Abstract
It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Δ9-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; β-adrenergic receptor kinase (β-ARK) using low molecular weight heparin (LMWH), a β-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Δ9-THC. The PKG inhibitor KT5823, the β-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of Δ9-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in Δ9-THC-tolerant mice.
Δ9-THC is the major psychoactive component in marijuana. There are two known cannabinoid receptors: CB1, primarily in the central nervous system (Felder et al., 1993), and its amino-terminal variant, the CB1A receptor (Shire et al., 1995); and the CB2 receptor found on cells of the immune system (Munro et al., 1993). Δ9-THC produces psychoactive effects through binding to CB1 receptors (Ledent et al., 1999; Zimmer et al., 1999; Buckley et al., 2000) that have been cloned (Matsuda et al., 1990; Gerard et al., 1991; Munro et al., 1993). CB1 and CB2 receptors have specific antagonists (Rinaldi-Carmona et al., 1994, 1998).
CB1 and CB2 receptors are G protein-coupled receptors (GPCRs) linked to a Gi/o protein, which when activated inhibits the activity of adenylyl cyclase (Howlett and Fleming, 1984). Upon agonist binding, the βγ subunit dissociates from the α subunit of the Gi/o protein (Childers and Deadwyler, 1996). The α subunit inhibits adenylyl cyclase, whereas the βγ subunit has been linked to activation of other cellular events, such as activation of tyrosine kinases (TKs).
Tolerance develops to the in vivo and in vitro pharmacological effects of cannabinoids (Martin, 1985; Dill and Howlett, 1988; Mason and Welch, 1999). Receptor down-regulation is a possible mechanism of Δ9-THC tolerance (Oviedo et al., 1993;Rodriguez de Fonseca et al., 1994). Studies indicate that the cannabinoid receptor is rapidly internalized after binding of an agonist (Hsieh et al., 1999). However, Abood et al. (1993), found no alterations in cannabinoid receptor mRNA or protein levels in mouse whole brain homogenates after a chronic injection paradigm. Thus, the effects of long-term administration of Δ9-THC on receptor down-regulation are unclear. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. We hypothesized that modification of intracellular phosphorylations with several kinase inhibitors might attenuate the expression of THC-induced tolerance.
cAMP-Dependent Protein Kinase (PKA).
Acute administration of Δ9-THC decreases cAMP formation by inhibiting adenylyl cyclase and decreases PKA activity. Conversely, chronic cannabinoid exposure enhances the adenylyl cyclase activity, and increases cAMP levels and PKA activity in the same areas that CB1 receptor down-regulation is observed (i.e., cerebellum, striatum, and cortex) (Rubino et al., 2000). Thus, the adenylyl cyclase cascade seems to become constitutively active during tolerance.
PKG.
The cannabinoid levonantradol, but not dextronantradol, decreases basal and isoniazid-induced increases in cGMP (Leader et al., 1981). Thus, cannabinoids could alter cyclic-GMP formation (and thus PKG activity) in tolerance expression.
Protein Kinase C (PKC).
Δ9-THC increases the activity of brain PKC in vitro (Hillard and Auchampach, 1994; De Petrocellis et al., 1995). PKC seems to directly affect CB1 receptors. Phosphorylation of the CB1 receptor with PKC suppresses the modulation of calcium channels by cannabinoids (Garcia et al., 1998). Neurotransmitters that activate PKC restore the neuronal excitability and synaptic activity inhibited by cannabinoids. We hypothesized that PKC inhibitors might reverse Δ9-THC tolerance.
TKs.
CB1 receptor activation of the βγ subunit of G proteins can stimulate TKs. One target of activation by the βγ subunit is Src tyrosine kinase that has been shown to activate Ras, activating mitogen-activated protein kinase. CB1 and CB2 stimulation increases the activation of MAPK (Rinaldi-Carmona et al., 1998), which becomes tyrosine-phosphorylated in cannabinoid-treated cells, an effect blocked by TK inhibitors (Bouaboula et al., 1995). We tested the Src tyrosine kinase inhibitor PP1 (Daub et al., 1997) in mice for its effects on THC-induced antinociception and reversal of tolerance.
PI3-K.
PI3-K is an early intermediate of the Gβγ-mediated MAPK signaling pathway (Daub et al., 1997). Therefore, we proposed to block PI3-K and reduce the βγ subunit-mediated tyrosine phosphorylation of MAPK.
