0022-3565/99/2891-0312$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 289:312–320, 1999
Vol. 289, No. 1
Printed in U.S.A.
k- and m-Opioid Inhibition of N-Type Calcium Currents Is
Attenuated by 4b-Phorbol 12-Myristate 13-Acetate and Protein
Kinase C in Rat Dorsal Root Ganglion Neurons1
ANTHONY P.J. KING, KAREN E. HALL, and ROBERT L. MACDONALD
Departments of Neurology (A.P.J.K., R.L.M), Internal Medicine (K.E.H.), and Physiology (R.L.M.), University of Michigan, and Veteran Affairs
Medical Center (K.E.H.), Ann Arbor, Michigan
Accepted for publication December 2, 1998
This paper is available online at http://www.jpet.org
k- and m-opioid agonist inhibition of calcium currents (Macdonald and Werz, 1986; Moises et al., 1994a; Tsunoo et al.,
1986; Werz and Macdonald, 1984) is thought to mediate
opioid reduction of calcium-dependent neurotransmitter release from presynaptic terminals (Werz and Macdonald,
1984). k- and m-agonists selectively inhibited the same high
threshold calcium currents in rat dorsal root ganglion (DRG)
neurons (Moises et al., 1994b; Wiley et al., 1997). Although
N-type calcium current accounted for ;75% of the total opioid-sensitive calcium current, both k- and m-agonists also
inhibited P- and Q-type calcium currents (Moises et al.,
1994a; Wiley et al., 1997). Opioid inhibition of calcium current was mediated specifically by coupling to the G protein
a-subunit Gao (Gross et al., 1990a; Moises et al., 1994b;
Wiley et al., 1997). Activation of a number of other inhibitory
G protein-linked neurotransmitter receptors, including musReceived for publication August 24, 1998.
1
This study was supported by National Institutes of Health Grant DA
04122 to RLM. APJK is a recipient of a National Institutes on Drug Abuse
Postdoctoral Training Grant Fellowship 2T32DA07268.
bitol-related inhibition of calcium current. Similar effects were
seen with intracellular dialysis of PKM. Intracellular PKC-I did
not block opioid inhibition of calcium current but did reverse
PMA and PKM effects on opioid receptor coupling to calcium
channels. Because neither PMA nor PKM changed the proportion of v-CgTX-inhibited current, their effects were not due to a
decrease in the proportion of N-type current. After v-CgTX
treatment, there were no differences in the dynorphin A effects
on control and PMA- or PKM-treated neurons, suggesting that
PKC primarily affected coupling to N-type calcium channels.
These data suggest that in acutely dissociated rat dorsal root
ganglion neurons, endogenous PKC is required for maintenance of calcium current, may play a role in regulation of
neuronal calcium channels, and could be involved in tolerance
and/or cross-talk inhibition of opioid responsiveness.
carinic, adrenergic, somatostatin, and g-aminobutyric acidB
receptors, also inhibited calcium channels in a pertussis toxin-sensitive manner (Swartz, 1993). Although receptors demonstrate specificity for G protein a-subunits, the actual inhibition of calcium current may be mediated via the G protein
bg-subunits (Herlitze et al., 1996; Ikeda, 1996), possibly by
inhibiting calcium current through a direct interaction with
the a1 subunit of the calcium channel (De Waard et al., 1997;
Zamponi et al., 1997).
Protein kinase C (PKC) has been implicated in several in
vivo studies of opioid action. Intrathecal administration of
the phorbol ester 4b-phorbol 12-myristate 13-acetate (PMA)
blocked opioid-induced antinociception in rats (Zhang et al.,
1990; Narita et al., 1997). Intrathecal administration of PKC
inhibitors did not block opioid antinociception but did block
acute opioid tolerance (Narita et al., 1995). Furthermore,
chronic i.p. morphine increased PKC activity in the pons/
medulla in rats (Narita et al., 1994).
Although in vivo tolerance is often receptor specific (i.e.,
homologous), heterologous (i.e., cross-receptor) tolerance has
ABBREVIATIONS: PKC, protein kinase C; PKC-I, protein kinase C inhibitory peptide, PKC19 –31; PKM, protein kinase C catalytic subunit fragment;
PMA, 4b-phorbol 12-myristate 13-acetate; 4a-PdBu, 4a-phorbol 12,13-didecanoate; DMSO, dimethyl sulfoxide.
