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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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M), but not the selective CB2 receptor ligand SR144528 (1 M), inhibited the outward current. Pertussis toxin (100 ng ml-1 for 20 h) completely abolished the outward current.
M CP55,940.
M CP55,940 in a concentration-dependent manner.
M CP55,940.
M) and 8-Br-cyclic AMP (10 M) significantly reduced the outward current evoked by 10 M CP55,940.
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INTRODUCTION |
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To date three cannabinoid receptors, CB1, CB1A and CB2, have been identified. They belong to the superfamily of seven transmembrane-spanning region, Gi/Go protein- coupled receptors. CB1 receptors have been cloned (Matsuda et al. 1990) and found in the central and peripheral nervous systems and certain peripheral non-neuronal tissues. In the brain CB1 receptors are highly localised in the hippocampus, cerebellum and substantia nigra (Glass et al. 1997). They are also located presynaptically on peripheral nerves in the gut, vas deferens and bladder (Pertwee et al. 1996). However, it is now becoming apparent that CB1 receptors are not restricted to neuronal cells. They have been identified on certain immune cells (Parolaro, 1999) and smooth muscle cells (Filipeanu et al. 1997). Stimulation of CB1 receptors can cause inhibition of adenylyl cyclase (Childers & Deadwyler, 1996) which has been linked to a number of intracellular events including the inhibition of nitric oxide production (Waksman et al. 1999). In other preparations, CB1 receptors have been shown to stimulate MAP kinase through a Gi protein (Bouaboula et al. 1999), or control the activation of inwardly rectifying K+ channels (McAllister et al. 1999), and the inhibition of voltage-dependent Ca2+ channels (Twitchell et al. 1997). Other studies have described the coupling of CB1 receptors to the stimulation of phospholipase C (Ho et al. 1999).
In addition to these effects, some authors have reported changes in intracellular calcium concentration ([Ca2+]i) by cannabinoid compounds in both neuronal (NG108-15 cells, Sugiura et al. 1996) and non-neuronal preparations (endothelial cells, Mombouli et al. 1999). Filipeanu et al. (1997), using fura-2 fluorometry, described an increase in [Ca2+]i in DDT1MF-2 smooth muscle cells following CB1 receptor stimulation by the herbal CB receptor agonist 
9-tetrahydrocannabinol. DDT1MF-2 cells, derived from a Syrian hamster vas deferens carcinoma by Norris & Kohler (1974), possess Gq protein-coupled histamine receptors (H1) and purinergic receptors (P2Y) for which the signal transduction pathways have been studied in detail (cf. Molleman et al. 1991a). Activation of H1 and P2Y receptors in DDT1MF-2 cells increases the phosphatidyl inositol diphosphate (PIP2) turnover to inositol-(1,4,5)-trisphosphate (IP3) and diacylglycerol. The IP3-induced release of Ca2+ from the sarcoplasmic reticulum (SR) evokes a Ca2+-dependent K+ current that can be measured using the whole cell version of the patch clamp technique (DenHertog et al. 1992). DDT1MF-2 cells are convenient for studying receptor stimulation-linked increases in [Ca2+]i, as they do not express voltage-gated Ca2+ channels (Molleman et al. 1991b) . The aims of our study were to investigate the effect of CB receptor stimulation in DDT1MF-2 cells on Ca2+-dependent K+ current, to identify the source of the resulting increase in [Ca2+]i, and to determine the signalling pathways involved between CB receptor activation and the increase in [Ca2+]i. Part of this study has been published previously in abstract form (Begg et al. 1999).