G Protein-Coupled Receptor Kinase (GRK).
β-Adrenergic receptor desensitization involves rapid PKA and GRK phosphorylation. GRK phosphorylation in turn promotes β-Arrestin binding and receptor internalization (Seibold et al., 1998). We inhibited β-adrenergic receptor kinase (β-ARK), a type of GRK, with low molecular weight heparin (LMWH) to evaluate the chronic affects of Δ9-THC after inhibition of β-ARK.
Finally, to evaluate a biochemical correlate to behavioral tolerance, we evaluated the release of dynorphin in the spinal cord of THC-tolerant and -nontolerant rats. Tolerance to Δ9-THC induces a decrease in dynorphin release temporally correlated to decreased antinociception (Mason and Welch, 1999). We hypothesized that reversal of behavioral tolerance by a kinase inhibitor might also reverse the decrement in dynorphin release observed in THC-tolerant rats.
Materials and Methods
Animal Model of Δ9-THC Tolerance.
All studies using the tail-flick test were performed on male ICR mice. The mice were kept on a 12-h light/dark cycle and received food and water ad libitum. In the acute studies, mice weighed 16 to 25 g; in chronic studies, mice weighed 25 to 34 g upon testing. Mice were rendered tolerant to Δ9-THC over 7 days. The mice received twice daily s.c. injections of Δ9-THC (20 mg/kg) for 6 days and on day 7 just received the morning dose. On the morning of day 8, mice were challenged with an ED80 dose (i.t.) of Δ9-THC for determination of tolerance. Rats were used for the spinal cord release of dynorphin to obtain sufficient cerebrospinal fluid for testing. Male Sprague-Dawley rats, weighing between 350 and 400 g, obtained from Harlan (Indianapolis, IN), were housed in plastic cages, two rats per cage, and maintained on a fixed 12-h light cycle at a temperature of 22 ± 2°C. Water and food (Harlan Rat Chow) were provided ad libitum. Rats were rendered tolerant to Δ9-THC using the doses and time course as for mice and were also challenged on day 8 with the ED80 dose of Δ9-THC (i.t.) for tolerance determination.
Intrathecal Injections.
Intrathecal injections were performed in mice following the protocol of Hylden and Wilcox (1983). Unanesthetized mice were injected with 5 μl of drug between the L5 and L6 area of the spinal cord with a 30-gauge needle. In studies using rats, the pentobarbital-anesthetized rats were placed in stereotaxis, and an incision was made on the atlanto-occipital membrane to expose the cisterna magna. A catheter of polyethylene-10 tubing was inserted through the exposed cisternal cavity, caudally, into the subarachnoid space of the spinal cord. The catheter contained an artificial cerebrospinal fluid, composed of 125 mM Na+, 2.6 mM K+, 0.9 mM Mg2+, 1.3 mM Ca2+, 122.7 mM Cl−, 21.0 mM HOC−, 2.4 mM HOP
Measurement of Dynorphin.
Measurement of dynorphin A(1-17) was accomplished using a dynorphin A(1-17)-specific radioimmunoassay kit obtained from Peninsula Laboratories (Belmont, CA). The reconstituted samples were analyzed in duplicate. The manufacturer reports cross-reactivity of dynorphin A(1-17) antibody as 100% versus dynorphin A(1-24), a parent compound, and less than 2% versus smaller peptide fragments. We found no cross-reactivity of the antibody to dynorphin A(1-8), dynorphin A(1-13), dynorphin B, anandamide, or morphine. Only the linear portion of the radioimmunoassay standard curve, between 0.1 and 64 pg/ml of the standard dynorphin peptide, was used to calculate dynorphin concentration. The cerebrospinal fluid from individual rats was analyzed for dynorphin concentrations using at least six rats per test group. The rats were evaluated in the tail-flick test before the removal of cerebrospinal fluid for testing. Thus, the behavioral effects of each rat can be compared with the dynorphin levels in that individual rat's spinal fluid.
Tail-Flick Test.
Mice and rats were tested for antinociception by the tail-flick procedure (D'Amour and Smith, 1941). Reaction times of 1.5 to 4 s were used for the control, whereas a time of 10 s was used for the cutoff to prevent tissue damage. Antinociception was quantified as the percentage of maximum possible effect (% MPE) formula: % MPE = 100 × [(test − control)/(10 − control)] (Harris and Pierson, 1964). % MPE values were calculated for each animal, using at least six animals per dose, for which mean effect and S.E.M. were calculated for each dose. At least three doses of each test drug or combination of drugs were used to generate dose-response curves.
