1Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120; and 2Department of Physiology and Cell Biology/MS352, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Zhang, Yi-Hong,
J. L. Kenyon, and
G. D. Nicol.
Phorbol Ester-Induced Inhibition of Potassium Currents in Rat
Sensory Neurons Requires Voltage-Dependent Entry of Calcium.
J. Neurophysiol. 85: 362-373, 2001.
The whole
cell patch-clamp technique was used to examine the effects of protein
kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents
(IK) in cultured capsaicin-sensitive
neurons isolated from dorsal root ganglia from embryonic rat pups and
grown in culture. PDBu, in a concentration- and time-dependent manner, reduced IK measured at +60 mV by
~30% if the holding potential (Vh)
was 20 or
47 mV but had no effect if
Vh was
80 mV. The PDBu-induced
inhibition of IK was blocked by
pretreatment with the PKC inhibitor bisindolylmaleimide I and
IK was unaffected by 4-
phorbol,
indicating that the suppression of IK
was mediated by PKC. The inhibition of
IK by 100 nM PDBu at a
Vh of
50 mV was reversed over
several minutes if Vh was changed to
80 mV. In addition, intracellular perfusion with 5 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA) or pretreatment with
-conotoxin GVIA or
Cd2+-Ringer, but not nifedipine, prevented the
PDBu-induced suppression of IK at
50
mV, suggesting that a voltage-dependent influx of calcium through
N-type calcium channels was necessary for the activation of PKC. The
potassium channel blockers tetraethylammonium (TEA, 10 mM) and
4-aminopyridine (4-AP, 3 mM and 30 µM) reduced IK, but only TEA attenuated the
ability of PDBu to further inhibit the current, suggesting that the
IK modified by PDBu was sensitive to
TEA. Interestingly, in the presence of 3 mM or 30 µM 4-AP, 100 nM
PDBu inhibited IK when
Vh was
80 mV. Thus 4-AP promotes the
capacity of PDBu to reduce IK at
80
mV. We find that activation of PKC inhibits
IK in rat sensory neurons and that
voltage-dependent calcium entry is necessary for the development and
maintenance of this inhibition.
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INTRODUCTION |
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To understand the regulation of
the sensitivity of sensory neurons, we have investigated the signaling
pathways activated by sensitizing agents. Although recent work has
focused on mechanisms activated by cAMP and protein kinase A
(Cui and Nicol 1995; Evans et al. 1999
),
other pathways can mediate the physiological responses to sensitizing
agents by mechanisms that are only poorly understood. For example,
bradykinin, a potent inflammatory mediator, produces an inward current
in C-type sensory neurons (nonmyelinated slowly conducting axons)
through the activation of protein kinase C (PKC) (Burgess et al. 1989
). In addition,
Schepelmann et al. (1993)
demonstrated that phorbol
ester (an activator of PKC) (Mellor and Parker 1998
;
Nishizuka 1984
, 1986
) directly stimulated as well as
sensitized the excitation of primary afferents in the knee joint to
mechanical manipulation. This enhancement of neuronal activity would
suggest that the release of neurotransmitter from sensory neurons might
be facilitated. Indeed the release of substance P and calcitonin
gene-related peptide from isolated sensory neurons grown in culture was
augmented by pretreatment with the phorbol ester, phorbol 12,13 dibutyrate (PDBu), and this enhancement was blocked by inhibition of
PKC by staurosporine (Barber and Vasko 1996
). These
results suggest that activation of PKC can modulate the sensitivity of
sensory neurons to stimulation. Such observations raise the question as
to the nature of the cellular mechanisms whereby PDBu-induced
activation of PKC gives rise to the enhanced excitability of sensory neurons.
Previous work has implicated several mechanisms by which the activation
of PKC can facilitate membrane excitability. In hippocampal slices,
exposure to phorbol 12,13-diacetate increased the number of action
potentials evoked by a depolarizing current and also suppressed the
slow afterhyperpolarization, suggesting that this phorbol ester
inhibited potassium currents in these CA1 neurons (Baraban et
al. 1985). Also treatment with phorbol esters led to a
broadening of the action potential in mouse sensory neurons (Werz and Macdonald 1987
). However, the actions of PKC
and its possible modulation of the isolated
IK in mammalian sensory neurons have
not been examined. We have shown previously that the excitability of
embryonic rat sensory neurons was enhanced by prostaglandin E2, through activation of the cyclic AMP
signaling pathway, and that this involved the suppression of a delayed
rectifier-like IK (Evans et al.
1999
; Nicol et al. 1997
). Because phorbol esters facilitate both the activation of articular primary afferents (Schepelmann et al. 1993
) and the release of
neuropeptides (Barber and Vasko 1996
), we examined the
possibility that the activation of PKC, similar to the activation of
protein kinase A (PKA), inhibits IK.
Also we investigated the mechanism of action for PKC wherein we find a
novel requirement for voltage-gated Ca2+ channel
activity. Preliminary results have appeared in abstract form
(Zhang and Nicol 1998
).
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METHODS |
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Isolation and culture of embryonic rat sensory neurons
The procedures for isolation and culture of rat sensory neurons
have been described previously (Vasko et al. 1994).
