Department of Physiology and Pharmacology, Wake Forest University, Winston Salem, North Carolina 27157
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ABSTRACT |
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Hampson, Robert E.,
Jian Mu, and
Sam A. Deadwyler.
Cannabinoid and Kappa Opioid Receptors Reduce Potassium K
Current via Activation of Gs Proteins in Cultured
Hippocampal Neurons.
J. Neurophysiol. 84: 2356-2364, 2000.
The current study showed that potassium K current
(IK), which is evoked at depolarizing
potentials between 30 and +40 mV in cultured hippocampal neurons, was
significantly reduced by exposure to the CB1 cannabinoid receptor
agonist WIN 55,212-2 (WIN-2). WIN-2 (20-40 nM) produced an average
45% decrease in IK amplitude across
all voltage steps, which was prevented by SR141716A, the CB1 receptor
antagonist. The cannabinoid receptor has previously been shown to be
Gi/o protein-linked to several cellular
processes; however, the decrease in IK
was unaffected by modulators of Gi/o proteins and
agents that alter levels of protein kinase A. In contrast, CB1
receptor-mediated or direct activation of Gs
proteins with cholera toxin (CTX) produced the same decrease in
IK amplitude as WIN-2, and the latter
was blocked in CTX-treated cells. Gs protein
inhibition via GDP
S also eliminated the effects of WIN-2 on
IK. Consistent with this outcome,
activation of protein kinase C (PKC) by arachidonic acid produced
similar effects to WIN-2 and CTX. Kappa opioid receptor agonists, which also reduce IK amplitude via
Gs proteins, were compared with WIN-2 actions on
IK. The kappa receptor agonist U50,488
reduced IK amplitude in the same
manner as WIN-2, while the kappa receptor antagonist, nor-binaltorphimine, actually increased
IK amplitude and significantly reduced
the effect of co-administered WIN-2. The results indicate that CB1 and
kappa receptor activation is additive with respect to
IK amplitude, suggesting that CB1 and
kappa receptors share a common Gs protein
signaling pathway involving PKC.
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INTRODUCTION |
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Activation of brain cannabinoid
(CB1) receptors in different types of cells and neurons can inhibit
adenylyl cyclase (Bidaut-Russell et al. 1990;
Howlett 1998
), reduce N and P/Q type calcium currents (Mackie and Hille 1992
; Twitchell et al.
1997
), enhance inward rectification (Garcia et al.
1998
; Henry and Chavkin 1995
), and suppress
potassium "M" (IM), and "D"
(ID) currents (Mu et al. 1999b
; Schweitzer 2000
) while altering the
voltage dependence of potassium A current,
IA (Deadwyler et al. 1993
,
1995
). Several of these effects are blocked by pertussis toxin
(PTX) and mimicked by the nonhydrolyzable GTP substrate GTP
S,
indicating that the CB1 receptor is linked to these processes via
inhibitory (Gi/o) G proteins (Deadwyler et
al. 1993
; Garcia et al. 1998
; Mackie and
Hille 1992
). Recent studies have shown that CB1 receptors bind
to both Gi/o and Gs protein
subunits, but under normal conditions, only Gi/o
proteins are activated (Felder et al. 1998
). However, when Gi/o proteins are fully "saturated" via
activation of other Gi/o protein-linked
receptors, CB1 receptor coupling to Gs proteins is "unmasked" as shown by increased adenylyl cyclase
activity and cAMP accumulation (Felder et al. 1998
;
Glass and Felder 1997
).
Comparison between CB1 and kappa opioid receptors reveals many
similarities, including inhibition of adenylyl cyclase via Gi/o proteins (Aghajanian and Wang
1987; Eriksson et al. 1992
; Hescheler et
al. 1987
; Konkoy and Childers 1993
), alteration
in the voltage dependence of IA
(Simmons and Chavkin 1996
), decrease in amplitude of a
potassium current described as IA but
that closely resembled ID
(Müller et al. 1999
), and increased analgesia
(Welch and Stevens 1992
). In contrast, potassium "K"
current (IK) a non- or slowly
inactivating outward potassium current, exhibited in cultured
hippocampal cells (Storm 1990
; Velumian et al.