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METHODS |
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Cell culture
The DDT1MF-2 cell line was a gift from Dr S. A. Nelemans (University of Groningen, Netherlands), and a CHO (Chinese hamster ovary) cell line stably transfected with the human CB1 receptor was a gift from Dr C. T. O'Shaughnessy (GlaxoWellcome UK). The cells were cultured at 37 oC in closed 25 cm2 flasks and plated on 9.6 cm2 6-well plates and grown to near confluency in an atmosphere of 5 % CO2-95 % O2. DDT1MF-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum, penicillin (50 g ml-1), streptomycin (50 g ml-1) and L-glutamine (2 mM), harvested by means of a plastic cell scraper and plated on glass coverslips for electrophysiology. CHO cells were grown in DMEM-F12 Ham's medium supplemented with 9 % fetal calf serum, 1 mg ml-1 neomycin, L-glutamine (2 mM) and 500 g ml-1 hygromycin, and harvested by means of trypsinisation. All culture media and supplements were obtained from Gibco UK.
Patch clamp
Glass pipettes were made using Clark (UK) GC150TF-10 capillaries, fire polished and backfilled with intracellular solution (for composition see 'Solutions'), giving a typical resistance of 3-7 M
. Chloride-coated silver wire connected the pipette filling fluid to the probe input. The probe of the patch clamp amplifier (Axopatch-1D, Axon Instruments, USA) was mounted on a course manipulator (MC35A, Narishige, Japan) and directed to the cells by means of a hydraulic micromanipulator (MHW-3, Narishige). The data acquisition was performed using a digital interface (Digidata 1200, Axon Instruments) connected to a Viglen personal computer (UK), and the software pCLAMP 6 (Axon Instruments). This software was also used for offline data analysis.
All experiments were performed at room temperature (22 oC). Whole cell patch clamp measurements under voltage clamp were performed after gigaseal formation (Hamill et al. 1981), followed by disruption of the membrane under the pipette by a suction pulse. The process was monitored by applying a test pulse (10 mV, 5 ms, 100 Hz) to the cell which allowed the analysis of a resting leak current and enabled optimal capacitive transient cancellation. Cells were clamped at a holding potential of -60 mV, which is close to the resting membrane potential for these cells (Molleman et al. 1989). During experimentation the holding potential was set to -30 mV to enhance any outward current. In one type of experiment a ramp protocol was used: the membrane potential was stepped to -130 mV for 50 ms then increased to -30 mV over a 1 s period. The reversal potential of the outward current was calculated by subtracting the ramp response under control conditions from the ramp response in the presence of cannabinoids.
Western blot analysis
Western blotting was carried out as described previously (Baydoun & Morgan, 1998) using antibodies raised against the first 77 residues of the amino terminal of the rat CB1 receptor (Tsou et al. 1998). Briefly, cell lysates (20 g protein per lane) were separated by SDS-PAGE, transferred onto 0.2 m nitrocellulose membrane (Anderman and Co., Kingston-upon-Thames, UK) and blocked for 1 h at room temperature in 100 mM NaCl, 10 mM Tris, 0.1 % (v/v) Tween-20, pH 7.4 (STT) containing 5 % (w/v) non-fat dried milk. Membranes were then incubated overnight with either the anti-CB1 antibody alone (1:1000 dilution in STT containing 5 % (w/v) non-fat dried milk) or with antibody pre-incubated with its fusion protein (2 g). Antibodies and fusion proteins were a gift from Dr K. Mackie (University of Washington, Seattle, WA, USA). Blots were washed with STT (6 X 10 min) and incubated with a 1:10 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. Following further washing (6 X 10 min) in STT, immunoreactive bands were visualised using an ECL detection system (Amersham, UK).
Solutions
Electrophysiological measurements were performed in extracellular solution (ECS) containing (mM): NaCl 125, KCl 6, MgCl2 2.5, NaH2PO4 1.2, Hepes 10, glucose 11, sucrose 67, CaCl2 1.2, pH 7.35. The intracellular solution (ICS) contained (mM): NaCl 5, KCl 142, MgCl2 1.2, Hepes 20, glucose 11, K-ATP 5, Na-GTP 0.1, pH 7.2. All drugs were administered in the ECS, superfused at a rate of 4 ml min-1 with the exceptions of thapsigargin, which was added to the ICS, and pertussis toxin, which was added to the cell culture medium 20 h before recording. After each experiment the apparatus was thoroughly washed with dilute hydrochloric acid, ethanol and distilled water to avoid any carry-over of cannabinoids.