Briefly, timed-pregnant rats were rendered unconscious with
CO2 and killed by cervical dislocation. The
dorsal root ganglia were dissected from each embryo (E15-E17), and
sensory neurons were dissociated from the ganglia with 0.025% trypsin
(37°C, 25 min) and mechanical agitation. The cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Grand Island,
NY) supplemented with 2 mM glutamine, 50 µg/ml penicillin and
streptomycin, 10% (vol/vol) heat-inactivated fetal bovine serum, 50 µM 5-fluoro-2'-deoxyuridine, 150 µM uridine, and 250 ng/ml 7S-nerve
growth factor (Harlan Bioproducts for Science, Indianapolis, IN).
Approximately 150,000 cells/ml were plated in a collagen-coated culture
dish containing small plastic cover slips. Cultures were maintained at
37°C in a 5% CO2 atmosphere, and the medium
was changed every second day. All procedures have been approved by the
Animal Use and Care Committee of the Indiana University School of Medicine.
Recording procedures
Recordings were made using the whole cell patch-clamp technique
as previously described (Evans et al. 1999;
Hamill et al. 1981
; Nicol et al. 1997
).
Briefly, a cover slip with the sensory neurons (4-6 days in culture)
was placed in a recording chamber where the neurons were bathed in
normal Ringer solution of the following composition (in mM): 140 NaCl,
5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH at 7.4 with NaOH. Recording pipettes typically had resistances of 2-5 M
when filled with the following solution (in mM): 140 KCl, 5 MgCl2, 4 ATP, 0.3 GTP, 2.5 CaCl2, 5 EGTA (calculated free
Ca2+ concentration of ~100 nM), and 10 HEPES,
at pH 7.2 with KOH. We have not corrected for the junction potential of
3.7 mV (Barry 1994
) and expect that the actual membrane
potentials are 3-4 mV more negative than those listed. In experiments
using bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, tetrapotassium salt), the concentration of KCl was lowered accordingly.
To isolate IK, the cells were
superfused with 140 mM N-methyl-glucamine chloride Ringer
(NMG, an equimolar substitution for NaCl), pH 7.4 with KOH. We found
that superfusion with NMG significantly depolarized the resting
membrane potential in sensory neurons to 47 ± 0.3 (SE)
mV from a control level of
56 ± 0.5 mV (n = 59)
in normal Ringer solution. The membrane voltage was held at three
different Vhs to ascertain the
contribution of various potassium channel populations to the total
IK. Initially the voltage was held at
the zero-current potential for that particular neuron. A voltage-step
protocol was used to examine the activation of IK, wherein voltage steps of 300 ms
were applied at 5-s intervals in +10 mV increments to a maximum of +60
mV. Vh was then changed to
80 mV,
and the voltage-step protocol was repeated; this sequence was then
repeated for a Vh of
20 mV. In some
cases, recordings at each holding potential were performed in different neurons.
Whole cell currents were recorded from sensory neurons with an Axopatch
200 (Axon Instruments, Foster City, CA) patch-clamp amplifier; the data
were acquired and analyzed using pCLAMP6 (Axon Instruments). Both
capacitance and series resistance compensation were used; however, no
compensation was made for leak currents. The average uncompensated
series resistance was 3.5 ± 0.1 M (range 2.3-5.3 M
,
n = 44). The maximum voltage error resulting from the
uncompensated series resistance was calculated for each cell and
averaged 7.4 ± 0.4 mV (n = 44). Cells were
superfused continuously during exposure to the various test compounds.
In experiments using 10 mM TEA, the extracellular NMG concentration in
the Ringer solution was reduced to 130 mM. In contrast, 4-aminopyridine
(4-AP) was added directly to the Ringer solution. These agents were
bath applied for 3-4 min prior to the acquisition of additional
current recordings. The results presented in this report were obtained from neurons that were sensitive to capsaicin (see criteria described in Evans et al. 1999
; Holzer 1991
). All
experiments were performed at room temperature (~23°C).
Data analysis
All values represent the means ± SE . The voltage
dependence for activation of the outward
IK was fitted with the Boltzmann relation (see Evans et al. 1999). Statistical
differences between the control currents and those obtained under
various treatment conditions were determined by using a
repeated-measures ANOVA (RM ANOVA) with a Tukey post hoc test; for the
comparison of control to a single drug treatment, a Student's paired
t-test was used. Values of P < 0.05 were
judged to be statistically significant.
Chemicals
All chemicals were obtained from Sigma Chemical (St. Louis, MO).
PDBu, 4 phorbol, and capsaicin were dissolved in
1-methyl-2-pyrrolidinone (HPLC grade) to obtain concentrated stock
solutions, which were then diluted with Ringer solution to the
appropriate concentration. We have demonstrated previously that the
vehicle, 1-methyl-2-pyrrolidinone, had no effect on either the
activation or steady-state inactivation curves obtained for potassium
currents (Nicol et al. 1997
).
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RESULTS |
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Ability of PDBu to inhibit IK depends on the holding potential
To investigate the role of PKC in the modulation of
IK, sensory neurons were exposed to
the phorbol ester, PDBu. Previous studies demonstrated that different
populations of potassium currents can be isolated by using different
Vhs to inactivate specific types of
currents (e.g., Belluzzi et al. 1985a,b
). Using this approach, sensory neurons were held at different
Vhs and then treated with PDBu to
determine the possible actions of PKC on the activation of specific
types of IK. We chose three different Vhs:
20 mV was used to isolate the
delayed rectifier-like IK, the resting
potential of the neuron was used to observe the effects of PDBu under
physiological conditions, and
80 mV was used to examine both the
delayed rectifier-like IK and any
rapidly-inactivating type IK (e.g.,
IA-like currents). As shown in Fig.
1, depolarizing voltage steps from a
Vh of
20 mV (A) elicited
a series of IK that exhibited little
time-dependent inactivation, whereas when the neuron was held at more
hyperpolarized levels of Vh,
IK showed greater extents of
inactivation during test depolarizations (B and
C). These results suggest that when
Vh was held at
20 mV, the elicited
IK consisted primarily of a
delayed-rectifier like current. The application of 100 nM PDBu to
sensory neurons caused a time-dependent suppression of
IK, when
Vh was either
47 (the resting or
0-current potential, Fig. 1B) or
20 mV (Fig.
1A). When held at
47 mV, PDBu reduced
IK in this particular neuron (measured
at +60 mV) from a peak value of 2.52 to 1.77 nA (a 30% reduction). A
similar result was obtained in a different neuron held at
20 mV (0.81 to 0.54 nA). In contrast, when a different neuron was held at a
Vh of
80 mV, the elicited
IK was unaffected by PDBu (Fig.
1C).
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The actions of 100 nM PDBu on IK are
summarized in Fig. 2. As shown in Fig.
2A, after a 20-min exposure to PDBu, outward currents obtained at +60 mV (Vh,20 mV) were
reduced significantly (Student's paired t-test) from a
control value of 0.69 ± 0.1 to 0.48 ± 0.06 nA
(n = 7; also see Fig. 4, 100 nM PDBu). This inhibition
developed slowly; IK was lowered to
0.61 ± 0.08 and 0.55 ± 0.07 nA after 2- and 10-min
applications, respectively (data not shown). In cells held at
20 mV,
PDBu significantly reduced IK recorded
at potentials positive to
50 mV (Student's paired
t-test), and we attribute this effect to the inhibition of
membrane potassium conductance described in the following text. In
addition, measurements of membrane resistance (between the
60- and
40-mV steps) indicate that after a 20-min exposure to 100 nM PDBu the
input resistance was increased significantly to 416 ± 51 M
(n = 7, paired t-test) from a control value
of 318 ± 39 M
. Similarly, when
Vh was held at the resting potential
(which averaged
47 ± 0.6 mV, n = 11), PDBu
significantly suppressed IK in a
time-dependent manner (see Fig. 2B) for voltage steps
positive to
40 mV. This suppression was observed in all 11 neurons
examined and ranged from 16 to 45% inhibition. Also the resistance
measured between the
80- and
40-mV steps under control conditions
was 350 ± 36 M
(n = 11), whereas after a
20-min exposure to PDBu, the resistance increased significantly to
421 ± 47 M
(paired t-test). To determine if PDBu
altered the voltage dependence of activation of
IK, the conductance-voltage relationship was examined and is shown in Fig. 2C. These
average results were fit with the Boltzmann relation. When neurons were held at a Vh of
47 mV, only the
value of G/Gmax was reduced
significantly after PDBu treatment for voltage steps positive to
30
mV (at +60 mV the value was decreased to 0.71 ± 0.03), whereas
V0.5 (control
3 mV versus PDBu
6
mV) and k (control and PDBu both 14 mV) were unaffected.
However, in recordings from four different neurons, IK was unaffected by PDBu when the
neurons were held at
80 mV (see Figs. 2D and 4). The
conductance-voltage relationship is shown in Fig. 2E wherein
the Boltzmann fitting parameters were not altered significantly by PDBu
(G/Gmax at +60 mV was
0.95 ± 0.03); V0.5 (control
4
mV versus PDBu
6 mV) and k (control and PDBu both 14 mV)
were nearly identical to that obtained at
47 mV.
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The component of total IK that was
inhibited by PDBu was determined by subtracting the currents remaining
after 20 min in 100 nM PDBu from their respective control traces; these
results are shown in Fig. 3. Figure
3A, left, illustrates the PDBu-sensitive IK obtained from a representative
neuron held at 47 mV; this current exhibited little time-dependent
inactivation. Figure 3A, middle, shows the
average current-voltage relation for the PDBu-sensitive IK obtained from 11 neurons.
Activation of IK begins at
around
20 mV, and the peak current obtained at +60 mV had a mean
value of 0.71 ± 0.12 nA. Figure 3A, right,
demonstrates the conductance-voltage relation with the Boltzmann fit
wherein V0.5 was 4 mV and k
was 12 mV. The panels in Fig. 3B represent the
PDBu-sensitive IK obtained at a
Vh of
20 mV. The current traces from
a representative neuron exhibit no inactivation; the average peak
current obtained at +60 mV was 0.21 ± 0.05 nA (n = 7). These results suggest that the PDBu-sensitive current exhibited
the properties characteristic of a delayed rectifier-like
IK.
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The concentration dependence for the inhibition of
IK also was examined; these results
are summarized in Fig. 4. At a
concentration of 1 nM, PDBu had no effect on
IK, whereas 10 nM PDBu inhibited IK by 21 ± 2 and 15 ± 3%
when neurons were held at 20 and
48 mV, respectively. As in the
preceding text, 10 nM PDBu was without effect at
80 mV. These results
indicate that the inhibition of IK by
PDBu depends on concentration, time, and the holding voltage. In
addition, this suppression does not result from a shift in the
voltage-dependence of activation.
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The possibility that the inhibitory actions of PDBu were mediated by
the activation of PKC rather than some nonspecific effect of the
phorbol was examined by using an inhibitor of PKC as well as an
inactive analog of the phorbol. Sensory neurons were pretreated with an
inhibitor of PKC, bisindolylmaleimide I (BIM) (Toullec et al.
1991), to ascertain whether the PDBu-induced inhibition of
IK was prevented by inactivation of
PKC. As shown in Fig. 5, the inhibition
of IK produced by 100 nM PDBu at both
20 and
45 mV was blocked by pretreatment with 1 µM BIM. However,
IK obtained for voltage steps to +50
and +60 mV for the Vh of
45 mV (see Fig. 5B) was reduced by a small although significant amount
after a 20-min exposure to PDBu in the presence of BIM. For example, the step to +60 mV elicited an average
IK of 1.84 ± 0.18 nA after a
10-min treatment with 1 µM BIM, whereas after a 20-min exposure to
100 nM PDBu in the presence of BIM, IK
was reduced significantly to 1.62 ± 0.20 nA (n = 3). After treatment with PDBu in BIM, the fraction of current remaining
at +60 mV was (0.88 ± 0.02 vs. 0.71 ± 0.03, n = 3). After treatment with PDBu in BIM, the fraction of the current remaining at +60 mV (0.88 ± 0.02, n = 3) was significantly larger than that remaining in
the absence of BIM (0.71 ± 0.03, n = 11). As
described in the preceding text, neither BIM nor PDBu had any effect at
a Vh of
80 mV (Fig. 5C).
Exposure to the inactive analogue of PDBu, 4
-phorbol, failed to
alter IK over a 20-min time course at
any of the three Vhs (data not shown).
For example, at Vhs of
50 and
20
mV, IK was 2.36 ± 0.39 and
0.49 ± 0.12 nA (+60-mV step), respectively, after a 20-min
treatment with 100 nM 4
-phorbol compared with a values of 2.44 ± 0.47 and 0.59 ± 0.16 nA (n = 4) for the
control condition. Therefore these findings suggest that the inhibition
of IK produced by PDBu results from the direct activation of PKC rather than by a nonspecific mechanism.
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Voltage and calcium dependence of the PDBu-induced inhibition
The inability of PDBu to suppress
IK elicited from 80 mV was
unexpected and we examined the voltage dependence of PDBu action using
the protocol summarized in Fig.
6A.
IK was activated by depolarizations to
+60 mV. The holding potential was set initially to
50 mV, shifted to
80 mV, and returned to
50 mV. Hyperpolarization increased the
amplitude of IK (in this cell from
2.89 to 3.61 nA) due to the removal of inactivation. With
Vh set to
50 mV, addition of 100 nM
PDBu caused a slowly developing inhibition of
IK. After a 20-min exposure to PDBu,
shifting Vh to
80 mV caused
IK to increase in amplitude until it
equaled that elicited by depolarization in the control condition. Thus
hyperpolarization reversed the inhibition of
IK caused by PDBu at a
Vh of
50 mV. This recovery from
inhibition was fully reversed when Vh
was again set to
50 mV as IK
declined to 0.71 ± 0.01 of its control value (Fig.
6A). In three cells studied using this protocol, a 20-min exposure to 100 nM PDBu significantly reduced
IK to 0.80 ± 0.02 of control
value (paired t-test) when
Vh was
50 mV (Fig. 6C). Shifting Vh to
80 mV resulted in the
recovery of IK to control values over
~6-8 min, i.e., currents elicited from
80 mV in PDBu were not
significantly different from those in control (0.96 ± 0.06 of the
control, paired t-test, see Fig. 6D). Thus at a
Vh of
50 mV, PDBu caused an
inhibition of IK that developed over tens of minutes but could be reversed and reinstated relatively rapidly
(10 min). A plausible explanation for this is that the slow development
of inhibition reflects the PDBu-induced translocation of PKC to the
plasma membrane and assembly of the complexes necessary for activation
of the enzyme (Bazzi and Nelsestuen 1987
;
Kishimoto et al. 1980
; Mosior and Epand
1994
; Mosior and Newton 1995
). Once in place,
PKC can be activated and deactivated quickly by changes in calcium
activity resulting from the opening and closing of nearby
voltage-dependent calcium channels (see following text). In this
scheme, the rapid reversal and return of inhibition reflects the time
course of phosphorylation/dephosphorylation of the PKC substrate
(possibly the IK channels themselves)
mediated by PKC and local phosphatases.
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The mechanism underlying the voltage dependence of the inhibition of
IK is unlikely to be a direct
interaction between membrane potential and PKC. A plausible mechanism
is a depolarization-induced calcium influx at 50 mV that raises
intracellular calcium
([Ca2+]i) thereby
enhancing the activity of PKC. Although the inclusion of 5 mM EGTA in
our standard pipette solution will control bulk [Ca2+]i, the relatively
slow ON rate for calcium binding by this buffer will allow
significant changes in [Ca2+]i
near active calcium channels (Stern
1992
). Accordingly, we investigated the idea that calcium
influx via voltage-gated calcium channels is necessary for the
inhibition of IK by PDBu. We first dialyzed neurons with 5 mM BAPTA (with no added calcium) instead of 5 mM EGTA. Although the affinity for calcium is nearly the same for these
two buffers, the association rate constant for BAPTA is 400-1,000
times than that for EGTA (Adler et al. 1991
; Stern 1992
). The faster ON rate for calcium
binding by BAPTA would control
[Ca2+]i much closer
to the calcium channels (Stern 1992
; also see
DISCUSSION), suggesting that BAPTA might inhibit activation
of PKC that was dependent on calcium influx. BAPTA was allowed to
perfuse intracellularly for 5 min before any recordings were attempted.
The magnitude of IK recorded in cells
dialyzed with BAPTA was not significantly different from that in cells
dialyzed with EGTA (data not shown). However, in neurons dialyzed with
BAPTA, IK was unaffected by a 20-min
exposure to PDBu whether the cells were held at
50 mV (0.99 ± 0.03 of control, n = 4, Fig. 6C) or
80 mV
(1.01 ± 0.03 of control, Fig. 6D). Thus inhibition of
IK by PDBu was abolished by the
addition of a rapid intracellular calcium buffer.
The results obtained with BAPTA imply that an increase in
[Ca2+]i is critical
in the PKC-induced inhibition of IK.
We next tested the hypothesis that calcium influx via voltage-gated
calcium channels was necessary for the inhibition of
IK by the activation of PKC by using
agents that inhibit identified components of calcium influx. We first
examined the responses of five neurons pretreated with 200 µM
CdCl2, a nonselective inhibitor of voltage-gated
Ca2+ channels (Kostyuk and Krishtal
1977; Lansman et al. 1986
; Swandulla and
Armstrong 1989
). Cd2+ did not have a
significant effect on the amplitude of
IK (data not shown). Furthermore
in the presence of Cd2+,
IK was unaffected by exposure to 100 nM PDBu for 20 min whether the cells were held at
50 mV (0.96 ± 0.03 of control, Fig. 6C) or
80 mV (1.04 ± 0.05 of
control, Fig. 6D). Thus calcium influx via voltage-gated
Ca2+ channels was necessary for the inhibition of
IK by the activation of PKC.
Evans et al. (1996) showed that 20-30% of the total
calcium current in rat sensory neurons was mediated by L-type
Ca2+ channels that were inhibited by 1 µM
nifedipine, whereas ~70% was mediated by N-type
Ca2+ channels that were inhibited by 100 nM
-conotoxin GVIA (CTx). Accordingly, we examined the effect of PDBu
on IK in the presence of nifedipine or
CTx using the voltage protocol of Fig. 6A to identify the
calcium channels that lead to the activation of PKC. Nifedipine (1 µM) did not effect the peak IK
elicited from either
50 or
80 mV (data not shown). In the presence
of nifedipine at a Vh of
50 mV, a
20-min exposure 100 nM PDBu significantly reduced
IK (0.87 ± 0.02 of control,
n = 3, Fig. 6C). The inhibition by PDBu in
the presence of nifedipine was not significantly different from the
inhibition observed in our standard conditions. Last, at a
Vh of
80 mV, PDBu failed to reduce
IK in the presence of nifedipine and
was similar to results obtained in our standard conditions (Fig.
6D). Thus calcium influx via L-type
Ca2+ channels was not necessary for the
inhibition of IK by the activation of PKC.
In contrast, treatment with 300 nM CTx prevented the inhibition of
IK by PDBu. Results from a
representative sensory neuron are shown in Fig. 6B. With
Vh set to 50 mV, the peak
IK was unaffected by a ~3-min
exposure to CTx. In the presence of CTx, a 20-min exposure to 100 nM
PDBu had no effect on the amplitude of
IK. Similarly, CTx or PDBu did not
affect IK when
Vh was
80 mV. In five neurons
studied in the presence of CTx, IK was
unaffected by exposure to 100 nM PDBu for 20 min whether the cells
were held at
50 mV (0.97 ± 0.01 of control, Fig. 6C)
or
80 mV (1.05 ± 0.03 of control, Fig. 6D). These
data, in combination with the results obtained with BAPTA, indicate
that calcium influx via N-type Ca2+ channels was
necessary for the inhibition of IK by
the activation of PKC.
Pharmacological characterization of the PDBu-suppressed IK
The inhibitors, TEA and 4-AP, were used to examine the nature of
the potassium currents that were modulated by PDBu (Thompson 1977). The different holding voltages were used in combination with the pharmacological agents to isolate delayed rectifier-like currents from rapidly inactivating currents.
Component of IK inhibited by activation of PKC is blocked by TEA
The inhibition of IK by PDBu was
limited to ~30% of the total current, suggesting that specific
components of IK may be subject to
regulation by PKC. To investigate the nature of these components, we
examined the effects of PDBu in the presence of specific
K+ channel blockers. Figure
7 shows
IK elicited from a representative neuron at Vhs of 20,
47, and
80
mV under control conditions (left) and in the presence of 10 mM TEA, an inhibitor of delayed rectifier K+
channels (middle). At each of the holding potentials, TEA
significantly reduced IK at test
potentials positive to
20 mV (RM ANOVA). The extent of inhibition at
+60 mV was 62 ± 4% when Vh was
20 mV, 70 ± 4% at a Vh of
47 mV, and 50 ± 6% at a Vh of
80 mV (n = 6). Figure 7, right, shows the
time course of this TEA-sensitive current obtained by subtracting the
TEA traces from their respective control traces. The
TEA-sensitive currents activated rapidly and showed little
inactivation during the depolarizing steps, properties typical of
delayed rectifier-like IK.
|
We next examined the sensitivity of the components of
IK remaining in the presence of 10 mM
TEA to activation of PKC by a 20-min exposure to 100 nM PDBu. Results
obtained from six neurons are summarized in Fig.
8 and Table
1. At a
Vh of 20 mV, addition of 10 mM TEA
inhibited IK at test potentials
positive to
20 mV (see Fig. 8A, left). In the
presence of TEA, addition of PDBu did not cause a significant further
reduction in IK. However, to reduce
the variability in the current measurements with these different
pharmacological treatments, the values of
IK were normalized to the peak current
obtained at +60 mV under control conditions (see Fig. 8A,
right). In the presence of TEA, PDBu caused a small but
significant reduction in the normalized values of
IK only for voltages positive to +40
mV (RM ANOVA). When held at either
47 (see Fig. 8B) or
80 mV (see Fig. 8C) in the presence of TEA, PDBu produced
no further inhibition of IK (see Table
1). Thus at each of the Vhs tested,
addition of PDBu did not significantly reduce
IK in the presence of 10 mM TEA
(n = 6, RM ANOVA). This implies that TEA and activation
of PKC inhibited current flow through the same population of
K+ channels, a conclusion that is supported by
the similar time- and voltage dependencies of the PDBu-sensitive
currents (Fig. 3) and the TEA-sensitive current (Fig. 8).
|
|
Component of IK inhibited by activation of PKC is not blocked by 3 mM 4-AP
A number of voltage-gated K+ channels are
inhibited by 4-AP with individual channels showing characteristic
sensitivities to this compound (see reviews by Chandy and Gutman
1995; Mathie et al. 1998
). Accordingly, we
investigated the relationship between the PDBu-sensitive component of
IK and currents inhibited by
relatively high (3 mM) and low (30 µM) concentrations of 4-AP. The
effects of 3 mM 4-AP for the three different
Vhs, are illustrated for a
representative neuron in Fig. 9.
IK was elicited from holding potentials of
20,
47, and
80 mV under control conditions
(left) and in the presence of 3 mM 4-AP (middle).
4-AP had little effect on IK elicited
from a Vh of
20 mV but significantly
inhibited IK elicited from more
negative holding potentials (see following text). The 4-AP-sensitive
difference currents obtained at each holding potential are shown in
Fig. 9, right. Interestingly, the time course of these
currents did not show a marked inactivation associated with
IA-like K+ currents
(Connor and Stevens 1971
; Neher 1971
),
indicating that 3 mM 4-AP inhibits more slowly inactivating components
of IK in these sensory neurons.
|
We next examined the sensitivity of the components of
IK remaining in the presence of 3 mM
4-AP to activation of PKC by a 20-min exposure to 100 nM PDBu. Results
obtained from 10 neurons are summarized in Fig.
10 and Table 1. When held at 20 mV,
4-AP produced a small although significant reduction in
IK elicited for only those voltage
steps between
40 and 0 mV (Fig. 10A, left, RM
ANOVA). For more depolarized steps, 4-AP had no significant effect; for
example, at +60 mV the current remaining was 95 ± 6%
(n = 10) of the control. In contrast, 4-AP
significantly decreased IK by 44 ± 4% when neurons were held at
46 mV (n = 10, Fig.
10B, significant for voltages positive to
40 mV, RM ANOVA)
and by 48 ± 2% at
80 mV (n = 9, Fig.
10C, significant for voltages positive to
40 mV, RM
ANOVA).
|
The addition of 100 nM PDBu in the presence of 4-AP, at a
Vh of 20 mV, reduced
IK by an amount comparable to that
observed in the absence of 4-AP (see Table 1). For both the absolute
(Fig. 10, left) and normalized currents (right),
PDBu significantly inhibited IK in the
presence of 4-AP for voltages positive to
30 mV (RM ANOVA). At
Vhs of
46 and
80 mV, PDBu, in the
presence of 4-AP, caused a modest but significant reduction in the
normalized values of IK (see Fig. 10,
B and C, right, Table 1). For these
Vhs, PDBu significantly inhibited
IK for voltages positive to +20 mV (RM ANOVA). Thus in contrast to TEA, we find little overlap between the
PDBu-sensitive component of IK and the
component inhibited by 3 mM 4-AP.
4-AP (30 µM) permits inhibition of IK by PDBu at 80
mV
We examined if the capacity of PDBu to inhibit
IK was modified by a lower
concentration of 4-AP. The results obtained with 100 nM PDBu in the
absence and presence of 30 µM 4-AP are summarized in Fig.
11 and Table 1. At a
Vh of 20 mV, 30 µM 4-AP
significantly reduced IK for voltages
positive to
20 mV (RM ANOVA). At +60 mV this amounted to a
suppression of only 9 ± 2% (n = 10, see Fig.
11A). At
47 mV, 4-AP significantly decreased
IK for all voltages positive to
40
mV and this corresponded to a reduction of 14 ± 1% at +60 mV
(n = 10, see Fig. 11B). At a
Vh of
80 mV, 30 µM 4-AP
significantly suppressed IK (between
40 and +60 mV, Fig. 11C); at +60 mV,
IK was inhibited by 16 ± 3%
(n = 10).
|
Exposure to 100 nM PDBu in the presence of 30 µM 4-AP further
suppressed IK by a similar amount at
all Vhs. At a
Vh of 20 mV, the PDBu-induced
inhibition of IK was 17 ± 3%
(see Fig. 11A and Table 1) and was similar to that observed
with 3 mM 4-AP. The normalized currents are shown in Fig.
11A (right) wherein PDBu significantly suppressed
IK for values positive to
40 mV (RM ANOVA). The results obtained with PDBu at
47 mV were quite similar to
those obtained at
20 mV (see Fig. 11B and Table 1),
although at this Vh, the percent
inhibition by PDBu was greater that that observed with 3 mM 4-AP. The
normalized currents are shown in Fig. 11B (right)
where current values positive to
20 mV were significantly different
from those in 4-AP (RM ANOVA). In contrast to the control and TEA
recordings described in the preceding text, PDBu produced further
inhibition of IK at a
Vh of
80 mV in the presence of 30 µM 4-AP (Fig. 11C and Table 1). For the normalized
current, only values positive to +20 mV were significantly different
from in 4-AP alone (RM ANOVA). Thus these results demonstrate that the
IK, which is suppressed by PDBu, is
not sensitive to either concentration of 4-AP.
Taken together, these findings indicate that PDBu modulates an
IK that is sensitive to TEA but not
4-AP. Also our observations suggest that high and low concentrations of
4-AP somehow "unmask" or enable PDBu to inhibit
IK at a
Vh of 80 mV, whereas PDBu does not
exhibit this capacity under control conditions or in the presence of TEA.
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DISCUSSION |
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We have demonstrated that the phorbol ester, PDBu, suppressed
IK in nociceptive sensory neurons in a
manner that depended on the holding voltage. These actions of PDBu were
mediated by PKC because the suppression of
IK was prevented by inhibition of PKC,
whereas 4- phorbol had no effect. PDBu did not reduce IK in the presence of TEA, suggesting
that these treatments inhibit the same component of the current. We
also found that 4-AP inhibited a current component that was distinct
from that inhibited by TEA and largely unaffected by PDBu.
Our observations that the activation of PKC resulted in the suppression
of IK suggest that modulation of this
current by intracellular signaling pathways is an important mechanism
in the regulation of sensory neuron sensitivity to different modalities
of peripheral stimulation. Our findings are consistent with previous
observations wherein exposure to PDBu depolarized unmyelinated rat
vagus nerve (Rang and Ritchie 1988) and caused both a
direct excitation as well as sensitization of different classes of
articular afferent fibers in the knee joint of the cat
(Schepelmann et al. 1993
). Similarly, PDBu (50 nM)
caused a direct release of substance P and calcitonin gene-related
peptide from rat sensory neurons grown in culture, whereas lower
concentrations (10 nM) sensitized the potassium- or
capsaicin-evoked release of these peptides (Barber and Vasko
1996
). These results indicate that the PKC signaling cascade
and its regulation is important in mediating the enhancement of the
sensitivity of sensory neurons to stimulation.
The suppression of IK by PDBu appeared
to be mediated by the activation of a calcium-dependent isoform of PKC
rather than novel or atypical isoforms (Mellor and Parker
1998) because the inhibition of
IK by PDBu was prevented by
intracellular BAPTA or pretreatment with
-conotoxin, a specific
blocker of N-type voltage-dependent calcium channels. These results
suggest that, in the presence of PDBu, there may be sufficient calcium
influx at the more depolarized holding potentials (
47 and
20 mV) to activate PKC, whereas at
80 mV, the lower influx of calcium does not
permit activation of PKC. Indeed this contention is supported by
electrophysiological measurements of calcium currents in sensory neurons of mouse and rat dorsal root ganglia. Current-voltage curves
indicate that in these neurons the calcium current begins to activate
at voltages positive to
60/
50 mV (Evans et al. 1996
; Gross and Macdonald 1989
; Regan et al.
1991
; Scroggs and Fox 1992
). The calcium
dependency for the activation of PKC is consistent with the recent
results of Kondratyuk and Rossie (1997)
that the phosphorylation of synaptosomal sodium channels by PKC was increased by
depolarization and that this phosphorylation depended on the presence
of extracellular calcium. Furthermore Gross and Macdonald (1989)
found that PDBu inhibited the calcium current (N and L types) in mouse sensory neurons at a holding voltage near the resting
membrane potential, whereas phorbol ester was not effective at
80 mV.