1997
), is decreased by kappa opioid receptor stimulation via a
PTX-insensitive, Gs protein-linked pathway
(Baraban et al. 1995
; Piros et al. 1996
). The present study tested whether cannabinoids would also affect IK amplitude, and if so, whether
Gs proteins were involved. This would implicate a
potential common signaling pathway for the modulation of
IK by both types of receptor.
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METHODS |
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Cell culture
The preparation of hippocampal neurons in culture was similar to
that described in several previous reports (Deadwyler et al.
1993, 1995
; Hampson et al. 1995
). Hippocampi
from fetal (E-18) rats (Zivic-Miller) were incubated with neutral
protease (dispase 1, Boehringer Mannheim Biochemica, 2 U/ml) for 40-50
min at 37°C. After stopping the enzymatic reaction with 1.0 mM
NaEDTA, cells were dissociated by gentle trituration via two
flame-polished Pasteur glass pipettes and plated at a density of
3-4 × 105 cells per 35-mm dish. The
plating medium consisted of 59% Dulbecco's modified Eagle's medium
(DMEM, 1×), 19.5% F-12 nutrient mixture (HAM, 1×), 10% fetal bovine
serum (FBS), 10% horse serum (HS), 1% L-glutamine (200 mM), all from GIBCO BRL. Cultures were grown at 37°C in a humidified
5% CO2 incubator. After 48 h, half of the
medium was replaced by "feeding" medium, which consisted of 98%
Neurobasal medium, 2% B-27 supplement, 0.25% L-glutamine
(200 mM), 0.1% 2-mercaptoethanol (all purchased from GIBCO BRL), and 25 mM KCl. Seventy-two hours after plating, 50% of the medium was
again replaced with feeding medium, and the cultures were treated with
0.75 µM cytosine
-D-arabinofuranoside (Ara-C, SIGMA) to prevent proliferation of glia. The culture medium was then changed
every 3 days for the remainder of the experiment. All experiments were
performed on cultured cells after 7-15 days incubation.
Recording methods
The whole cell patch-clamp procedure was similar to that
reported previously for recording potassium currents (Deadwyler
et al. 1993; Mu et al. 1999b
). Briefly, patch
electrodes were prepared from 1.5-mm OD, 1.1-mm ID borosilicate glass
capillaries to produce 1- to 2-µm (2-5 M
) tip openings.
Electrodes were filled by suction and backfilling with a standard
intracellular solution of (in mM) 140 KCl, 11 EGTA, 1 CaCl2, 2 MgCl2, 2 ATP, and
20 HEPES buffer (Sigma) plus 200 µM GTP. During recording,
hippocampal cells (7-15 days) were taken from the incubator washed and
constantly perfused with medium consisting of (in mM) 140 NaCl, 5 KCl,
2.5 CaCl2, 2 MgCl2, 10 glucose, and 20 HEPES with 1 µM tetrodotoxin (TTX; Sigma) added to
block voltage-gated sodium channels. Fast-inactivating A and D outward
potassium currents, were blocked with 4-aminopyridine (4-AP, 5 mM)
leaving only the noninactivating K current (Storm 1990
;
Velumian et al. 1997
; Wu and Barish
1992
). Cultured cells were perfused with heated (37°C) and
oxygenated (95% O2-5%
CO2) bathing medium throughout the experiment.
Sealing the pipette to the neuron and obtaining access to the whole
cell followed previously described patch-clamp procedures (Deadwyler et al. 1993, 1995
; Doerner et al.
1988
; Hamill et al. 1981
; Mu et al.