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ABSTRACT
In rat dorsal root ganglion neurons, activation of k- and m-opioid receptors decreases N-type calcium current, whereas a
constitutively active form of protein kinase C (PKC; i.e., PKM, a
PKC catalytic subunit fragment) increases N-type calcium current. PKC also attenuates inhibition of calcium current by several G protein-linked neurotransmitter systems. We examined
the effects of activation of endogenous PKC by 4b-phorbol
12-myristate 13-acetate (PMA) and dialysis of cells with PKM
and a pseudosubstrate inhibitor PKC(19 –31) (PKC-I) on k- and
m-opioid-mediated inhibition of calcium current, calcium current amplitude, and rundown. PMA modestly increased peak
calcium current and substantially reduced calcium current “rundown,” effects blocked by PKC-I. In contrast, PKC-I decreased
calcium current and increased current rundown. PMA attenuated morphine-, dynorphin A-, and U50,488- but not pentobar-
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PMA and PKM Reduce Dynorphin A Inhibition of Calcium Current
Materials and Methods
Preparation of Acutely Dissociated Neurons. DRG neurons
were prepared from Sprague-Dawley rats, 14 to 50 days of age, of
both sexes using a technique similar to that described previously
(Gross et al., 1990b). Briefly, after removal from the spinal column,
thoracic DRG neurons were minced, incubated with collagenase and
trypsin, and then triturated to separate the cells. Cells were then
plated onto uncoated 35-mm culture plates or plates coated with
rat-tail collagen in minimal essential media supplemented with 16.5
mM NaHCO3, 28.2 mM glucose, and 10% fetal calf serum. Cultures
were incubated at 37°C in 95% air, 5% CO2. Recordings were made
at room temperature, typically within 10 h of plating.
Drugs and Enzymes. Constitutively active PKC catalytic subunit (PKM) purified from bovine brain (Woodgett and Hunter, 1987)
was a kind gift of Dr. Michael Browning (University of Colorado,
Denver, CO). Protein kinase C inhibitory peptide (PKC-I), PKC19 –31,
and dynorphin A were obtained from RBI (Natick, MA). Fetal calf
serum was from Life Technologies, Inc. (Grand Island, NY). Morphine, U50,488, PMA, nifedipine, v-conotoxin from Conus geographus (GVIA), collagenase, trypsin, and all other chemicals were from
Sigma Chemical Co. (St. Louis, MO).
Solutions and Drug Application. Stock solutions of PKM (1
mM) and PKC-I (4 mM) were stored at 280°C. Just before use,
solutions of PKM, PKC-I, and/or vehicle were prepared. PKM and
PKC-I were diluted into internal pipette solution (see below) at 40
nM or 4 mM, respectively, and kept on ice until use. To prepare a
solution of both PKM and PKC-I, the stock solution of PKC-I was
diluted to 4 mM into the working solution of PKM, and the mixture
was incubated at 37°C for 15 min to inactivate PKM. When using
solutions containing PKM or PKC-I, the recording pipette tip was
filled with internal recording solution, and then back-filled with the
peptide-containing solution. Stock solutions of all other drugs were
diluted into external solution on the day of use. Nifedipine was
dissolved in dimethyl sulfoxide (DMSO) (10 mM) on the day used
(final DMSO concentration was less than the 1:1000). Stock solutions
of dynorphin A and v-conotoxin GVIA (1 mM) were dissolved in
filtered distilled water, lyophilized in aliquots, and stored at 220°C
until the day used. Stock solutions of morphine were prepared in
distilled water (10 mM) and of PMA in DMSO (100 mM) and were
stored at 220°C until used. Opioids were applied to cells using a
modified U-tube application system (Greenfield and Macdonald,
1996) using a solenoid-controlled 10-s application of drug, followed
by vacuum reuptake. PMA and v-conotoxin GVIA were applied by
pressure ejection from a blunt-tipped (20 – 40-mm opening) pipette
positioned ;25–50 mm from the neuron.
Whole-Cell Patch Clamp Recording Techniques. Voltageclamp recordings were made using the whole-cell variant of the
patch-clamp recording technique (Hamill et al., 1981) using an Axopatch 1-B amplifier (Axon Instruments, Foster City, CA), and glass
recording pipettes, micropipette tip resistances of 1–2 megaohms,
and seal resistances of greater than 1 gigaohm. Patch clamp micropipettes were pulled from Labcraft micro-hematocrit capillary tubes
(Curtin Matheson Scientific, Inc., Houston, TX) using a P-87 Flaming-Brown micropipette puller (Sutter Instrument Co., San Rafael,
CA). Signals were low pass filtered at 2 kHz using an 8-pole Bessel
filter then digitized, recorded, and analyzed using pCLAMP6 software (Axon Instruments).
Cells were removed from the 5% CO2 incubator, and the medium
was replaced with external recording medium (67 mM choline chloride, 100 mM tetraetylammonium chloride, 0.8 mM MgCl2, 5.3 mM
KCl, 5.6 mM glucose, 10 mM HEPES, and either 5 mM BaCl2 or
CaCl2, 325–330 mOsM) with a pH of 7.35. Patch clamp micropipettes
of 3–10 megaohms were filled with internal solution (140 mM CsCl,
5.3 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, 5 mM
MgATP, and 0.1 mM LiGTP). The pH was adjusted to 7.3 with CsOH
after addition of ATP and GTP, and the final osmolality was 300 –310
mOsM, 10 –15% lower than the external solution.