Our observations provide information about the spatial organization of
the calcium channels and PKC. We found that PDBu caused a PKC-dependent
inhibition of IK in cells dialyzed
with 5 mM EGTA but not with 5 mM BAPTA. These observations can be
understood in terms of the different capacities of EGTA and BAPTA to
control the free calcium concentration near calcium channels. Using the length constant formulation, diffusion coefficients, and association rate constants provided by Stern (1992), one calculates
that in 5 mM EGTA, the concentration of free calcium falls with
distance from a channel with a length constant of ~370 nm. In 5 mM
BAPTA, the length constant is only 11 nm. Thus in 5 mM BAPTA, a
molecule of PKC >44 nm (4 length constants) from an open plasma
membrane channel will not be subject to an increase in calcium
concentration above bulk free calcium. In 5 mM EGTA, the corresponding
distance is 1.5 µm. Accordingly our observations imply that, on
average, the PKC molecules regulating
IK are farther than ~40 nm and
nearer than ~1 µm from the source of activating calcium.
Exposure to phorbol esters can lead to an increased excitability as
reflected by the enhanced number of action potentials elicited by a
depolarizing current pulse in pyramidal neurons of the hippocampal
slice (Baraban et al. 1985). Consistent with this idea,
we demonstrated that activation of PKC suppressed the peak
IK in mammalian sensory neurons; this
enhanced excitability may contribute to the sensitization of
neuropeptide release. Our findings are supported by the results of
others wherein PDBu and other phorbol esters inhibit
IK in a variety of neuronal systems. In cultured hippocampal pyramidal neurons, the phorbol ester, phorbol
12,13 diacetate, suppressed a persistent
IK by ~30% (Doerner et al.
1988
). Similarly, phorbol esters inhibited the
IsK heterogously expressed in
Xenopus oocytes (Busch et al. 1992
) and the
slow IK activated by acetylcholine in
chick cochlear ganglia (Yamaguchi and Ohmori 1993
). In
observations similar to ours, Shipston and Armstrong
(1996)
reported that PDBu suppressed the BK-type
IK in
GH4C1 cells and that
pretreatment with TEA prevented the actions of PDBu. Phorbol esters
also inhibited the currents conducted by the cloned and expressed
potassium channels Kv1.2 and Kv1.5 (Vogalis et al. 1995
)
as well as Kv3.1 (Kanemasa et al. 1995
).
The identity of the potassium channel(s) modulated by PKC remains to be
determined. Our findings indicate that the inhibitory effect of PDBu is
prevented by pretreatment with the potassium channel blocker TEA. Under
control conditions, IK also was
suppressed by 30 µM and 3 mM 4-AP; however, the 4-AP-sensitive
current differed from that inhibited by TEA in two respects. First, the
inhibition by 4-AP was greater at more negative holding potentials,
suggesting that the 4-AP-sensitive current exhibited a
voltage-dependent inactivation that was not observed for the
TEA-sensitive current. Second, in the presence of 4-AP, PDBu further
reduced IK, whereas in the presence of
TEA, PDBu was without effect. Based on this sensitivity to TEA, one
could speculate on the nature of the gene product modulated by phorbol
ester; Kv1.1, 1.6, 2.1, and 3 fit the TEA sensitivity. However, it is
unlikely to be Kv3 based on the rapid inactivation exhibited by this
gene product. Additional studies, which are currently ongoing, are
necessary to further elucidate the specific types of potassium
channel(s) that are modulated by phorbol esters as well as inflammatory
prostaglandins. Interestingly, in the presence of either concentration
of 4-AP, PDBu partially inhibited the remaining
IK elicited from a
Vh of 80 mV. The mechanism behind
this action is unclear. Although it is well established that the block
produced by 4-AP can be relieved with depolarized voltage steps
(Castle et al. 1994
; Hermann and Gorman
1981
; Stephens et al. 1994
; Yeh et al.
1976
), this cannot account for the ability of PDBu to now
inhibit IK at this hyperpolarized
Vh. It is possible that an
IK, which is PDBu insensitive but 4-AP
sensitive, dominates the total current that is active at a
Vh of
80 mV and that this current
must be removed before the effects of PDBu can be observed at this
particular Vh. Thus inhibition of this
current by 4-AP "unmasks" the PDBu-sensitive IK. Also, this PDBu-insensitive,
4-AP-sensitive current might be inactivated at the more depolarized
holding potentials because the extent of inhibition produced by PDBu at
20 and
47 mV in the presence of the low concentration of 4-AP is
similar to that observed in the absence of 4-AP. However, 4-AP, at each
concentration, inhibited the total IK
by similar amounts at the
47 and
80 mV holding potentials.
Alternatively in the absence of 4-AP, PDBu had no effect on
IK when
Vh was
80 mV because there may have been insufficient calcium influx to support activation of PKC. Thus a
possibility is that 4-AP increased the calcium influx and led to the
activation of PKC. The cellular mechanisms giving rise to the
facilitation and the interrelationship between 4-AP and PDBu clearly
are complex and will be areas for future investigation.
In summary, our findings demonstrate that the phorbol ester-induced activation of PKC suppresses a potassium current in rat sensory neurons. The reduction in this current may contribute to the enhanced excitability and the augmented release of neuropeptides observed after activation of PKC. Therefore the PKC signaling pathway plays an important role in modulating the excitability of sensory neurons and may contribute to the enhanced neuronal sensitivity during inflammation.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-30527 to G. D. Nicol.
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FOOTNOTES |
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Address for reprint requests: G. D. Nicol, Dept. of Pharmacology and Toxicology, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46202-5120 (E-mail: gnicol{at}iupui.edu).
Received 19 May 2000; accepted in final form 15 September 2000.
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REFERENCES |
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