates that the
magnitude of the PDBu-dependent response was probably slightly underestimated. The results of individual experiments are illustrated in Fig. 6.

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Fig. 6.
GCl observed
following a second exposure to isoproterenol (in presence or absence of
phorbol ester) is normalized to that observed during an initial
exposure to isoproterenol in absence of drug. Dashed line represents no
change in normalized
GCl relative to
initial exposure of isoproterenol alone. Average of all cells in each
group is illustrated by a solid line through data points. There is a
statistically significant difference between groups
(P < 0.01; one-way ANOVA).
* Results obtained in presence of PDBu are statistically
different from those obtained in presence of 4 -PDD or absence of any
phorbol ester (P < 0.05; Bonferroni
t-test).
n, No. of cells.
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As to whether the order of kinase activation affected the magnitude of
the PKC-dependent response, the effect that preactivation of PKC had on
the subsequent response to isoproterenol was not statistically
different from the magnitude of the response that acute activation of
PKC had after the current had already been activated by isoproterenol.
However, it should be noted that there was greater variability in the
response to isoproterenol following preactivation of PKC (see Fig. 6),
with the response to isoproterenol being increased by up to 150% in
some cells.
Is PKC activity obligatory for PKA-dependent stimulation of cCFTR?
In our hands, activation of endogenous PKC alone elicits no measurable
macroscopic Cl
conductance
in guinea pig ventricular myocytes (see Fig. 1). However, PKC
activation does potentiate PKA-dependent stimulation of the cCFTR
Cl
current (see Figs. 2 and
5). This raises the interesting question of whether basal PKC activity
might also affect PKA-dependent regulation of cCFTR in native cardiac
myocytes, in a manner similar to the effect that basal PKC has been
reported to have on eCFTR channel activity in heterologous expression
systems (19). To test this hypothesis, we examined the response to
isoproterenol in the presence and absence of 10 µM chelerythrine, a
highly selective inhibitor of PKC. A protocol similar to that used in
Figs. 5 and 6 was followed when investigating the effect of PKC
inhibitors. Cells were briefly exposed to 100 nM isoproterenol alone,
followed by exposure to the PKC inhibitor alone for 5 min, after which time 100 nM isoproterenol was reintroduced in the continued presence of
inhibitor. In this way, the effect of the PKC inhibitor on the
isoproterenol-activated current could be compared with the current
activated by isoproterenol alone in the same cell. This protocol is
depicted in the experiment illustrated in Fig.
7A. After
exposure to 10 µM chelerythrine, the magnitude of the
isoproterenol-stimulated current was reduced by 79 ± 7.7%
(n = 11).

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Fig. 7.
PKC inhibitors significantly attenuate activation of cCFTR by
isoproterenol. A: time course of
changes in membrane current recorded during 100-ms voltage-clamp steps
to +50 mV applied once every 3 s. Magnitude of current elicited by 100 nM isoproterenol is significantly smaller in presence of 10 µM
chelerythrine (CHEL) than it is in its absence.
B:
GCl of
isoproterenol-activated Cl
current recorded in presence of a PKC inhibitor is normalized to that
recorded in presence of isoproterenol alone, before addition of
inhibitor. BIS, bisindolylmaleimide. Dashed line at
top represents no change in normalized
GCl relative to
initial exposure of isoproterenol alone. Dashed line at
bottom represents complete abolition
of current. Average of all cells in each group is illustrated by a
solid line through data points. There is a statistically significant
difference between groups (P < 0.0001; one-way ANOVA). * Significant difference between each
inhibitor group vs. control group (P < 0.05; Bonferroni t-test).
Difference between 2 groups treated with inhibitor is not significant
(P > 0.05; Bonferroni
t-test).
n, No. of cells.
|
|
The ability of chelerythrine to inhibit isoproterenol activation of the
macroscopic Cl
current in
native cardiac myocytes is consistent with the effect that this drug
had on PKA-stimulated eCFTR channel activity (19). However, because our
experiments relied on
-adrenergic receptor stimulation of cAMP
production and because chelerythrine has been reported to increase
phosphodiesterase activity (10), we also studied the effect of another
highly selective PKC inhibitor, bisindolylmaleimide. After exposure to
30 nM bisindolylmaleimide, the magnitude of the
isoproterenol-stimulated current was reduced by 52 ± 10%
(n = 8). Although there is no
statistical difference between the magnitude of the effects of
bisindolylmaleimide and chelerythrine, both effects are significantly
different from the average results obtained in the time control
experiments (Fig. 7B). These
findings are consistent with the idea that basal PKC activity in
cardiac myocytes does modulate PKA-dependent activation of cCFTR.
 |
DISCUSSION |
PKC alone is not sufficient to activate cCFTR.
Results of the present study demonstrate that we are unable to elicit a
measurable macroscopic Cl
current from guinea pig ventricular myocytes by stimulating endogenous PKC activity. This is consistent with our previous work (24), and it is
also consistent with the effect that PKC has on eCFTR channel activity.