Data Analysis. Leak current was estimated as the inverse of the
current generated by hyperpolarizing commands of equal value to
those used to depolarize the neurons. These were digitally subtracted
from total currents to give leak-subtracted barium or calcium currents. Statistical comparisons of the effects of drugs, peptides, and
PKM on peak current and on current rundown were performed using
a two-tailed Student’s t test. Comparisons between dynorphin A
inhibition of calcium currents before and after treatment with drugs
in the same cell were analyzed using a paired-sample t test.
Results
PMA Increased Calcium Channel Current. Effects of
phorbol esters and PKM on calcium channels and k-opioid
signaling were studied using a tight-seal, whole-cell voltage
clamp protocol on the somata of acutely dissociated rat DRG
neurons. Barium was used as a charge carrier through calcium channels, and internal calcium was buffered with
EGTA. Currents were elicited by voltage steps to 0 mV from
a holding potential of 280 mV. Under these conditions, primarily transient high-threshold, voltage-dependent calcium
channel currents were activated (Moises et al., 1994a). External application of 250 nM PMA to the neurons increased
total barium current, with maximal increase within 3 min
(Fig. 1, A and B). In contrast, after treatment of neurons with
vehicle (1:1000 DMSO) or 250 nM 4a-phorbol 12,13-didecanoate (4a-PdBu), which does not activate PKC, barium
current decreased or “ran down” slightly (Fig. 1, A and B).
The average maximal increase in current was to 116 6 4% of
current before application of PMA (Fig. 1C). The actual increase in barium current induced by PMA appeared to be
somewhat larger than this, because currents in both vehicleand 4a-PdBu-treated neurons ran down about 10% within 3
min (Fig. 1C). The largest PMA-induced increase recorded
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also been observed in opioid coupling to calcium channels
(Kaneko et al., 1997). d-Opioid receptors recently also have
been shown to be heterologously desensitized by N-methylD-asparate in a PKC-dependent manner (Fan et al., 1998).
Although PKC does not appear to be involved in agonistinduced receptor phosphorylation (Pei et al., 1995) and thus
may not be directly involved in homologous desensitization,
it may be important in mediating cross-tolerance between
different opioid receptors and/or other neurotransmitters
such as N-methyl-D-aspartate.
Activation of PKC with phorbol esters reduced calcium
current inhibition by inhibitory G protein-linked neurotransmitter receptor agonists in several rat neuronal types
(Swartz, 1993; Wilding et al., 1995; Zhu and Ikeda, 1994), but
PKC did not appear to be involved in the actual signaling
pathway leading to inhibition of calcium current induced by
these agonists (Swartz, 1993). Because multiple neurotransmitter systems can activate PKC, PKC attenuation of kand/or m-opioid receptor coupling to calcium channels could
be a potential mechanism for the observed PKC effects on
systemic opioid action. However, although PKC has been
shown to attenuate inhibition of calcium currents by other
Gi-linked neurotransmitter, it is not known whether activation of PKC can block opioid-induced inhibition of calcium
currents. To investigate this possibility, we examined the
effects of PKC on m-opioid (morphine) and k-opioid (dynorphin A and U50,488) inhibition of calcium channel currents
in rat DRG neurons.
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was to 136% of control current, and increases in current were
seen in 8 of 11 neurons tested. Thus, the PMA-induced increase in calcium current reported here was consistent with
effects reported previously of PMA in DRG neurons (Swartz,
1993), although the magnitude of the increase was smaller
than that reported in other rat neuronal types such as cerebral cortical and superior cervical ganglion neurons (Swartz,
1993). The observed effect of PMA was also smaller than that
produced by internal application of PKM, which we have
previously shown maximally increased peak calcium current
to .200% of control in DRG neurons (Hall et al., 1995).
PMA Decreased and PKC-I Increased Rate of Calcium Channel Current Rundown. We reported previously
that calcium currents elicited from DRG neurons routinely
ran down over time; currents reached a peak at 2–5 min and
then progressively decreased for the remainder of the recording (Hall et al., 1995). This phenomenon appeared to be due
to dialysis out of the neuron of cellular components required
for maintaining functional calcium channels. As rundown
was slowed by inclusion of ATP in the recording pipette (Hall
and Macdonald, unpublished data), it appeared likely that a
loss of protein kinase activity was involved. All data reported
here were obtained from recordings with 5 mM ATP in the
pipette. In control neurons, current declined to 50% of peak
value within ;20 min of patch rupture and was usually
,10% of peak within 40 min of patch rupture (Fig. 2, M). We
have shown previously that intracellular PKM increased
peak current without affecting rundown. In contrast, while
Fig. 2. PMA decreased and PKC-I increased the rate of calcium channel
current rundown. Barium currents were evoked by a 200-ms pulse to 0
mV from a Vh of 280 mV with barium as a charge carrier in the presence
of 3 mM nifedipine in control (E, n 5 6), PMA-treated (F, n 5 5),
PKC-I-treated (M, n 5 5), and PKC-I and PMA-treated (f, n 5 5) DRG
neurons. Rates of current rundown were compared by dividing inward
currents at each time point (-Ibarium) by the maximum inward current
(-Ibariummax) attained during the experiment. The rundown period was
defined as the period following maximum current. PMA treatment caused
a significant decrease in the rate of rundown in control and PKC-I treated
neurons, whereas PKC-I increased the rate of rundown in control neurons. (Data were analyzed using a two-tailed Student’s t test. Values were
shown as mean 6 S.E.M.
external PMA only modestly increased “peak” calcium current, it substantially decreased the rate of current rundown.