Materials.
Doses for all drugs used were predetermined in naı̈ve animals using the maximal dose without toxicity. Time points were determined in naı̈ve animals to ascertain the point at which each drug had its peak effect. Dimethyl sulfoxide (100%, DMSO) was purchased from Sigma-Aldrich (St. Louis, MO). KT5720, purchased from Calbiochem (La Jolla, CA), was prepared in 100% DMSO and was injected i.t. at a dose of 2.7 μg/mouse 15 min before drug or vehicle (i.t.). The tail-flick test was then conducted 15 min after the second injection. KT5823, purchased from Calbiochem, was prepared in 100% DMSO and injected i.t. at a dose of 2.5 μg/mouse 15 min before drug or vehicle (i.t.). The tail-flick test was then conducted 15 min after the second injection. Dibutyryl-cAMP (10 μg/mouse) and dibutyryl-cGMP (5 μg/mouse) were purchased from Calbiochem and were prepared in distilled water (dH2O) and injected i.t 15 min before the i.t. injection of drug or vehicle. Fifteen minutes later, the tail-flick test was conducted. Δ9-THC was obtained from the National Institute on Drug Abuse and was prepared in 100% DMSO for acute tests and in 1 part ethyl alcohol (Aaper Alcohol and Chemical, Shelbyville, KY), 1 part Emulphor, and 18 parts 0.9% normal saline (Baxter, Deerfield, IL) (1:1:18) for tolerance studies. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and was prepared in 100% DMSO and injected i.t. 15 min before drug or vehicle (i.t.). The tail-flick test was then conducted 15 min after the second injection. Bisindolymaleimide I, HCl (bis; Calbiochem) was prepared in dH2O and injected i.t. (5 μg/mouse) or (0.5 μg/mouse) 15 min before drug or vehicle (i.t.). The tail-flick test was then conducted 15 min after the second injection. LMWH was purchased from Sigma-Aldrich and was prepared in dH2O and injected i.t. (30 μg/mouse) 15 min preceding the i.t. injection of drug or vehicle. The tail-flick test was then conducted 15 min after the second injection. PP1 purchased from Alexis Corporation (Läufelfingen, Switzerland) was prepared in 100% DMSO and injected i.t. 10 min before the i.t. injection of drug or vehicle, with the administration of the tail-flick test 15 min after the second i.t. injection.
Statistical Analysis.
Analysis of variance was used to determine significant differences between control and treatment animal groups followed by Dunnett's t test. These calculations were performed using StatView, version 512+ (BrainPower, Inc., Agoura Hills, CA). p values of less than 0.05 were deemed significant. Parallelism of the dose-response curves was determined by the methods of Tallarida and Murray (1987). Potency ratios were determined using the methods of Colquhoun (1971).
Results
The i.t. administration of KT5720, a PKA inhibitor, at a dose of 2.7 μg/mouse in 100% DMSO vehicle (i.t.), significantly (p < 0.05) reversed Δ9-THC antinociceptive tolerance in a dose-dependent manner, as determined by the tail-flick test. There was a leftward shift of the dose-response curve. The ED50 in the Δ9-THC-tolerant mice was shifted from 80 μg/mouse (95% confidence limits, 62–102) to 8.6 μg/mouse (95% confidence limits, 4.7–16) in the KT5720-treated mice. The lines were parallel and the potency ratio was 9.3 (Fig.1).
The PKG inhibitor KT5823, at a dose of 2.5 μg/mouse in 100% DMSO vehicle (i.t.), had no effect on Δ9-THC antinociceptive tolerance (2% MPE in the tolerant mice compared with 7% in the tolerant animals treated with KT5823; Fig.2 A). A higher dose of KT5823 (5 μg/mouse) had no greater effect. Pretreatment with 10 μg/mouse produced lethality.