Although exogenous PKC alone has been reported to stimulate activity of
eCFTR in heterologous expression systems, the magnitude of such
responses is small compared with the effect that PKA has on the same
channels (3, 4, 30). Furthermore, activation of endogenous PKC alone
has been reported to have no measurable effect on eCFTR constitutively
expressed in epithelial cells (37).
The fact that we see no response when activating PKC alone would appear
to be inconsistent with the effect that others have reported in cardiac
myocytes. We can dismiss the possibility that the concentration of
cytosolic free Ca2+ in our
experiments did not permit activation of PKC. Although some isozymes of
PKC are Ca2+ dependent, they are
not prevalent in adult cardiac myocytes (25, 29). Even if they are
involved, phorbol ester activation of these isozymes does not
necessarily require Ca2+ (22).
Furthermore, we failed to see PKC-dependent activation of a
Cl
current even when we
used the perforated patch technique, which does not disturb cytosolic
Ca2+ (see Fig. 1), and when we
buffered cytosolic Ca2+ at a level
that should have permitted activation of
Ca2+-dependent isoforms of PKC
(24). Finally, we can infer that PDBu was activating PKC because we
found that it did stimulate the delayed rectifier
K+ current, an effect that was
reversed by the PKC inhibitor bisindolylmaleimide. In addition, PDBu
does affect the cCFTR Cl
current in the presence of isoproterenol, an effect that is not mimicked by the inactive phorbol 4
-PDD.
Perhaps the most plausible explanation for the discrepancy between our
results with phorbol ester alone and those of others is that basal PKA
activity is variable in different preparations. Our present work
demonstrates that PKC enhances the cCFTR
Cl
current in the presence
of submaximal PKA-dependent stimulation of channel activity (see Fig.
4). Therefore, if basal PKA activity is high enough to maintain even a
minimal degree of channel activation, then acute stimulation of PKC
would be expected to elicit a response.
PKC modifies PKA-dependent regulation of cCFTR.
Although we do not find any evidence that activation of PKC alone
stimulates the cCFTR Cl
current, activation of PKC in the presence of concurrent PKA-dependent stimulation does elicit a significant response. This suggests that PKA
and PKC are exerting their effects at distinct phosphorylation sites.
Our assumption has been that these sites are on the channel protein
itself. However, there are other potential explanations. One
possibility is that, in the presence of
-adrenergic receptor stimulation, PKC enhances cAMP production or inhibits cAMP breakdown. However, such a mechanism is not consistent with the fact that activation of PKC enhances the cCFTR
Cl
current even in the
presence of a supramaximally stimulating concentration of isoproterenol
(Fig. 2). It is also not consistent with the fact that activation of
PKC does not affect the threshold for
-adrenergic stimulation of the
current (Fig. 4).
Another possible explanation for the effects that we have observed is
that PKC is facilitating PKA-dependent channel phosphorylation by
directly stimulating PKA or by inhibiting phosphatase activity. Although we cannot rule out either of these possibilities, a simpler interpretation might be that PKC is directly phosphorylating the channel protein itself. A recent study has indicated that some eCFTR
PKA phosphorylation sites are stimulatory and others are inhibitory
(36). Therefore, it is plausible that PKC phosphorylation may have an
allosteric affect on the subsequent phosphorylation of other sites by
PKA. That is, PKC phosphorylation may facilitate PKA-dependent
phosphorylation of stimulatory sites or suppress PKA-dependent
phosphorylation of inhibitory sites. For PKC to affect channel function
in this way would suggest that it is causing a conformational change in
the tertiary structure of the protein. This question has actually been
addressed, and it was determined that PKA, but not PKC, causes a
conformational change (9). However, these studies used a construct
limited to the regulatory domain, which contains 90% of the consensus
sites for PKA phosphorylation but only 24% of those for PKC. In
addition, the impact on the protein when exposed to both kinases
concurrently was not investigated.
Is PKC obligatory for cCFTR activation?
An important aspect of the role of PKC in regulating cCFTR activity
relates to the question of whether PKC is mandatory for channel
activation, as has been suggested for eCFTR (19). We have found that
the PKC inhibitors chelerythrine and bisindolylmaleimide both attenuate
isoproterenol-dependent activation of cCFTR currents, which is
consistent with the idea that basal PKC activity plays an important
role in regulating PKA-dependent responses. However, this
interpretation assumes that chelerythrine and bisindolylmaleimide do
not directly inhibit PKA activity. These two compounds were chosen on
the basis of their selectivity for inhibition of PKC. Both are 200 times more potent as inhibitors of PKC than as inhibitors of PKA. The
concentration of chelerythrine that causes 50% inhibition (IC50) of PKC is 0.7 µM,
whereas the IC50 for PKA is 170 µM (15). For bisindolylmaleimide, the
IC50 for PKC is 0.01 µM, whereas the IC50 for PKA is 2 µM (31).