Current from neurons treated with external PMA ran down
at a slower rate than from control neurons (Fig. 2, f), typically with currents not reduced to 50% of peak until 30 – 40
min after patch rupture. In some neurons treated with PMA,
currents as large as 50% of peak were recorded as long as 70
min after patch rupture. This suggested potential differences
in effects of activation of endogenous PKC by PMA and introduction of a constitutively activated kinase into the neuron.
The effect of PMA to protect the currents from rundown
was abolished by intracellular application of PKC-I (Fig. 2,
F). As we have reported previously, intracellular application
of PKC-I alone increased the rate of current rundown (Fig. 2,
E). Currents from neurons treated with PKC-I ran down
faster than those from control cells in the absence of PMA
(50% reduction in 9 –10 min). These data suggested a role for
endogenous PKC in maintaining functional calcium channels
in acutely dissociated DRG neurons under whole-cell patch
clamp conditions. Rundown of calcium channel current was
also observed when 5 mM calcium was used as the charge
carrier, and application of PMA slowed rundown to a similar
extent (data not shown). PMA also had a similar effect on
rundown of peak calcium currents recorded from neurons in
the presence of external solution containing 150 mM sodium
chloride (data not shown).
PMA Attenuated Dynorphin A Inhibition of Calcium
Channel Currents. Morphine and Dynorphin A, through
interaction with m- and k-opioid receptors, respectively, inhibit calcium channel currents through a rapid, reversible,
voltage-dependent, pertussis toxin-sensitive mechanism
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Fig. 1. PMA increased current through calcium channels in rat DRG
neurons. Calcium channel currents were evoked with a 200-ms pulse to 0
mV from a holding potential of 280 mV with barium as the charge
carrier. Currents from three separate neurons treated with either PMA
(f), 4a-PdBu (Œ), or vehicle (1:1000 DMSO, M) are shown in A as peak
currents plotted as a function of time after patch rupture and in B as
superimposed traces at 0 – 4 min after treatment. Peak barium current
amplitude was measured at 15 ms after pulse. In C, relative change in
peak amplitude was plotted as a function of time after treatment with
PMA (f), 4a-PdBu (Œ), or vehicle (1:1000 DMSO, M). Points represent
means 6 S.E.M. Change in peak amplitude was calculated by dividing
the (-Ibarium) at each point by the current before treatment (-Ibariumt 5 0).
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PMA and PKM Reduce Dynorphin A Inhibition of Calcium Current
Fig. 3. PMA attenuated m- and k-opioid inhibition of calcium channel
current. In A, barium currents evoked by a 200-ms pulse to 0 mV from a
Vh of 280 mV were shown as peak current plotted as a function of time
after patch rupture and as superimposed traces before and immediately
after 10-s applications of 3 mM dynorphin A (arrows marked by Dyn) and
250 nM PMA (bar). B, inhibition (shown as percentage of change in
amplitude from currents immediately before application of agonists) of
barium current by 3 mM dynorphin A (Dyn, n 5 11), 3 mM U50,488 (U50,
n 5 4), 3 mM morphine (Morph, n 5 8), and 100 mM pentobarbital
(PentoB, n 5 4) before (light column) and 3 min after (dark column)
treatment of neurons with 250 nM PMA.
ium current by 35% and did not slow apparent activation rate
(data not shown). PMA had no effect of pentobarbital inhibition of barium currents (Fig. 3B).
To verify that the PMA-induced attenuation of dynorphin A
action was due to activation of endogenous PKC, PKC-I peptide
(4 mM) was included in the recording pipette to inhibit cellular
PKC. This technique has been shown to block various cellular
effects of PMA, including attenuation of norepinephrine and
baclofen inhibition of calcium channel currents in rat neurons
(Swartz, 1993). Neurons were treated with intracellular PKC-I
and then tested for dynorphin A inhibition of barium currents
before and after external application of 250 nM PMA (Fig. 4A).
The initial application of dynorphin A reduced barium current
by 21% (Fig. 4A), which indicated that activation of PKC was
not required for coupling of k-opioid receptors to calcium channels. After a 1-min recovery, the neuron was treated with 250
nM PMA, which did not appear to slow current rundown. After
2 min of PMA treatment, 3 mM dynorphin A was reapplied to
the neuron, which led to a reversible 18% inhibition of barium
current (Fig. 4A). Four neurons treated with intracellular
PKC-I and tested in this manner showed no significant difference in dynorphin A inhibition before or after PMA treatment
(Fig. 4B).