Drugs
All drugs were obtained from Sigma UK, unless stated otherwise. CP55,940 was obtained from Tocris UK. SR141716 and SR144528 were a kind gift from Sanofi Recherche, France. All cannabinoid drugs were dissolved in ethanol, shielded from light and kept at -20 oC. Histamine was dissolved in distilled water and kept at 4 oC. PD90589 was obtained from Calbiochem UK, dissolved in DMSO, shielded from light and kept at -20 oC. Recombinant charybdotoxin and recombinant iberiotoxin were obtained from Alomone Labs, Israel, and dissolved in a buffer consisting of 0.1 % bovine serum albumin, 0.1 mM NaCl, 0.01 % EDTA and 0.1 % Tris. 8-Br-cyclic AMP was obtained from Roche UK, and dissolved in distilled water. U73122 was dissolved in ethanol and kept at -20 oC. Pertussis toxin and SQ22,526 were dissolved in distilled water and kept at 4 oC.
Statistical analysis
Values are presented as means ± standard error of the mean. Student's unpaired t test was used to assess significant differences between values when comparing two groups. A one-way ANOVA test was performed, followed by a post hoc Dunnett test to assess significant differences between control and multiple test values. Significance was assumed if P < 0.05.
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RESULTS |
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Effect of cannabinoid application on membrane current
The synthetic CB receptor agonist CP55,940 evoked a concentration-dependent transient outward current in DDT1MF-2 cells (Fig. 1). A peak of 558 ± 47 pA was evoked by 10 M CP55,940 after 124 ± 6 s (n = 6), and further increases in concentration did not produce any further increase in outward current.
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A, a sample trace showing the outward current evoked by 10
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The selective CB1 receptor ligand SR141716, at a concentration of 1 M, delayed the onset of the outward current evoked by 10 M CP55,940 (peak occurred at 332 ± 9 s, a significant increase of 268 % from control, n = 6), and reduced the current to 133 ± 21 pA (a significant inhibition of 77 %, n = 6) (Fig. 2). SR141716, at a concentration of 1 M evoked no response by itself, but at the higher concentration of 10 M, it produced an outward current (peak 537 ± 47 pA, n = 6). The selective CB2 receptor ligand SR144528, at a concentration of 1 M, had no effect on the size of the outward current (514 ± 25 pA, n = 5), or the latency of the peak of the outward current (peak occurred at 131 ± 8 s, n = 5).
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M CP55,940
A, sample trace of outward current evoked by 10
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To identify the G protein involved in the CB receptor- mediated response, DDT1MF-2 cells were incubated with culture medium containing pertussis toxin (100 ng ml-1) for a period of 16 h. This pre-treatment completely abolished the outward current evoked by 10 M CP55,940 (Fig. 5, n = 6). In contrast, the outward current evoked by 10 M histamine was not affected by the pre-treatment with pertussis toxin (663 ± 25 pA, n = 6 vs. controls; 650 ± 65 pA, n = 6).
Identification of the CB receptor using Western blotting
Antibodies raised against the amino terminus of the rat CB1 receptor (Tsou et al. 1998) were applied to blots made with whole cell lysates of DDT1MF-2 cells, with CHO cells stably transfected with the rCB1 receptor as a positive control. The result shows a clear labelling in the region of 63 kDa in both DDT1MF-2 cells and CHO CB1 cells (Fig. 3). If the antibody was pre-incubated with its fusion protein, this labelling was completely absent (Fig. 3).
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Cells lysates obtained from DDT1MF-2 cells were separated by SDS-PAGE electrophoresis and probed with a polyclonal anti-CB1 antibody. Lysates obtained from CHO cells over-expressing the rCB1 receptor protein were used as positive controls. Lanes represent 1: lysates from DDT1MF-2 cells, 2: lysates from CHO cells. A, result when the primary antiserum was pre-incubated with fusion protein. B, result without pre-incubation with fusion protein.
