The Roles of Several Kinases in Mice Tolerant to Delta-9-Tetrahydrocannabinol

It has been suggested that the CB1 G-protein-coupled receptor is internalized following agonist binding and activation of the second messenger pathways. The process of desensitization and resensitization is intimately involved with receptor internalization. Phosphorylation alters tolerance to cannabinoids thus contributing to tolerance. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. These findings led us to look at which kinase(s) may be involved in cannabinoid tolerance. We therefore hypothesize that by preventing phosphorylation of the CB1 receptor, we may reverse tolerance. We evaluated our hypothesis by testing the role of several kinases in tolerance: protein kinase A (PKA), protein kinase C (PKC), protein kinase C (PKG). Beta Adrenergic Receptor Kinase (β-ARK), Phosphatidylinositol 3-kinase (PI3K) and the src family tyrosine kinase. We also looked at cAMP and cGMP analogs. We evaluated PKA using KT5720, a PKA inhibitor; PKC using bisindolylmaleimide I, HCI] (bis), a PKC inhibitor; PKG using KT5823, a PKG inhibitor; β-ARK using Low molecular weight heparin (LMWH), a β-ARK inhibitor; PI3K using LY294002, a PI3K inhibitor and PP1 a src family tyrosine kinase inhibitor. The cAMP analog was dibutyryl-cAMP and the cGMP analog was dibutyryl-cGMP. ICR mice were rendered tolerant to △9- tetrahydrocannabinol (△9-THC) by administering injections of 20mg/kg △9-THC s.c. every 12 hours for 6.5 days. The mice were subsequently challenged 24 hours later with an ED8O of △9-THC at 20μg/mouse (i.t.). Antinociception was measured by the tail-flick test, %MPE’s and ED5O’s were calculated. The PKG inhibitor, KT5823, showed no significant change in %MPE. The β-ARK inhibitor, LMWH, showed no significant change in the %MPE. The PI3K inhibitor, LY294002, showed no significant change in the %MPE. Inhibition of PKC, by bis had no effect on tolerance, but at a higher dose attenuated the antinociceptive effect of △9-THC in non-tolerant mice. PPl, the src family tyrosine kinase inhibitor, reversed tolerance. KT5720, the PKA inhibitor reversed △9- THC tolerance. These data support a role for PKA and tyrosine kinase in phosphorylation events in THC tolerant mice. (Supported by NIDA grants K02DA00186 and P5ODA05274)