Blackwell Publishing Journal Backfiles 1879-2005
Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 ± 0.2 nM) ≈β-endorphin (Ki = 0.7 ± 0.5 nM) ≈ morphine (Ki = 0.8 ± 0.5 nM) ≈ [d-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 ± 0.5 nM) ⋙ U50,488 (Ki = 910 ± 0.78 nM) 〉 [d-Pen2,5]-enkephalin (Ki = 3,170 ± 98 nM) 〉 dextrorphan (Ki = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 ± 0.5 nM) to a lower-affinity state (IC50 = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.
Type of Medium: