PKCε Regulates Behavioral Sensitivity, Binding and Tolerance to the CB1 Receptor Agonist WIN55,212-2
Guanine nucleotides regulate binding of opiate agonists to membrane receptors by increasing agonist dissociation rates. The current study demonstrates that the ability of guanosine 5'-triphosphate (GTP) and its nonhydrolyzable analogue guanylyl-5'-imidodiphosphate (Gpp(NH)p) to inhibit opiate agonist binding to rat brain membranes can be altered by two methods: by preincubating with EDTA, and by preincubating at pH 4.5. EDTA pretreatment increased the potency of Gpp(NH)p in inhibiting [3H]morphine binding by 4-fold, with little apparent change in the maximum effect of Gpp(NH)p or on levels of binding itself. The effect of EDTA pretreatment was blocked by prior incubation of membranes with excess calcium or manganese but could not be reversed by any divalent cation if the EDTA incubation was longer than 10 min. EDTA pretreatment increased the effects of GTP on dissociation rates of agonists. Pretreatment of membranes at pH 4.5 increased the ability of guanine nucleotides to regulate agonist binding by increasing the maximum effect of Gpp(NH)p from 50% to 80% inhibition of [3H]morphine binding with minor increase in potency of Gpp(NH)p. The actions of EDTA and low pH pretreatments were additive when both were conducted on the same membranes. These results suggest that modification of brain membranes can alter the interaction of receptors with guanine nucleotide-regulatory components which may lead to changes in post-receptor membrane events.