1999b
). Voltage-clamp recordings and command voltage steps were
performed with an AxoPatch 1D amplifier and TL-1-LabMaster controller
(Axon Instruments) connected to a PC-compatible computer. Whole cell
records were acquired and stored on magnetic disk using pClamp CLAMPEX
software (Axon Instruments). Pipette tip junction potentials were
continuously monitored and compensated as necessary at the amplifier
prior to breakage of the seal. Access (series) resistance was typically
2-5 M
, and series resistance compensation was usually not necessary
because of low pipette resistance. Leakage correction and capacitance
compensation (typically 10-30 pF) utilized dialed-in compensation
adjustment at the amplifier, as well as a P/
4 subtraction procedure
within the acquisition program. Cells were voltage-clamped and held
near resting membrane potential (
50 mV). Activation of
IK utilized a protocol consisting of a multiple depolarizing pulses (
40 to +40 mV) preceded by a single prepulse step to
40 mV, which inactivated residual fast inactivating potassium currents. Measurement of current amplitudes and time constants utilized pClamp software.
Drug preparation
WIN 55,212-2 (Research Biochemicals, Natick, MA), was prepared
daily from a 10 mM stock solution in ethanol and diluted with extracellular bathing medium, and the ethanol was evaporated under a
constant stream of nitrogen (Deadwyler et al. 1993). The
drug was applied via pressure pipette (10- to 50-µm tip opening)
controlled by a solenoid valve (Picospritzer II, General Valve)
modified to eject a steady stream of drug-containing media over the
surface (Carbone and Lux 1987
). The drug solution was
titrated to the same osmolality as the extracellular bathing medium
(Adams and Nonner 1990
). Equivalent bath concentrations
corresponding to the pressure pipette applications of WIN 55,212-2 were
verified and are reported in the text (Mu et al. 1999b
).
Due to the lipophilicity of the drug, a 30-s ejection via the
application pipette was followed by a washout period of at least 2 min
(Deadwyler et al. 1993
). Previous studies demonstrated
that the effects of pressure pipette applications of WIN 55,212-2 were
rapid (approximate 10-s onset) and were reversed after perfusion of
bathing medium for 2 min. All current traces were obtained during the
drug application period (Deadwyler et al. 1995
). No
effect was observed on whole cell currents with application of
vehicle-only solution. Controls consisted of vehicle-only applications
to neurons that never were exposed to drugs, as well as pre- and
postdrug measurements of IA and ID within the same treated neurons.
To elucidate cannabinoid receptor coupling to G proteins, culture
dishes (6-15 days) were prepared with PTX (10 µg per dish) or
cholera toxin (CTX, 10 µg/ml) 18 h prior to testing. The
recording pipette solution also contained PTX or CTX for dialyzing cell cytoplasm during recording. Control cells were recorded from the same
batches of culture dishes with no PTX or CTX added. To inhibit Gs protein activation, GDPS (400 µM), a
nonhydrolyzable GDP analogue, was added to the recording pipette
solution. Again control data were obtained from neurons in the same
culture dish not dialyzed with GDP
S. All results were from cells
exposed to only one of the above conditions unless otherwise specified.
The CB1 receptor-specific antagonist, SR141716A (provided by Sanofi
Reserche, Montepelier, France), was prepared as a 1 mM stock solution
in ethanol and diluted in bathing medium to 300 nM, and the ethanol was
evaporated under a stream of nitrogen. Both the kappa
receptor-selective agonist, U50,488, and the antagonist
nor-binaltorphimine (nor-BNI) were made up as 2 µM stock solutions in
bathing medium and applied in either 1.0 or 2.0 µM concentrations via
extracellular pressure pipette in the same manner as WIN 55,212-2. These concentrations were not calibrated in bath solutions, and
therefore are reported as pipette delivery concentrations.
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RESULTS |
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Identification of IK in cultured hippocampal neurons
In media containing TTX (1 µM) and 4-AP (5 mM) to eliminate
other potassium currents, a moderate outward current,
IK (0.98 ± 0.04 nA; mean ± SE), was slowly activated (time constant = 50-100 ms) in
response to a depolarizing voltage step to +40 mV from a brief holding
potential of 40 mV (Fig. 1, control).