The PKC inhibitor bis, at a dose of 0.5 μg/mouse administered i.t., did not affect the antinociceptive tolerance in mice. The % MPEs in the tolerant groups treated with bis compared with vehicle-treated mice were not significantly different (20 ± 15 versus 14 ± 6.0, respectively) (Fig. 2B). At an increased dose of 5 μg/mouse, there was not a significant shift in the ED50 values of the tolerant mice treated with bis compared with tolerant mice treated with vehicle [41 (32–51) versus 80 (50–129); Fig.3], although clearly there was a trend toward a rightward shift in the dose-effect curve. The dose-effect curves for mice pretreated with bis versus those pretreated with vehicle were not parallel in the tolerant mice. Interestingly, in the nontolerant mice, there was an attenuation of the antinociceptive effect of Δ9-THC. There was a significant (p < 0.05) rightward shift in the dose-response curve of Δ9-THC. The ED50 was shifted from 7 (5–11) in the vehicle-treated nontolerant animal to 26 (16–45) in the bis-treated nontolerant animal. The dose-effect curves were parallel and the potency ratio was 3.6.
LY294002, a phosphatidylinositol-3-kinase inhibitor, administered i.t. in 100% DMSO vehicle at a dose of 0.1 μg/mouse, did not significantly alter Δ9-THC antinociceptive tolerance in mice (Fig. 2C). The LY294002-treated tolerant mice had a % MPE of 10 ± 10 compared with the tolerant vehicle-treated mice who had a % MPE of 2 ± 1. At higher doses, LY294002 produced an antinociceptive effect that confounded use for tolerance reversal studies.
LMWH, which inhibits β-ARK, at a dose of 30 μg/mouse administered i.t., did not affect the antinociceptive tolerance in the mice. The % MPE in the Δ9-THC-tolerant group treated with LMWH (5 ± 2) was not significantly different from the vehicle-treated THC-tolerant group (14 ± 6.0) (Fig. 2D).
We also evaluated PP1, a Src family tyrosine kinase inhibitor. Because the βγ subunit of the cannabinoid receptor interacts with tyrosine kinase to activate MAPK, we wanted to determine what would happen if this pathway was disrupted. At a dose of 0.0001 μg/mouse, in 100% DMSO vehicle administered i.t., PP1 significantly (p < 0.05) reversed Δ9-THC antinociceptive tolerance in mice. The 0.0001 μg/mouse dose was shown to be inactive (% MPE 4 ± 1) in the tail-flick test in naı̈ve mice, and in the nontolerant group the % MPE (44 ± 20) did not differ from that of vehicle-pretreated mice (% MPE, 45 ± 19) (Fig.4). At doses of 0.001 μg/mouse and higher, PP1 shows a variable antinociceptive affect that confounded studies at the higher doses.
We also evaluated enhancement of tolerance. Because PKA inhibition reversed tolerance, we evaluated the ability of a cAMP analog to enhance tolerance. The challenge dose of Δ9-THC was increased to 100 μg/mouse i.t. to get approximately 50% MPE in the tolerant mice. Dibutyryl cyclic-GMP at 5 μg/mouse administered i.t. did not significantly enhance tolerance (36 ± 20% MPE in the tolerant animals compared with 46 ± 23% MPE in the db-cGMP-treated animals) (data not shown). Higher doses (30 and 50 μg/mouse) of db-cGMP had intrinsic antinociceptive effects. Dibutyryl cyclic-AMP at doses of 10 to 50 μg/mouse administered i.t. also did not enhance Δ9-THC antinociceptive tolerance (55 ± 14% MPE in the tolerant animals compared with 46 ± 23% MPE in the db-cAMP-treated animals for the 10 μg/mouse group). db-cAMP did not produce intrinsic antinociception at the doses tested.
A study was performed to address the hypothesis that KT5720 reversal of tolerance might restore dynorphin release by 300 μg/rat Δ9-THC to levels observed in nontolerant rats. Doses and time points for KT5720 administration and tail-flick testing were as those in the mouse. Figure 5A indicates THC-stimulated dynorphin release was significantly depressed in THC-tolerant rats (25 ± 7 pg/ml in non-THC-tolerant rats versus 6 ± 3 pg/ml in THC-tolerant rats). Administration of KT5720 before Δ9-THC did not alter dynorphin release in nontolerant rats (22 ± 6 pg/ml) compared with the nontolerant Δ9-THC only group. However, KT 5720 significantly reversed the Δ9-THC-stimulated decrease in dynorphin observed in Δ9-THC-tolerant rats (levels of dynorphin were raised to 32 ± 8 pg/ml). Figure 5B shows the behavioral response (tail-flick test) in the same rats. The rats were tolerant to Δ9-THC as indicated by “a”. KT5720 significantly reversed tolerance as indicated by “b”. Thus, as the behavioral response returned to nontolerant levels, the release of dynorphin increased to nontolerant levels.