Effect of Blockade of N-Type Current on Reduction
by PMA of Calcium Channel Current Inhibition by
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(Taussig et al., 1992; Moises et al., 1994b). Acutely dissociated DRG neurons are a heterogeneous preparation, with
populations of dynorphin- and/or morphine-sensitive neurons, and furthermore, dynorphin A can have effects through
m- as well as k-opioid receptors. However, populations of
dynorphin-sensitive and morphine-insensitive DRG neurons
have been observed (Moises et al., 1994a); and studies using
m-specific
H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2
(CTAP), b-funaltrexamine) and k-specific (norbinaltorphimine) antagonists also indicated that the effects of dynorphin
A and morphine on calcium currents at the concentrations
used in this study were due primarily to interactions with
k-opioid and m-opioid receptors, respectively (Moises et al.,
1994a; Wiley et al., 1997; King and Macdonald, unpublished
observation). k-Opioid receptor inhibition of calcium channel
currents was probably mediated through pathways similar or
identical with those of m-opioid receptors, which have been
shown to be rapid and membrane delimited (Wilding et al.,
1995).
k-Opioid agonists inhibited voltage-activated calcium currents by 20 – 40% in DRG neurons and produced delayed
activation kinetics (Moises et al., 1994a). In the experiments
reported here, dynorphin A reduced calcium current an average of 26 6 4% in the presence of the L-type calcium
channel blocker nifedipine. Because nifedipine has been demonstrated not to decrease opioid-sensitive currents (Moises et
al., 1994a), 3 mM nifedipine was routinely added to increase
the relative proportion of opioid-sensitive current in the neuron tested. Dynorphin A inhibition of total barium current in
rat DRG neurons was attenuated by extracellular application
of 250 nM PMA (Fig. 3). In the neuron shown, an initial
application of 3 mM dynorphin A for 10 s rapidly and reversibly reduced total peak barium current from 1.9 to 1.4 nA
(26% inhibition) (Fig. 3A). This inhibition of the barium current was associated with two distinct kinetic components,
slowing of the activation rate and inhibition of steady-state
current. After the current completely recovered from the
effect of dynorphin A, 250 nM PMA was applied to this
neuron, and after 3 min of treatment with PMA, 3 mM dynorphin A was applied again for 10 s. This application of dynorphin A decreased the current from 1.9 to 1.8 nA, i.e., a 5%
inhibition, and the attenuated dynorphin A inhibition in the
presence of PMA did not result in a slowing of the activation
rate (Fig. 3A). PMA reduced the inhibition of subsequent
applications of dynorphin A in the same neuron an average of
80% (p , .005, Fig. 3C). This effect of PMA was not due to a
tachyphalaxis resulting from multiple applications of dynorphin A, because multiple applications of dynorphin A after 1
min or more recovery in the absence of PMA showed ,10%
reduction in efficacy (data not shown). This effect was also
not due to effects of time or rundown, because the percentage
of inhibition of current by dynorphin A in control neurons
(not treated with PMA) was not significantly different at 2– 4
min and 10 –15 min after seal rupture (data not shown).
As has been shown previously (Moises et al., 1994a), 1 mM
morphine and U50,488 also inhibited barium current (Fig.
3B) and exhibited apparent slowing of activation (data not
shown). Similar to its effects on dynorphin, PMA attenuated
the inhibitory effects of morphine and U50,488 by 80% and
78%, respectively (Fig. 3B). In contrast, 100 mM pentobarbitol, which appears to act as an open-channel blocker of calcium channels (Gross and Macdonald, 1988), inhibited bar-
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Dynorphin A. DRG neurons contain multiple high-threshold calcium current subtypes, including N-, L-, Q-, P- and
R-types (Moises et al., 1994a; Wiley et al., 1997). N-type
current accounted for about 75% of the total dynorphin Asensitive current in rat DRG neurons, although dynorphin A
also inhibited a portion of the Q-, P-, and R-current (Wiley et
al., 1997). To gain information as to the specific calcium
channels being affected by PMA, we examined the effect of
the N-channel blocker v-conotoxin GVIA on the ability of
PMA to attenuate dynorphin A inhibition of calcium currents
(Fig. 5). Dynorphin A (3 mM) was applied to DRG neurons to
determine the total dynorphin A-sensitive current (Fig. 4A,
traces 1 and 2). Dynorphin A reduced the current from 2.7 to
2.2 nA (21%). After recovery, the neuron was treated with
250 nM PMA for 2 min, and dynorphin A was reapplied,
which reversibly inhibited current from 2.5 to 2.4 nA (a 7%
decrease) (Fig. 5A, traces 3 and 4). After recovery for 1 min,
N-channels were blocked with v-conotoxin GVIA, which reduced the basal current by 52% from 2.5 to 1.2 nA, and then
dynorphin A was reapplied. Dynorphin A only reduced the
current from 1.2 to 1.1 nA (Fig. 5A, traces 5 and 6), a decrease
which represented 5% of the original current and a 9% inhibition of the residual current. A similar protocol was followed
to determine the levels of dynorphin A- and v-conotoxin
Fig. 5. Effect of PMA and v-conotoxin GVIA on dynorphin A inhibition of
calcium channel currents. In A, barium currents evoked by a 200-ms
pulse to 0 mV from a Vh of 280 mV in the presence of 3 mM nifedipine
were shown as peak current plotted as a function of time after patch
rupture and as superimposed traces at times indicated by arrows. Application of dynorphin A (3 mM) for 10 s reversibly reduced total highthreshold barium current. Application of PMA (250 nM) for 5 min had
little effect on total barium current but substantially reduced the fraction
of current inhibited by dynorphin A. Application of v-conotoxin GVIA (3
mM) for 30 s irreversibly reduced total whole-cell barium current but had
little effect on the fraction of current inhibited by dynorphin A. B, dynorphin A inhibition (shown as percentage of change) of barium current in
control (n 5 4) and PMA-treated (n 5 4) neurons before and after
treatment with v-conotoxin GVIA (3 mM). PMA treatment significantly
(p , .01) decreased dynorphin A inhibition of barium current. There was
no difference in dynorphin A inhibition in control and PMA neurons after
treatment with v-GVIA, and v-GVIA did not further reduce the fraction
of current inhibited by dynorphin A in PMA-treated neurons.