The IK activation protocol (Fig. 1,
inset) revealed a mean one-half activation voltage
(V1/2) of approximately 5.1 ± 1.5 mV
measured across 20 cells whose resting potential averaged
50 mV
(Table 1). Addition of tetraethylammonium
(TEA, 25 mM) markedly reduced the sustained outward potassium current
at all voltage steps. The current was resistant to TEA at 10 mM
concentrations; this, along with the activation protocol, distinguishes
it from IM (Schweitzer
2000
; Storm 1990
). Based on these pharmacologic
and physiologic manipulations and information from previously
characterized potassium currents in hippocampal neurons in culture
(Deadwyler et al. 1993
, 1995
; Hampson et al.
1995
; Mu et al. 1999a
; Velumian et al.
1997
; Wu and Barish 1992
), the current shown in
Fig. 1 was identified as IK
(Storm 1990
).
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Effects of a cannabinoid receptor activation on IK
IK amplitude was reduced 25%
(Fig. 2, inset) from control
amplitude (see preceding text) to 0.76 ± 0.05 nA
[F(1,173) = 9.40, P < 0.01, n = 20, Table 1] following exposure to low concentrations (20 nM) of WIN
55,212-2 (WIN-2) in all neurons tested. Further reduction in
IK amplitude (46%, Fig. 2,
inset) was obtained at higher cannabinoid (40 nM)
concentrations [WIN-2 = 0.53 ± 0.06 nA,
F(1,173) = 19.07, P < 0.001, Table 1].
Figure 1 shows that the reduction in
IK amplitude following exposure to
WIN-2 was uniform across all voltage steps, and control amplitudes were recovered within 5 min after washout. The effect of differing concentrations of WIN-2 (5-80 nM) on
IK activation is plotted in Fig. 2 as
G/Gmax, calculated from
I/Imax [i.e.,
GK = IK/(Vm EK) with
EK ~
100 mV], then fitted to a
Boltzmann function: G/Gmax = 1/{1 + exp[(V1/2
Vm) × 1/k]}. If
Imax remained constant at all doses of
WIN-2, then the effect of WIN-2 would be to alter V1/2 (Deadwyler et al. 1993
,
1995
). Increases or decreases in Imax produce no change in
V1/2, but such changes alter the slope factor, 1/k, of the fitted Boltzmann activation curves. The
decreasing slopes (range of k = 8.9-37.4) of the
activation curves (Fig. 2) reflect a WIN-2 concentration-related
decrease in maximum IK steady-state
amplitude at every voltage step in the activation protocol
[F(5,173) = 18.42, P < 0.001] with
no significant change in the voltage dependence for activating
IK (mean
V1/2 change = 1.8 ± 1.7 mV).
Exposure to the CB1 receptor antagonist SR141716A (300 nM) 5 min prior
to application of WIN-2 (40 nM) completely blocked the decrease in
IK amplitude [mean = 0.96 ± 0.08 nA, F(1,173) = 0.54, P = 0.46, Table 1].
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Cannabinoid reduction in IK requires Gs proteins
Prior studies have demonstrated that CB1 receptors are linked to
IA and ID via inhibitory
Gi/o proteins. Although reported elsewhere
(Deadwyler et al. 1993, 1995
; Hampson et al.
1995
), the linkage is summarized in Table 1 by the following
results: application of WIN-2 (40 nM) produced a 15- to 20-mV positive shift in voltage dependence for steady-state inactivation
(V1/2, mV). A similar 15-mV positive shift
was produced when Gi/o proteins were stimulated
by GTP
S (600 µM) with no further effect produced by addition of
WIN-2 [Table 1, all F(1,214) > 10.35, P < 0.001]. However, inactivation of the G
proteins with GDP
S (400 µM) did not block WIN-2 effects on
IA, suggesting that CB1 receptors were still capable of activating the Gi/o proteins.