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Characteristics of the outward current
A voltage ramp protocol was used to determine the reversal potential for the outward current evoked by CB receptor stimulation. The protocol was performed at the peak of the outward current evoked by 10 M CP55,940 and revealed that the reversal potential for the outward current was -84 ± 5 mV (n = 6) (Fig. 4). The K+ equilibrium potential, calculated using the Nernst equation, was -80 mV. The ramp protocol also showed that the current did not rectify in the range of membrane potentials studied (-130 to -30 mV).
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Graph was calculated by subtracting the ramp response (-130 to -30 mV over 1 s) under control conditions from the ramp performed at the peak of the response to 10
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Replacement of the K+ ions in the ICS with Cs+ ions, which non-specifically block K+ channels, completely blocked the outward current evoked by 10 M CP55,940. DDT1MF-2 cells that were exposed to the high conductance Ca2+-dependent K+ channel toxin iberiotoxin (10 nM) (Galvez et al. 1990) showed a strongly reduced outward current evoked by 10 M CP55,940 (40 ± 15 pA, a significant reduction of 93 % from control, Fig. 5, n = 4). Another high conductance Ca2+-dependent K+ channel toxin, charybdotoxin (10 nM) (Miller et al. 1985), completely abolished the outward current evoked by 10 M CP55,940 (Fig. 5, n = 4).
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M CP55,940
A, sample trace of outward current evoked by 10
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Role of calcium
To establish the contribution of Ca2+ from the extracellular space to the cannabinoid-induced response, experiments were performed with Ca2+-free ECS. Removal of Ca2+ from the ECS completely abolished the outward current evoked by 10 M CP55,940 (n = 8), even without the addition of EGTA to the ECS to chelate residual Ca2+. The addition of 0.1 mM Cd2+, which non-specifically blocks Ca2+ channels, to the ECS that contained normal Ca2+ also completely abolished the outward current evoked by 10 M CP55,940 (n = 3).
To establish the contribution of Ca2+ from intracellular stores to the cannabinoid-induced response, experiments were carried out with the blocker of the SR Ca2+-ATPase thapsigargin (0.1-10 M) (Treiman et al. 1998), applied to the ICS. Thapsigargin concentration-dependently evoked an outward current with a maximum of 612 ± 65 pA at 10 M (Fig. 6, n = 6). DDT1MF-2 cells that were exposed to iberiotoxin (10 nM) showed a strongly reduced outward current evoked by 10 M thapsigargin (122 ± 35 pA, a significant reduction of 80 % from control, Fig. 6, n = 3). Charybdotoxin (10 nM) completely abolished the outward current evoked by 10 M thapsigargin (Fig. 6, n = 3). The presence of thapsigargin reduced the outward current evoked by 10 M CP55,940 in a concentration-dependent manner (Fig. 7) with complete reduction at 10 M thapsigargin (n = 6). Ten micromolar thapsigargin also significantly but not completely reduced the outward current evoked by 10 M histamine (119 ± 35 pA, n = 6).
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M thapsigargin
A, sample trace of the sustained outward current evoked by 10
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M CP55,940
A, a sample trace of the effect of thapsigargin (0.1
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Inhibition of signal transduction pathways
To examine the involvement of phospholipase C in the cannabinoid-evoked response, DDT1MF-2 cells were exposed to the potent phospholipase C inhibitor U73122 for 15 min (Bleasdale et al. 1990). U73122 inhibited the outward current evoked by 10 M histamine in a concentration-dependent manner (Fig. 8), but had no effect on the outward current evoked by 10 M CP55,940.
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M CP55,940 ( ) or 10 M histamine ( )
Significant difference from control: *P < 0.01, †P < 0.001 and ‡P < 0.0001.
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To investigate the role of MAP kinase in the cannabinoid-evoked response, PD98059, a selective MAP kinase inhibitor (Alessi et al. 1995), was administered to DDT1MF-2 cells for 15 min. PD98059 inhibited the outward current evoked by 10 M CP55,940 in a concentration-dependent manner (Fig. 8). At the higher concentration of 10 M, PD98059 also inhibited the outward current evoked by 10 M histamine.