GVIA-sensitive currents in neurons not treated with PMA. In
the absence of conotoxin, dynorphin A inhibited an average of
22 6 2% of the current before and 5 6 2% of the current after
PMA treatment (n 5 11); after v-conotoxin GVIA treatment,
dynorphin A inhibited an average of 6 6 1% of the current in
control (n 5 4) and 6 6 2% of the current in PMA treated (n 5
4) neurons (Fig. 5B). Although the effects of dynorphin A on
the residual calcium currents after PMA were small, there
appeared to be no difference in dynorphin sensitivity in control and PMA-treated neurons after v-conotoxin GVIA treatment (Fig. 5B). v-Conotoxin GVIA also did not appear to
further decrease the dynorphin A-sensitive current when
applied after PMA (Fig. 5B, compare striped columns). Furthermore, when PMA was applied after v-conotoxin GVIA,
the small residual dynorphin-sensitive current was not further reduced (data not shown).
These data suggested that PMA did not decrease the effi-
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Fig. 4. PKC-I blocks PMA attenuation of dynorphin A inhibition of
calcium channel current. In A, barium currents evoked by a 200-ms pulse
to 0 mV from a Vh of 280 mV from neurons dialyzed with 4 mM PKC-I in
the recording pipette were shown as peak current plotted as a function of
time after patch rupture and as superimposed traces before and immediately after 10-s applications of 3 mM dynorphin A (arrows marked by
Dyn) and 250 nM PMA (bar). B, inhibition of barium current (shown as
percentage of change in amplitude from currents immediately before
application of dynorphin) in neurons dialyzed with 4 mM PKC-I by 3 mM
dynorphin A before (light column) and 3 min after (dark column) treatment of neurons with 250 nM PMA. (n 5 4, NS.)
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PMA and PKM Reduce Dynorphin A Inhibition of Calcium Current
Fig. 6. Dynorphin A inhibition of calcium current was decreased in
PKM-treated neurons. A, superimposed current traces before and immediately after 10-s treatment with dynorphin A (3 mM) in control neurons
(Control), neurons dialyzed with PKM (40 nM) (PKM), and neurons
dialyzed with PKM (40 nM) and PKC-I (4 mM) (PKM1PKC-I). Currents
were evoked by a 200-ms pulse to 0 mV from a Vh of 280 mV, with
calcium as the charge carrier, in the presence of 3 mM nifedipine. B,
inhibition (shown as percentage of change) of barium current by 3 mM
dynorphin A in control (n 5 7, light column), PKM-treated (n 5 7, dark
column), and PKM 1 PKC-I-treated (n 5 6, striped column) neurons.
Data were normalized to the amplitudes of currents immediately before
application of dynorphin A.
v-conotoxin GVIA treatment, however, dynorphin A inhibition of calcium current was not significantly different in
control (5 6 4%) and PKM-dialyzed (6 6 2%) neurons (Fig.
7A, traces 3 and 4; Fig. 6B). Furthermore, in neurons dialyzed with PKM, treatment with v-conotoxin GVIA did not
appear to further decrease dynorphin A effects (Fig. 7B,
compare the striped columns). These findings suggested that,
like PMA treatment, PKM appeared to primarily affect k-opioid receptor coupling with N-type calcium currents. Also
similar to PMA, intracellular PKM did not decrease the proportion of N-type current. v-Conotoxin GVIA inhibited a
slightly larger proportion of calcium current in PKM-treated
neurons than in control neurons (data not shown).