Discussion
We sought to address the role of various kinases in Δ9-THC antinociceptive tolerance. We evaluated kinases that were downstream from the cannabinoid receptor (PKA, PI3-K, and TK), that may interact directly with the receptor (PKA, β-ARK, and PKC), and others that act in different pathways (PKC and PKG). When a ligand binds to a GPCR, as the cannabinoid receptor, there is a decreased affinity between the α and βγ subunits of the G protein, and they separate from one another. In the acute model of Δ9-THC exposure, the α subunit will produce a decrease in adenylate cyclase, decreases in cAMP, and decreases in PKA activation. There is also an associated opening of low-voltage potassium channels, leading to an efflux of potassium, and a modulation of calcium channels, leading to decreased calcium conductance. In animals chronically treated with THC, there is a compensatory increase in adenylate cyclase, cAMP, and PKA activation. The intrathecal administration of the protein kinase A inhibitor KT5720 reversed the antinociceptive tolerance to Δ9-THC in both mice and rats. Of significance, inhibition of PKA also reversed a biochemical correlate of tolerance, namely, dynorphin release. Thus, our data indicate that protein kinase A plays a role in the mechanism of Δ9-THC antinociceptive tolerance. The role of PKA in THC-induced tolerance is unknown. However, several possibilities for the site of action of PKA exist. PKA could be responsible for phosphorylating the CB1 receptor upon binding of a ligand to the receptor. PKA also could be increasing potassium conductance through phosphorylation of the potassium channel, causing the cell to become hyperpolarized. Other possible roles of PKA in THC-mediated tolerance include the possibility that PKA is rapidly and continuously phosphorylating the CB1 receptor if and when it is down-regulated into the cytosol in tolerant animals. The CB1 receptor seems to be rapidly internalized upon exposure to cannabinoids (Hsieh et al., 1999). Once the cell is “tolerant”, we propose that there will be a compensatory increase in production of PKA. Higher levels of PKA could be responsible for a continuous phosphorylation of the CB1 receptor while in the cytosol. This continued phosphorylation might facilitate the receptor down-regulation. Upon inhibition of PKA by KT5720, the receptor would in theory no longer remain phosphorylated and could therefore be recycled to the membrane where it would be capable of binding to the ligand again. If the phosphorylation of the receptor was maintained, and the receptor remained down-regulated, eventually it would be degraded, requiring mRNA for new protein synthesis.
Because PKA inhibition reverses cannabinoid antinociceptive tolerance, we hypothesized that a cAMP analog might enhance tolerance. In a nontolerant neuron exposed to cannabinoids, cAMP is decreased, but in the presence of forskolin, which increases cAMP, or Cl-cAMP, a cAMP analog, antinociception is attenuated (Cook et al., 1995). However, dibutyryl-cAMP did not enhance tolerance. Perhaps we were not able to increase levels of c-AMP to a level consistent with enhancement of tolerance.
We also evaluated kinases involved in the Gβγ-mediated signaling pathway. PI-3 kinase and tyrosine kinase work downstream from the βγ subunit of the GPCR, and these kinases are generally associated with growth and differentiation. With the membrane-destabilizing activity of cannabinoids and release of free arachidonic acid, such kinases might play a role in antinociception. LY294002 is a specific PI3-K inhibitor. PI3-K is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases (Vlahos et al., 1994). Pertussis-sensitive GPCRs and TKs may converge or share a common pathway upstream from the activation of MAPK. Our goal was to determine whether by blocking a kinase or kinases in the pathway leading to MAPK activation, we could reverse tolerance. Upon blocking PI3-K, tolerance was not affected. One caveat in our results was that we could not use higher doses of LY294002 due to its intrinsic analgetic effects. Thus, our results with PI3-K blockade are inconclusive. However, the blockade of the Src family tyrosine kinase with PP1 reversed tolerance. Thus, by blocking a tyrosine kinase, we may be inhibiting downstream actions of the βγ subunit. The βγ subunit may be necessary to maintain a tolerant state by activation of MAPK. Further studies need to be conducted looking for the role of the βγ subunit and tyrosine kinases and their role in central cannabinoid effects.
