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.
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
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