Chronic Morphine-Induced Changes in μ-Opioid Receptors and G Proteins of Different Subcellular Loci in Rat Brain

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Abstract
Prolonged exposure to opioid agonists can induce adaptive changes resulting in tolerance and dependence. Here, rats were rendered tolerant by subcutaneous injections of increasing doses of morphine from 10 to 60 mg/kg for 3, 5, or 10 consecutive days. Binding parameters of the μ-opioid receptor in subcellular fractions were measured with [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Although the density of surface μ-sites did not change after the 5-day morphine treatment, up-regulation of synaptic plasma membrane binding was detected after the 10-day drug administration. In contrast, the number of μ-binding sites in a light vesicle or microsomal fraction (MI) was elevated by 68 and 30% after 5 and 10 days of morphine exposure, respectively. The up-regulated MI μ-sites displayed enhanced coupling to G proteins compared with those detected in saline-treated controls. Pertussis toxin catalyzed ADP ribosylation, and Western blotting with specific antisera was used to quantitate chronic morphine-induced changes in levels of various G protein α-subunits. Morphine treatment of 5 days and longer induced significant increases in levels of Gαo, Gαi1, and Gαi2 in MI fractions that are part of an adaptation process. Up-regulation of intracellular μ-sites may be the result of post-translational changes and in part de novo synthesis. The results provide the first evidence that distinct regulation of intracellular μ-opioid receptor G protein coupling and G protein levels may accompany the development of morphine tolerance.