In addition to kinases downstream to the α and βγ subunits, we also tested other kinases involved in a variety of cellular processes. β-ARK is known to phosphorylate the β2-adrenergic receptor and is a potential candidate for phosphorylation of the cannabinoid receptor before internalization. Hsieh et al. (1999) noted that the CB1 receptor is internalized after a pathway grossly similar to the one used by the β2-adrenergic receptor. If such were the case, blocking β-ARK with LMWH might be expected to prevent receptor phosphorylation and possibly desensitization or down-regulation and reverse tolerance. Because LMWH did not reverse tolerance, it seems that the cannabinoid receptor is may not be phosphorylated by β-ARK. However, due to solubility and toxicity issues we may not have been able to increase the dose of LMWH to levels needed to block β-ARK.
PKC may act directly on the CB1 receptor and/or downstream from the receptor. It has been shown that cannabinoids increase brain protein kinase C activity in vitro (Hillard and Auchampach, 1994). Our data indicate that using two different doses of the PKC inhibitor did not alter tolerance to cannabinoids. However, we did observe at the higher dose of PKC inhibitor that the effects of Δ9-THC in nontolerant animals were significantly attenuated. Hillard and Auchampach (1994) showed that cannabinoids increase the levels of PKC in rat brain and that these increased levels are responsible for reestablishing neuronal excitability. Because inhibiting PKC attenuates the effects of cannabinoid-induced antinociception, it is likely that increased levels of PKC may be at least partially responsible for cannabinoid-induced antinociception.
In summary, the data presented indicate that by inhibiting PKA and Src tyrosine kinase, Δ9-THC antinociceptive tolerance can be reversed. It seems likely that these two kinases work independently of one another. The other kinase inhibitors for PKG, PKC, PI3-K, and β-ARK, did not alter tolerance at the doses testable. One has to take these negative data in the context of the possibility that the drug did not achieve high enough levels in the whole animal to inhibit the kinases. Thus, positive data as with PKA and Src TK indicate potential sites for Δ9-THC modulation, whereas negative data remain rather inconclusive. However, the higher dose of PKC inhibitor was shown to attenuate the antinociceptive effects of Δ9-THC in the nontolerant mice, which indicates that the inhibitor very likely reaches its site of action at a concentration that is active. However, although negative results must be interpreted with caution, positive results also need to be interpreted with caution as to the site of action of the drugs used. Our data indicate that the PKA and tyrosine kinase inhibition have prominent roles in tolerance to cannabinoids. The drugs used to inhibit such kinases (KT5720 and PP1, respectively) are the most selective drugs available. Such inhibitors likely act on PKA and TK at various sites intracellularly. It is intriguing that reversal of tolerance is a rapid process and occurs using drugs that do not alter the acute effects of THC. A similar effect has been shown for KT5720-induced reversal of tolerance to morphine (Bernstein and Welch, 1998). It takes several days to develop tolerance. The ability to reverse tolerance so rapidly is surprising and opens the door to a plethora of questions to be answered as to the plasticity of the neuronal system during the tolerance process. Certainly, the ability to reverse tolerance to any drug has profound clinical implications. A future direction would be to evaluate the phosphorylation state of the receptor. If PKA were responsible for the initial desensitization or maintaining the down-regulated state of the receptor, we would expect to see the receptor in the phosphorylated state in tolerant animals. We would also expect to see a dephosphorylated receptor in tissue that had been treated with the PKA inhibitor immediately before harvest. Additionally, and of greater clinical importance, will be to determine the duration of the reversal of tolerance with the kinase inhibitors.
Footnotes
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This study was supported by the National Institute on Drug Abuse Grants DA05274 and KO2-DA00186, the National Institute on Drug Abuse Center for Drug Abuse Research, and 2PODA097789.
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DOI: 10.1124/jpet.102.044446
- Abbreviations:
- Δ9-THC
- Δ9-tetrahydrocannabinol
- CB
- cannabinoid receptor
- TK
- tyrosine kinase
- PKA
- cAMP-dependent protein kinase
- PKG
- cGMP-dependent protein kinase
- PKC
- protein kinase C
- MAPK
- mitogen-activated protein kinase
- GRK
- G protein-coupled receptor kinase
- β-ARK,β-adrenergic receptor kinase
- LMWH, low molecular weight heparin
- PI3-K
- phosphatidylinositol-3 kinase
- % MPE
- percentage of maximum possible effect
- DMSO
- dimethyl sulfoxide
- bis
- bisindolymaleimide
- dH2O
- distilled water
- db
- dibutyryl
- GPCR
- G protein-coupled receptor
- Received September 17, 2002.
- Accepted January 24, 2003.
- The American Society for Pharmacology and Experimental Therapeutics