Chronic opioid treatment may uncouple opioid receptors and G-proteins: evidence from radiation inactivation analysis

Radiation inactivation (target size analysis) was used in this study to determine whether uncoupling of opioid receptor and G-protein is a contributing mechanism to opioid tolerance. Male Sprague-Dawley rats (160-260 g) were rendered tolerant to morphine or [D-Ala², D-Leu⁵]enkephalin (DADLE) by multiple i.p. or i.c.v. injections twice a day for 6 or 5 days. Control rats were injected with saline instead of opioids. The animals were killed, the midbrains excised and pooled together for each group. The washed P₂ membranes were suspended in buffer and irradiated with 1-10 Mrad doses of ⁶⁰Co irradiation, following which μ- or δ-opioid receptor binding activity of each sample was assayed. The molecular weight of the receptor was calculated from a standard irradiation curve constructed using several enzyme markers of known molecular weight. We found that the functional molecular size of μ-opioid receptor significantly decreased from 349 kDa to 228 kDa after 6 days of chronic morphine treatment, while, the molecular size of δ-opioid receptor decreased from 303 kDa to 223 kDa after 5 days of chronic DADLE treatment. These results are consistent with the uncoupling of opioid receptor from G-protein during chronic opioid treatment.