When cultures were pretreated for 18 h with PTX (10 µg/ml),
which inactivates Gi/o proteins, the effects of
WIN-2 on IA were blocked (Table 1). Pretreatment with CTX (10 µg/ml), however, produced the opposite effect, a 10-mV negative shift in
IA inactivation
(V1/2 =
83.8 ± 2.7 mV,
P < 0.01, Table 1). This was consistent with an
increase in cAMP due to stimulation of adenylyl cyclase and is similar to previously noted effects of cAMP analogues on
IA (Deadwyler et al.
1995
). In contrast, the positive shift in
V1/2 for
IA inactivation produced by WIN-2 was
not blocked by pretreatment with CTX
(V1/2 =
70.8 ± 1.3 mV) because
even though the resultant V1/2 was not significantly different from control, it was still significantly shifted [F(1,214) = 10.97, P < 0.001] from CTX alone. These results indicate
that CB1 receptor linkage to the IA
channel required activation of Gi/o as opposed to
Gs proteins.
In the current study, PTX treatment did not alter the
WIN-2-induced reduction of IK
amplitude [0.62 ± 0.11 nA, F(1,173) = 2.11, P = 0.15, Fig.
3, Table 1]. Similarly, dialyzing cells with GTPS (600 µM), to activate G proteins had no effect on its own (0.96 ± 0.04 nA) nor did it not block the decrease in
IK steady-state amplitude produced by
WIN-2 [0.56 ± 0.05 nA, F(1,173) = 18.28, P < 0.001, Table 1,
Fig. 3]. Further, the decrease in IK
produced by WIN-2 was reversed to control levels within 3-5 min on
washout, indicating no permanent effect of GTP
S on
IK. However, pretreatment of cultures
with cholera toxin (CTX, 1 µg/ml), which irreversibly activates
Gs proteins, resulted in a decrease in
IK amplitude [0.48 ± 0.09 nA,
F(1,173) = 21.8, P < 0.001], and application of WIN-2 had no further effect [0.52 ± 0.06 nA, F(1,173) = 1.74, P = 0.10, Table 1]. This was confirmed by dialyzing
cells with GDP
S (400 µM) to inhibit
Gs-protein stimulation (Fig. 3), which subsequently blocked the WIN-2-related decrease in
IK amplitude [0.96 ± 0.09 nA,
F(1,173) = 0.29, P = 0.59], thus the cannabinoid induced decrease in
IK amplitude appeared to be mediated
by stimulatory (Gs) and not inhibitory
(Gi/o) G proteins.
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Cannabinoid effects on IK do not involve protein kinase A
The reduction in IK amplitude by
cannabinoids was examined with respect to dependence on changes in
protein kinase A (PKA). Unlike IA
(Mu et al. 2000) (see also Table 1),
IK was insensitive to cellular
dialysis of the catalytic subunit (PKAc, 50 U) of PKA (mean = 0.98 ± 0.09 nA, Table 1) and, as indicated in Fig. 4, the amplitude reduction of
IK by WIN-2 was not changed [WIN + PKAc: mean = 0.52 ± 0.06 nA, F(1,173) = 20.02, P < 0.001]. This was also true of cells
treated with PTX [WIN-2 + PTX; mean = 0.62 ± 0.11 nA,
F(1,173) = 15.67, P < 0.001] or co-administered with IP-20 (6 µM), a specific inhibitor of
PKA [WIN-2 + IP-20: mean = 0.60 ± 0.07 nA,
F(1,173) = 16.54, P < 0.001]. The
resistance of the WIN-2 induced reduction in
IK to the preceding agents indicates that modulation of IK, although CB1
receptor dependent, did not act through the same signaling pathway
previously shown to modulate IA
(Deadwyler et al. 1995
; Mu et al. 2000
).
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Protein kinase C reduces IK amplitude
The absence of modification by PKAc, PTX, IP-20, and GTPS on
IK amplitude, coupled with its
sensitivity to Gs-protein activation (CTX and
GDP
S), coupling to a second messenger other than PKA. Although
manipulation of protein kinase C (PKC) activity had no effects on
IA or on WIN-2 modulation of
IA (Table 1), the preceding effects
suggested changes in PKC activity as a potential mediator of the
WIN-2-produced reduction in IK
amplitude. This was tested by activating PKC with arachidonic acid (AA)
as a stimulating agent (Luo and Vallano 1995
;
Nishizaki et al. 1999
; Shraga-Levine et al.