Discussion
External PMA and Dialysis with PKM Both Attenuated k- and m-Opioid Receptor Inhibition of Calcium
Channel Currents in DRG Neurons. We demonstrated
that in acutely dissociated rat DRG neurons, both external
treatment with the phorbol ester PMA and intracellular dialysis with activated PKC, PKM, attenuated k- and m-opioid
receptor-mediated inhibition of calcium currents. The PMA
attenuation of dynorphin A inhibition of calcium current was
similar to effects of PMA on several types of rat neurons on
inhibition of calcium currents by agonists of a2-adrenergic,
g-aminobutyric acidB, and muscarinic cholinergic receptors
(Swartz, 1993; Zhu and Ikeda, 1994). The effects of PMA on
calcium current inhibition appeared to be due to activation of
endogenous PKC rather than nonspecific actions on membranes or cellular proteins, because the effects of PMA were
blocked by dialysis of the neuron with the PKC-I peptide.
Endogenous PKC activity did not appear to be necessary for
dynorphin A inhibition of calcium currents because intracellular PKC-I did not block dynorphin A inhibition, consistent
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cacy of dynorphin A to inhibit the residual conotoxin-insensitive components (e.g., P-, Q-, and R-types) of barium current. These data suggested that PMA primarily affected
k-opioid receptor coupling to N-type calcium channels. The
observed PMA-induced decrease in dynorphin A inhibition of
barium current was not due to a decrease in the proportion of
N-type current present. There was no difference in v-conotoxin GVIA inhibition of barium current in control neurons
and neurons treated with 250 nM PMA (data not shown).
PKM Attenuated Dynorphin A Inhibition of Calcium
Channel Current. In addition to activating endogenous
PKC, phorbol esters could be accompanied by other “nonspecific” effects on calcium channels, receptors, or other cellular
proteins. For example, phorbol esters are known to alter the
cycling and down-regulation rate of multiple receptors and
ion channels and have been shown to recruit covert or previously inactive calcium channels in Aplysia neurons (Strong
et al., 1987). Phorbol esters activate multiple isoforms of
PKC, which may be differentially regulated in normal cells,
and there is evidence that a phorbol ester-insensitive PKC
isoform was involved in norepinephrine regulation of calcium
channels in chick DRG neurons (Boehm et al., 1996). Furthermore, there have been conflicting reports of the effects of
phorbol esters and other activators of PKC to either increase
(Swartz, 1993; Zhu and Ikeda, 1994), decrease (Diverse-Pierluissi and Dunlap, 1993; Werz and Macdonald, 1987), or have
no effect (Boehm et al., 1996) on calcium channel currents.
Because of these concerns, we examined the effect of intracellular application of purified, constitutively active PKC,
PKM, on dynorphin A inhibition of calcium channels. As
demonstrated previously with DRG neurons (Hall et al.,
1995), intracellular application of 20 nM PKM increased
peak calcium currents compared with those from control
neurons (3.4 6 0.4 nA PKM treated, n 5 6 versus 2.5 6 0.4 nA
control, n 5 7, p , .05); whereas intracellular application of
excess PKC-I (4 mM) with 20 nM PKM, decreased peak currents (1.5 6 0.3 nA, n 5 6, p , .05), likely as a result of
inhibition of endogenous PKC.
Dialysis of neurons with 20 nM PKM decreased dynorphin
A-mediated inhibition of calcium currents from 21 6 4% to
9 6 4% (Fig. 6, control (n 5 7) and PKM (n 5 6), p , .005).
Thus, intracellular treatment of neurons with purified PKC,
like activation of endogenous PKC with PMA, attenuated
dynorphin A inhibition of calcium current. When 4 mM PKC-I
was included in the recording pipette with 40 nM PKM, the
basal calcium current amplitude was reduced, but dynorphin
A inhibited calcium current by 23 6 1% (Fig. 6, PKM 1
PKC-I, n 5 14), not significantly different from Control and
p , .005 from PKM-treated neurons. Thus, the effect of PKM
on dynorphin A inhibition of calcium current was reversed by
PKC-I.
Effect of Blockade of N-Type Current on PKM Attenuation of Dynorphin A Inhibition of Calcium Channel
Current Inhibition. We next investigated effects of intracellular PKM on specific calcium current subtypes. Dynorphin A inhibition of calcium currents in control and PKMtreated neurons was compared before and after blocking
N-type currents with v-conotoxin GVIA. Before treatment
with v-conotoxin GVIA, dynorphin inhibition of calcium current was significantly smaller (p , .05) in neurons dialyzed
with 20 nM PKM (9 6 4%, n 5 6) than in control (23 6 3%,
n 5 7) neurons (Fig. 7A, traces 1 and 2; Fig. 6B). After
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King et al.
Vol. 289
2
with effects of PKC-I on morphine- and other agonist-induced
inhibition of calcium currents in rat neurons (Swartz, 1993;
Wilding et al., 1995).
To examine directly the effects of activated PKC on calcium
currents, we dialyzed neurons with PKM. PKM also attenuated k-opioid receptor-mediated inhibition of calcium currents in a manner similar to PMA treatment. These data
demonstrated that constitutively active PKC was capable of
blocking k-opioid receptor-mediated inhibition of calcium
currents, suggesting that endogenous PKC could be involved
in the physiological modulation of opioid receptor sensitivity.
