Cannabinoid and Kappa Opioid Receptors Reduce Potassium K Current via Activation of Gs Proteins in Cultured Hippocampal Neurons

Robert E. Hampson, Jian Mu, and Sam A. Deadwyler

Department of Physiology and Pharmacology, Wake Forest University, Winston Salem, North Carolina 27157


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GDPbeta 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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GTPgamma 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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INTRODUCTION
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 beta -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 MOmega ) 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 MOmega , 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, GDPbeta S (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 GDPbeta 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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INTRODUCTION
METHODS
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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Fig. 1. Effect of WIN 55,212-2 (WIN-2) on IK. Top left: current traces illustrate IK elicited at each of the voltage steps in the activation protocol (see inset). Top right: TEA (25 µM) in the bathing medium reduced IK at all voltage steps. Middle: current traces showing reduction in IK following exposure to 20 µM (left) and 40 µM (right) WIN-2. Bottom: IK recorded as in the preceding text following 5 min washout of WIN-2 (left, 20 µM; right, 40 µM). Calibration: 250 pA, 100 ms.


                              
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Table 1. Effects of cannabinoids and second-messenger modulators on IK and IA

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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Fig. 2. Activation curves (Boltzmann functions) illustrating concentration dependent reduction in IK by WIN-2. Each symbol represents the mean ± SE current as relative conductance (G/Gmax) elicited by a given depolarizing voltage step in the activation protocol calculated across several different cells (see Table 1). The associated fitted Boltzmann curve shows the systematic reduction in IK with no significant shift in voltage dependence. Inset: plot of log-dose vs. percent inhibition of IK amplitude, recorded in response to +40-mV depolarizing step. Range of concentration of WIN,55 212-2: 8.0-80.0 nM, applied via pressure pipette.

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 GTPgamma 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 GDPbeta 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 GTPgamma S (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 GTPgamma 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 GDPbeta 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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Fig. 3. Lack of influence of Gi/o proteins on WIN-2 reduction in IK. Mean ± SE IK amplitudes plotted across all cells tested in each condition (see Table 1). Current traces depict the effect on IK in pertussis toxin (PTX)-treated cells, cells dialyzed with GTPgamma S and cells exposed to cholera toxin (CTX). WIN-2, 40 µM. Calibration: 250 pA, 100 ms. Bar graph shows effects of various G-protein modulators on WIN-2 modulation of IK amplitude.

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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Fig. 4. Comparison of the effects of protein kinases A and C (PKA and PKC) and WIN-2 on IK and IA. Current traces depict different actions of PKA and PKC agents on IK (top) and IA (bottom). Effect of WIN-2 on IA is to shift voltage dependence is shown as a change in amplitude for the same depolarizing voltage step. - - -, control, PKA. ---, effect of catalytic subunit of PKA (PKAc, left), arachidonic acid (AA) stimulation of PKC (middle), or 40 nM WIN-2 (right) = effect PKAc. Graph plots means ± SE only of IK amplitude in cells treated with the indicated agents (see Table 1). WIN-2, 40 µM. Calibration, 250 pA, 100 ms.

Protein kinase C reduces IK amplitude

The absence of modification by PKAc, PTX, IP-20, and GTPgamma S on IK amplitude, coupled with its sensitivity to Gs-protein activation (CTX and GDPbeta 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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Fig. 5. Comparison of the effects of kappa opioid receptor compounds with WIN-2 on IK amplitude. Current traces illustrate the effects of kappa receptor agonist, U50,488 (U50) and antagonist [nor-binaltorphinmine (nor-BNI)] alone and in conjunction with WIN-2 at indicated concentrations. Note increase in IK amplitude above control level following application of nor-BNI (2 µM). Graph indicates IK means ± SE for all conditions tested. Note the 2 different concentrations of WIN-2 and U50 shown to illustrate additivity of cannabinoid and kappa receptor influences on IK. Calibration, 250 pA, 100 ms.


    DISCUSSION
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ABSTRACT
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METHODS
RESULTS
DISCUSSION
REFERENCES

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 GTPgamma 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 Delta 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 Delta 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).



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Fig. 6. Illustration of putative interaction between kappa opioid and cannabinoid receptors on IK amplitude. Left: filled diamonds illustrate the fact that kappa receptors appear to have endogenous ligand or constitutive tonic activity. Largest current trace reflects condition in which the antagonist (nor-BNI) alone is bound to the receptor (Fig. 5). Right: triangles show endogenous cannabinoids not bound to the CB1 receptor under control conditions because application of the antagonist alone (SR 141617A) has no effect on IK. Smallest current trace reflects reduction in IK amplitude via activation of either kappa of CB1 receptors. Median trace reflects control condition. Middle: interaction between both receptors is hypothesized to occur via stimulation of Gs protein and activation PKC (or possibly arachidonic acid stimulation of PKC), which alters IK (Fig. 4).

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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