1996
) and by testing specificity with a PKC inhibitor, staurosporine (Staur). Figure 4 shows the effects of each of these agents on IK amplitude. AA (1.0 µM
in pipette) reduced IK amplitude [0.64 ± 0.10 nA, F(1,173) = 14.8, P < 0.001, relative to control] to nearly the
same degree as WIN-2 (Table 1). The two drugs (WIN-2 and AA) if
co-applied at maximum effective concentrations, produced the same
effect as either drug alone [WIN-2 + AA: 0.62 ± 0.11 nA,
F(1,173) = 15.6, P < 0.001].
Administration of the PKC inhibitor staurosporine (Staur, 10 µM in
bath) had no effect by itself (0.94 ± 0.11 nA) but blocked the
effect of WIN-2 on IK amplitude
[WIN-2 + Staur: 0.92 ± 0.11 nA, F(1,173)
2.64, P = 0.10]. The effects of AA (1.0 µM) on
IK amplitude were not antagonized by
the CB1 receptor antagonist, SR141716A [0.66 ± 0.13 nA,
F(1,173) = 14.08, P < 0.001, Table 1], therefore the reduction in
IK amplitude by WIN-2 in Fig. 3
appeared to require increased PKC activity.
Interactions between CB1 and kappa opioid receptor modulation of IK amplitude
The kappa opioid receptor agonist U50,488 has also been
shown to reduce IK amplitude
(Baraban et al. 1995; Piros et al. 1996
), similar to the effects of WIN-2 demonstrated here. Table 1 shows that
the effects of the kappa receptor agonist U50,488 were also blocked by
addition of staurosporine to the bathing medium, and there was no
further effect of U50,488 following CTX ribosylation of
GS proteins. Thus kappa mediated effects on
IK, like CB1 receptors, likely
involved GS proteins and PKC signaling pathway
(Horn et al. 1994
; Kandasamy et al.
1995
). To determine whether this was the case, the
respective agonists for the two receptors (WIN-2 and U50,488) were
tested against the antagonist for the counterpart (Fig.
5). U50,488 produced a significant
decrease in IK [0.51 ± 0.04 nA,
F(1,173) = 20.46, P < 0.001] that was not altered by co-administration of SR141716A
(0.52 ± 0.06 nA). In contrast, application of the kappa receptor
antagonist nor-BNI produced a significant increase in
IK amplitude [1.14 ± 0.06 nA,
F(1,173) = 6.71, P < 0.01], suggesting a tonic activation of the kappa receptor and
subsequent decreased IK amplitude
under normal conditions in culture. Interestingly nor-BNI also greatly
attenuated the effects of WIN-2 on IK
amplitude [WIN + nor-BNI: 0.90 ± 0.08 nA, F(1,173) = 3.48, P = 0.15] as indicated
in Fig. 5. These results indicate that CB1 cannabinoid and kappa opioid
receptors may share a common signaling pathway involving PKC that is
linked to the conductance properties of the
IK channel.
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DISCUSSION |
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Prior reports from this laboratory have demonstrated that CB1
cannabinoid receptors enhance IA via
Gi/o protein linkage to cAMP and PKA sensitive
pathways that alter IA voltage
dependence (Deadwyler et al. 1993, 1995
). Cannabinoid
effects on IA are therefore presumed
to be mediated by inhibition of cAMP production (Bidaut-Russell et al. 1990
; Childers and Deadwyler 1996
;
Deadwyler et al. 1993
, 1995
; Howlett
1990
), reduced levels of cAMP-dependent PKA (Deadwyler et al. 1995
; Mu et al. 2000
), and a resultant
de-phosphorylation of the IA channel
(Mu et al. 2000
).