Involvement of cyclic AMP
To examine the involvement of adenylyl cyclase in the cannabinoid-evoked response, DDT1MF-2 cells were superfused with ECS containing the adenylyl cyclase inhibitor SQ22,536 (100 M) (Lippe et al. 1991) for a period of 15 min. SQ22,536 significantly reduced the outward current evoked by 10 M CP55,940 (Fig. 9), but had no effect on the outward current evoked by 10 M histamine. Administration of SQ22,536 (100 M) itself produced a transient inward current of -212 ± 54 pA (n = 4) (Fig. 9). This inward current was completely abolished when the intracellular K+ was replaced by the non-specific K+ channel blocker Cs+ (n = 4).
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M CP55,940 and 10 M histamine
A, sample traces showing the effect of the presence of 100
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DDT1MF-2 cells were superfused with ECS containing the membrane-permeable cyclic AMP analogue 8-Br-cyclic AMP (10 M) for 10 min. 8-Br-cyclic AMP significantly reduced the outward current evoked by 10 M CP55,940 or 10 M histamine (Fig. 10). Administration of 10 M 8-Br-cyclic AMP itself produced a transient outward current which peaked at 312 ± 45 pA (n = 6). This outward current was completely abolished when the intracellular K+ was replaced with the non-specific K+ channel blocker Cs+ (n = 4).
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M CP55,940 and 10 M histamine
A, sample trace showing the effect of the presence of 10
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DISCUSSION |
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Previous work has shown that activation of CB1 receptors in DDT1MF-2 cells evokes a rise in [Ca2+]i (Filipeanu et al. 1997). The aims of the present study were to see if the increase in [Ca2+]i, caused by CB1 receptor stimulation evokes a Ca2+-dependent K+ current, to establish the source of the increase in [Ca2+]i, and to determine the signalling pathway(s) between the CB1 receptor and Ca2+ mobilisation.
The synthetic cannabinoid receptor agonist CP55,940 evoked a concentration-dependent outward current that was inhibited by the CB1-selective ligand SR141716, but not by the CB2-selective ligand SR144528, suggesting that the response was mediated by CB1 receptors. This was confirmed by our observation that DDT1MF-2 cells possess a protein that binds antibodies raised against rat CB1 receptors. The molecular mass of the protein (~63 kDa) is consistent with values for the CB1 receptor reported in the literature (Tsou et al. 1998). At the lower concentration, SR141716 had no effect on membrane conductances, but at a tenfold higher concentration, SR141716 by itself elicited an outward current, possibly due to a direct effect on Ca2+ stores. High concentrations of SR141716 were shown to mobilise Ca2+ in endothelial cells through a pathway that did not involve the CB1 receptor (Mombouli et al. 1999). Our observation adds to the body of evidence that suggests that at high concentrations SR141716 is not a pure antagonist. Concentrations of CP55,940 required to elicit a response were several orders of magnitude higher than in tissue studies. One reason for this could be that long-term culture reduces the sensitivity of the receptor and/or signal transduction mechanism. This has been reported for P2 and H1 receptors in the same cell line (Molleman et al. 1990). A voltage ramp performed on the peak of the cannabinoid-induced current showed that the reversal potential was not significantly different from the calculated reversal potential for K+. The current was also blocked by the replacement of intracellular K+ by Cs+. Together these data show that the outward current evoked by CP55,940 is carried by K+. CB1 receptor stimulation in DDT1MF-2 cells leads to a rise in [Ca2+]i (Filipeanu et al. 1997), which raises the possibility that the outward current evoked by CP55,940 is a Ca2+-dependent K+ current. This is confirmed by our findings that the high conductance Ca2+-dependent K+ channel blockers iberiotoxin or charybdotoxin inhibited or completely abolished the cannabinoid-induced outward current. Previous work has shown that apamin, a toxin that blocks the family of small conductance Ca2+-dependent K+ channels, has no effect on the Ca2+-dependent K+ current evoked by a rise in [Ca2+]i in DDT1MF-2 cells (Molleman et al. 1989). Together these findings suggest the K+ current associated with CB1 receptor stimulation passes through large conductance Ca2+-dependent K+ channels, although within the membrane potential range studied (-130 to -30 mV) there was no outward rectification as expected from classic large conductance channels (Lattore et al. 1989).