The finding that both PKM and PMA attenuated k-opioid
receptor inhibition and increased basal calcium current was
significant in light of conflicting reports of actions of activators and inhibitors of PKC in various species and neuronal
types (Boehm et al., 1996; Diverse-Pierluissi and Dunlap,
1993; Swartz, 1993; Zhu and Ikeda, 1994). The present findings are in contrast to the report of Nomura et al., 1994,
which found that 2–3 min pretreatment with 1 mM PMA did
not affect inhibition of calcium currents by the m-agonist
5
D-Ala ,(Me)Phe ,Gly-(ol) in DRG neurons acutely dissociated from rats, 5–10 days of age. We saw comparable effects
of PMA to attenuate dynorphin and morphine action,
whether 5 mM barium or calcium was used as the charge
carrier (data not shown). The present study used rats 14 –50
days of age, which may account for the apparent differences
in PMA action.
Differential Effects of External PMA and PKM Dialysis on Peak Calcium Current and Current Rundown.
Although both PKM dialysis and external PMA application
increased calcium current, they did so with different efficacy.
Treatment of DRG neurons with PMA produced only a modest increase in peak calcium current (15%), which was in
contrast to larger effects of PMA (50 –100%) in rat cerebral
cortical, hippocampal, and superior cervical ganglion neurons (Swartz, 1993; Zhu and Ikeda, 1994). Dialysis of DRG
neurons with PKM resulted in a larger increase in basal
calcium current (;100%) than did PMA treatment, i.e., the
PKM-induced increase in calcium current in DRG neurons
was comparable to that induced by activation of endogenous
PKC by PMA in central neuronal types. This could reflect
differences in levels of PKC activity or efficacy between DRG
and central neurons. However, it is also possible that differences between effects of PMA and PKC may have been due to
the relatively high level of kinase activity in neurons dialyzed
with PKM, leading to the phosphorylation of nonphysiological substrates or other artifactual causes.
External treatment of neurons with PMA also acted differently than dialysis with PKM in terms of effects on current
rundown. As shown previously (Hall et al., 1995), dialysis of
DRG neurons with PKM, although increasing basal current,
had little or no effect on current rundown. However, PMA
treatment substantially reduced the rate of calcium current
rundown, whereas dialysis of neurons with PKC-I increased
the rate of current rundown. This suggested that at least in
whole-cell patch conditions used in these experiments, endogenous PKC activity was necessary for maintenance of
functional calcium currents. These findings also strengthened the conclusion that PKC may be involved in regulation
of calcium currents in a dynamic fashion.
PKC Primarily Attenuated k-Opioid Receptor Coupling to N-Type Calcium Currents. Rat DRG sensory
neurons contain low-threshold transient (T-type) calcium
current and multiple high-threshold calcium current subtypes. Using toxins that are specific blockers of current subtypes, L-, N-, P-, Q- and “R-” (toxin-resistant) subtypes of
high-threshold voltage-dependent calcium currents have
been reported in rat DRG neurons (Moises et al., 1994a;
Rusin and Moises, 1995; Wiley et al., 1997). Dynorphin A did
not inhibit L-type currents but did inhibit N-, P-, and Q-type
currents, although N-type current accounted for ;75% of the
total dynorphin A-sensitive current (Wiley et al., 1997). Neither PMA nor PKM decreased the proportion of N-type current, indicating that their effect to decrease dynorphin A
inhibition of calcium currents was not simply due to a selective removal of a large part of the dynorphin A-sensitive
calcium currents by PKC. Because PMA and PKM did not
block dynorphin A inhibition of total calcium current completely, it is possible that PKC selectively had an effect on
one or more subtype of calcium channel. Thus, the residual
dynorphin A inhibition seen in the presence of activated PKC
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Fig. 7. Effect of PKM and v-conotoxin GVIA on dynorphin A inhibition of
calcium currents. A, superimposed current traces of control and PKMdialyzed neurons before (trace 1) and immediately after (trace 2) 10-s
treatment with 3 mM dynorphin A; after treatment with 3 mM v-conotoxin GVIA (trace 3); and subsequently with 10-s 3 mM dynorphin A
(trace 4). Currents were evoked by a 200-ms pulse to 0 mV from a Vh of
280 mV with calcium as the charge carrier in the presence of 3 mM
nifedipine. B, comparison of dynorphin A inhibition (shown as percentage
of change) of calcium current (-Icalcium) in control neurons (n 5 6) and
neurons dialyzed with PKM (n 5 7) before and after treatment with
v-conotoxin GVIA (3 mM). Dynorphin A inhibition of Icalcium was reduced
in PKM-treated neurons (p , .05). There was no difference in dynorphin
A inhibition in control and PKM-treated neurons after treatment with
v-GVIA, and v-GVIA did not further reduce the fraction of current
inhibited by dynorphin A in PKM-treated neurons.
4
1999
PMA and PKM Reduce Dynorphin A Inhibition of Calcium Current
Acknowledgments
We thank Dr. Michael Browning and Ellen Dudek for the kind gift
of catalytic protean kinase C (PKM). We also thank Nadia Esmaeil
for assistance in preparing rat DRG neurons.
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