It is possible that the reduction in
IK amplitude demonstrated in the
present study also results from a change in phosphorylation status of
the IK channel. Voltage-dependent
potassium currents in several preparations have been shown to be
enhanced by phosphorylation of channel proteins (Chung and
Schlichter 1997; Egan et al. 1993
; Ivanina et al. 1994
; Xu et al. 1996
). A
reduction in phosphorylation as a result of CB1 receptor activation
could result in decreased IK amplitude
by a mechanism reciprocal to that described in the preceding text for
IA (Mu et al. 1999b
,
2000
). However, lack of blockade of WIN-2's effect on
IK by PTX and GTP
S (Fig. 3)
indicates that reduction of IK
amplitude did not involve stimulating a
Gi/o protein that is linked to the cAMP signaling
pathway (Table 1). Felder and co-workers (Felder et al.
1998
; Glass and Felder 1997
) showed that if
Gi/o protein binding is saturated by
co-application of a D2 receptor agonist, CB1
receptors consequently activate Gs proteins. This
mechanism is not a likely possibility in the present study because
resultant stimulation of Gs protein via CTX
(Fig. 3) mimicked the effect of CB1 receptor activation on IK amplitude, whereas CB1 activation
was still capable of altering the voltage dependence of
IA via stimulation of the
PTX-sensitive Gi/o protein pathway (Table
1).
The current study also confirmed prior reports that activation of the
kappa opioid receptor decreased IK
amplitude in cultured neurons (Baraban et al. 1995;
Simmons and Chavkin 1996
). Like cannabinoid receptors,
kappa opioid receptors are also linked to both Gs
and Gi/o proteins (Aghajanian and Wang
1987
; Eriksson et al. 1992
; Fan et al.
1998
; Hescheler et al. 1987
; Konkoy and Childers 1993
; Nah et al. 1993
). Furthermore,
kappa receptor modulation of IK
amplitude has been shown by others (in addition to this study) to be
PTX insensitive and Gs protein dependent
(Fan and Crain 1995
; Piros et al. 1996
)
and does not require a change in PKA or calcium-dependent PKC activity
(Baraban et al. 1995
).
Interactions between cannabinoid and opioid compounds were predicted
some time ago by Kaymakcalan (Ulku et al. 1980) whose studies suggested that the similarity between the pharmacological effects of both drugs may involve a common pathway. More recently, it
was demonstrated that the antinociceptive but not the behavioral effects of
9-THC in mice were blocked by
intrathecal administration of nor-BNI (Smith et al.
1994
). Another recent report showed that the
ED50 for antinociceptive effects of
9-THC was significantly reduced by
pretreatment with an inactive analog of morphine and that the effect
was prevented by both kappa and mu opioid antagonists (Reche et
al. 1998
). These results suggest that either cannabinoids act
directly on opioid receptors (Smith et al. 1994
) or they
modulate release of transmitter from opioid neurons (cf. Miller
and Walker 1995
). Because cannabinoid agonists have
very low affinity for all opioid receptor subtypes (Ali et al.
1989
) and because cannabinoid modulation of release of opioid peptides have only recently been reported in spinal cord (Mason et al. 1999
), neither of the preceding mechanisms, at this
point, is a probable basis for the cannabinoid and kappa opioid
receptor interactions on IK shown here.