In our experiments, we used concentrations that were 17 and 67 times
less than the IC50 values for
inhibition of PKA, respectively.
Other alternative explanations for the apparent inhibitory effects of
chelerythrine and bisindolylmaleimide must also be considered. For
example, it is possible that these compounds can directly block the
channel pore. Although there is no specific information about PKC
inhibitors directly blocking CFTR
Cl
channels, it should be
noted that bisindolylmaleimide does not exert such an effect on other
cardiac Cl
channels (8).
Another possibility is that PKC inhibitors interfere with ATP
interactions at one or both of the nucleotide binding domains (NBDs)
that are essential for the activity of this channel. This could be a
concern for bisindolylmaleimide, which acts as a competitive inhibitor
at the ATP binding site of PKC (31). However, ATP binding and
hydrolysis are believed to play a unique functional role at each of the
NBDs: one NBD is responsible for channel opening and the other effects
channel closing (1). Therefore, if bisindolylmaleimide were to
interfere with ATP interactions at the NBDs, it might be difficult to
predict what the net effect would be. A nonspecific interaction at an
NDB is not likely to explain the effect of chelerythrine because this
compound inhibits PKC activity independent of ATP binding (15).
Our working hypothesis has been that the mechanism underlying the
reduced responsiveness of isoproterenol in the presence of PKC
inhibitors and that responsible for the phorbol ester-induced increase
in the magnitude of PKA-dependent channel activity are one and the
same. Still, a possible interpretation of why PKC inhibitors attenuate
the isoproterenol response is that PKC affects cAMP levels, either by
stimulating cAMP production or by preventing its degradation. However,
PKC does not affect the isoforms of adenylate cyclase present in
cardiac muscle (17), and, if anything, PKC has been suggested to
stimulate phosphodiesterase activity in adult ventricular myocytes
(27). In any event, regardless of how PKC may modulate channel
activity, the fact remains that the presence of PKC inhibitors
significantly affects the overall cellular response to PKA-dependent
activation of the channel.
Although the PKC inhibitors significantly reduced the responsiveness of
the cells to isoproterenol, we cannot say that PKC activity was
completely inhibited. Therefore, we cannot conclude whether PKC is
absolutely obligatory for PKA-dependent activation of cCFTR. We can,
however, comment on the difference in the extent of attenuation seen in
the presence of bisindolylmaleimide and chelerythrine. One possible
explanation is that we used a concentration of chelerythrine that was
14 times the IC50 for inhibition
of PKC but a concentration of bisindolylmaleimide that was only 3 times
the IC50 for inhibition of PKC.
Another possibility is related to differences in the ability of each
compound to inhibit specific isozymes of PKC. Bisindolylmaleimide has
been shown to be less effective at inhibiting PKC
(21), the
predominant isoform in adult ventricular tissue (25, 29). However,
chelerythrine, at the same concentration and time of exposure that we
used, has been shown to completely abolish effects attributed to PKC
in cardiac myocytes (20). Therefore, if indeed the PDBu-induced potentiation of isoproterenol-stimulated
Cl
current is due to
activation of PKC
, we might expect chelerythrine to more effectively
inhibit the response.
Although we cannot conclude that PKC is absolutely mandatory for
channel activation by PKA, the fact that both inhibitors significantly
reduce the ability of isoproterenol to activate CFTR
Cl
current in these cells
suggests an underlying role for basal phosphorylation by PKC in order
for PKA to effectively activate the channel. Further studies
characterizing the distribution, function, and inhibitor profiles of
the PKC isozymes, as well as those investigating the point in the
signaling pathway affected by PKC, may clarify the regulatory role PKC
plays in modulating cCFTR activity.
In conclusion, besides suggesting that PKC regulates cCFTR and eCFTR in
a similar manner, our results help clarify the role that PKC plays in
regulating the activity of CFTR in cardiac myocytes. We show that,
although acute activation of PKC alone does not have a significant
effect, endogenous PKC activity does potentiate PKA-dependent
responses. Furthermore, our data are consistent with the idea that
perhaps the most important role of PKC lies in its basal activity,
which regulates subsequent PKA-dependent stimulation of cCFTR
Cl
channel function.
Therefore, the data presented here provide a functional example of a
kinase cross talk mechanism in which cellular responses are modulated
by the activity of different kinases.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Mitch Drumm for reading and critiquing the manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-45141, an American Heart Association Established Investigatorship, and a grant-in-aid from the Northeast Ohio Affiliate of the American Heart Association.
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. D. Harvey, Dept. of Physiology and
Biophysics, Case Western Reserve University, 2109 Adelbert Rd.,
Cleveland, OH 44106-4970.
Received 9 February 1998; accepted in final form 14 April 1998.
 |
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