The cannabinoid-induced outward current was completely abolished when the DDT1MF-2 cells were pre-treated with pertussis toxin. As expected, the Gq-coupled H1 histaminergic receptor-mediated rise in [Ca2+]i was not affected. This shows that the CB1 receptor is coupled to a Gi/Go protein. Gi/Go proteins also mediate rises in [Ca2+]i after CB1 activation in NG108-15 cells (Sugiura et al. 1996).
The removal of the extracellular Ca2+ or the addition of the calcium channel blocker Cd2+ to the ECS completely abolished the rise in [Ca2+]i through CB1 receptor stimulation by CP55,940. Therefore the rise in [Ca2+]i is completely dependent on the presence of extracellular Ca2+. This is dissimilar to the rise in [Ca2+]i evoked by histamine, which has a component independent of the presence of extracellular Ca2+ (Molleman et al. 1990).
Thapsigargin blocks the SR Ca2+-ATPase pump (Treiman et al. 1998) resulting in an emptying of Ca2+ stores, and a rise in [Ca2+]i. This rise in [Ca2+]i by thapsigargin evoked an outward current that was reduced by the high conductance Ca2+-dependent K+ channel blockers iberiotoxin and charybdotoxin showing that it was a Ca2+-dependent K+ current. Mobilisation of Ca2+ from thapsigargin-sensitive stores is the predominant source of the rise in [Ca2+]i associated with H1 receptor stimulation in DDT1MF-2 cells (DenHertog et al. 1992). Thapsigargin completely abolished the Ca2+-dependent K+ current evoked through CB1 receptor stimulation by CP55,940. This suggests that CB1 receptor stimulation mobilises Ca2+ from a thapsigargin-sensitive store, increasing the [Ca2+]i which evokes the observed Ca2+-dependent K+ current. Thapsigargin mobilises Ca2+ from the same store as that in which CB1 receptor stimulation mobilises Ca2+, so the presence of thapsigargin reduces the capacity of CB1 receptor stimulation to mobilise Ca2+. This is observed as a reduction in the outward current evoked by CP55,940. The predominant release of Ca2+ from stores, together with the sensitivity of the cannabinoid-induced response to extracellular Ca2+ suggests that there is a mechanism of capacitive Ca2+ entry into intracellular Ca2+ stores in DDT1MF-2 cells (VanderZee et al. 1995). Channels may exist that fill intracellular Ca2+ stores directly from the extracellular space, and the rise in [Ca2+]i by CB1 receptor stimulation may require this influx of Ca2+ to occur. Unsuccessful attempts were made to record this Ca2+ influx (M. Begg, M. E. Parsons & A. Molleman, unpublished observations), but recent publications have estimated that the level of Ca2+ influx may be too small to record using the patch clamp technique (Jackson et al. 1999).
Inhibition of the phospholipase C pathway by U73122 concentration-dependently inhibited the H1 receptor Ca2+-dependent K+ current, but did not affect the CB1 receptor-evoked Ca2+-dependent K+ current. This shows that the phospholipase C pathway is operational in DDT1MF-2 cells but is not involved in the rise in [Ca2+]i which evokes the CB1 receptor-mediated Ca2+-dependent K+ current.