The likelihood that CB1 and opioid receptors share a common
second-messenger pathway in the modulation of
IK amplitude is well supported by the
current findings and ancillary studies. The clear demonstration in the
present study that the actions of CB1 and kappa receptor agonists on
IK amplitude were additive was shown
when low concentrations of WIN-2 (20 nM) or U50,488 (1 µM), if
co-administered, produced near maximal reduction in IK amplitude in the same manner as
either drug alone at high concentration (Fig. 5). In addition, the
increase in IK amplitude
produced by nor-BNI (Fig. 5) suggests tonic activation of the kappa
receptor in primary cultures of hippocampal neurons. However,
application of SR141716A alone produced no similar increase in
IK amplitude nor did it alter the
effectiveness of the kappa receptor agonist, U50,488 (Fig. 5). Because
nor-BNI attenuated the effects of WIN-2 (Fig. 5, WIN-2 plus nor-BNI)
and because both CB1 and kappa receptors attenuated
IK through Gs
proteins and their effects were additive, it is possible that both
receptors acted via a common pathway. In this case, the attenuation of
WIN-2 effects on IK could also result
via a sequestration of Gs proteins by the kappa
receptor in the presence of nor-BNI. Nor-BNI acts as an inverse agonist as well as competitive antagonist, thus binding of nor-BNI to the kappa
receptor could result in effective inactivation of a limited pool of
Gs proteins available to link to either kappa or
CB1 receptors and hence a reduced effectiveness of the CB1 receptor.
Figure 6 illustrates the suspected nature
of the interaction between cannabinoid and kappa opioid receptors
showing convergence on AA-stimulated PKC as the shared substrate
controlling IK amplitude (Horn
et al. 1994; Kandasamy et al. 1995
;
Lahnsteiner and Hermann 1995
). However, if cannabinoids
produce their effects on IK via an
AA-PKC pathway (Fig. 6), the eventual consequence would be to
inactivate the CB1 receptor according to the recently described PKC-sensitive phosphorylation of a critical G protein linkage site on
the receptor (Garcia et al. 1998
).
|
In contrast to the preceding findings, Heath and Terrar
(2000) found in cardiac myocytes that potassium currents with
IK-like characteristics were increased
by elevations in PKC. Also, Zhu et al. (1998)
found that
AA elevated potassium currents that were not pharmacologically isolated
in hypothalamic neurons recorded in vitro. In the latter case the
concentrations of AA were nearly an order of magnitude larger (50 µM)
than used in the present study. Nevertheless these findings remain at
odds with the decrease in IK
demonstrated here and suggest that in different cells and under
different conditions (adult vs. developing neurons) the actions of PKC
on IK may differ.
The precise physiological consequences of convergent cannabinoid and
opioid inhibition of IK in hippocampal
neurons is not immediately apparent since cannabinoids have effects on
other potassium currents in these neurons as well (Deadwyler et
al. 1993; Mu et al. 1999b
; Schweitzer
2000
), all of which affect synaptic and cell firing properties
(Kirby et al. 1994
; Mu et al. 1999b
; Sullivan 1999
). In addition, the reported suppression of
LTP by both cannabinoids and opioids in hippocampal neurons
(Misner and Sullivan 1999
; Sandin et al. 1998
;
Terman et al. 1994
) suggests that both receptors alter
synaptic plasticity, perhaps via the effects demonstrated here on
voltage-dependent IK. It has been previously shown, and replicated in the current study, that
cannabinoids modulate potassium currents in hippocampal neurons, via
combinations of Gi/o and Gs
proteins, PKA-dependent and independent (possibly PKC) mechanisms
(Figs. 3-5). The presumption that CB1 receptors share a common
Gs-AA-PKC pathway for modulating
IK amplitude with kappa opioid
receptors provides insight at the cellular level as to how these two
receptor systems could act in synergy to modulate responses produced by
the activation of either receptor alone. Such synergism would
potentiate the action of submaximal doses of the ligands for each
receptor, or as in the case of kappa receptor antagonists, even
suppress ligand gated actions of the other receptor. Once demonstrated,
it is no longer sufficient to disregard such interactions in assessing
the effect of receptor-coupled signaling at the whole cell level.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Shou-Yuan Zhuang for technical assistance.
This work was supported by National Institute on Drug Abuse Grants DA-07625, DA-03502, and DA-00119 to S. A. Deadwyler.
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FOOTNOTES |
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Address for reprint requests: S. A. Deadwyler, Dept. of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston Salem, NC 27157 (E-mail: sdeadwyl{at}wfubmc.edu).
Received 14 February 2000; accepted in final form 18 July 2000.
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REFERENCES |
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