Other signal transduction pathways that have been implicated in CB receptor signalling were investigated. The MAP kinase pathway is operational in DDT1MF-2 cells (Robinson & Dickenson, 1999) and could also be coupled to CB1 receptor stimulation (Boulaboula et al. 1995). Our results show that blockade of this pathway by PD98059 concentration-dependently inhibited the Ca2+-dependent K+ current caused by CB1 receptor stimulation. MAP kinase blockade also inhibited the H1 receptor-mediated Ca2+-dependent K+ current, but to a lesser extent than seen with CP55,940. Thus the MAP kinase pathway plays a positive role in CB1 receptor signalling, and it is interesting to speculate on possible mechanisms. It could be that MAP kinase activity is required for Ca2+-dependent K+ channel activation or Ca2+ mobilisation. This would be consistent with the sensitivity of H1 receptor signalling to MAP kinase pathway blockade. However, we found different sensitivities of CB1 and H1 receptor signalling to inhibition of the MAP kinase pathway, which suggests that this pathway is the predominant pathway in the Ca2+-dependent K+ current evoked by CB1 receptor stimulation. Alternatively, MAP kinase modulates the pathways between receptor stimulation and the resulting Ca2+-dependent K+ current following CB1 receptor stimulation more strongly than following H1 receptor activation. Other studies on CB1 signal transduction processes have shown a requirement for MAP kinase to be activated in addition to the predominant signal transduction pathway for the cellular response to occur (Sanchez et al. 1998).
The adenylyl cyclase pathway was investigated because the pertussis toxin results implicated the involvement of a Gi/Go protein in the cannabinoid response. Application of 8-Br-cyclic AMP to DDT1MF-2 cells evoked a transient outward current. Conversely, the application of the adenylyl cyclase inhibitor SQ22,536, to inhibit the production of cyclic AMP evoked a transient inward current. Both responses were blocked by the replacement of K+ ions by Cs+ ions in the ICS, which suggests that there is an increase or a decrease in outward K+ current in response to a corresponding increase or decrease in cyclic AMP. The transient nature of the responses indicates that there is a compensatory mechanism that restores any disturbance caused by the changes in cyclic AMP concentration.
Paradoxically, our results show that the cannabinoid-induced K+ current is reduced in the presence of 8-Br-cyclic AMP or SQ22,536, and the histamine-induced K+ current is reduced by the presence of 8-Br-cyclic AMP only. These results could be explained if cyclic AMP asserts an inhibitory action on the CB1 and H1 signalling pathways, where the CB1 signalling pathway actually requires a reduction in cyclic AMP concentration. Cyclic AMP inhibition of the phospholipase C pathway has been shown in DDT1MF-2 cells (Sipma et al. 1995), consistent with our observation that the H1 receptor-mediated response is reduced in the presence of 8-Br-cyclic AMP. The CB1 receptor can be negatively coupled to adenylyl cyclase through a Gi/Go protein (Childers & Deadwyler, 1996) and in DDT1MF-2 cells this might be a requirement for signal transduction. Thus, the saturation of intracellular cyclic AMP levels by exogenous 8-Br-cyclic AMP would then inhibit the CB1-evoked Ca2+-dependent K+ current as adenylyl cyclase inhibition by CB1 receptor stimulation would not significantly reduce the concentration of cyclic AMP. The inhibition of the CB1 receptor-mediated response by SQ22,536 could arise from the compensatory mechanism that is acting when cyclic AMP is reduced. This compensatory mechanism, possibly the activation of non-cyclic AMP-dependent protein kinases, would not be sensitive to a CB1 receptor-induced reduction in cyclic AMP.
To conclude, our data suggest that CB1 receptor stimulation in DDT1MF-2 cells leads to a Ca2+-dependent K+ current, evoked by a Gi/Go protein-mediated rise in [Ca2+]i, for which both inhibition of adenylyl cyclase and activation of MAP kinase are required. The cannabinoid-induced outward current is a result of an increase in [Ca2+]i which is likely to arise from capacitive Ca2+ entry.
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Acknowledgements
We would like to thank Dr Ad Nelemans (University of Groningen, Netherlands) for the gift of DDT1MF-2 cells, Dr Celestine O'Shaughnessy (GlaxoWellcome, UK) for the gift of CHO CB1 cells, and Dr Kenneth Mackie (Washington University, Seattle, USA) for the gift of CB1 antibodies and fusion proteins.
Corresponding author
A. Mo lleman: Department of Biosciences, C. P. Snow Building, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK.
Email: a.molleman{at}herts.ac.uk
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