Absence of G-protein activation by μ-opioid receptor agonists in the spinal cord of μ-opioid receptor knockout mice

1. The ability of μ-opioid receptor agonists to activate G-proteins in the spinal cord of μ-opioid receptor knockout mice was examined by monitoring the binding to membranes of the nonhydrolyzable analogue of GTP, guanosine-5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS). 2. In the receptor binding study, Scatchard analysis of [³H][D-Ala²,NHPhe⁴, Gly-ol]enkephalin ([³H]DAMGO; μ-opioid receptor ligand) binding revealed that the heterozygous μ-knockout mice displayed approximately 40% reduction in the number of μ-receptors as compared to the wild-type mice. The homozygous μ-knockout mice showed no detectable μ-binding sites. 3. The newly isolated μ-opioid peptides endomorphin-1 and -2, the synthetic selective μ-opioid receptor agonist DAMGO and the prototype of μ-opioid receptor agonist morphine each produced concentration-dependent increases in [³⁵S]GTPγS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [³⁵S]GTPγS binding stimulated by specific δ₁-([D-Pen^2,5]enkephalin), δ₂-([D-Ala²]deltorphin II) or κ₁- (U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that μ-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that μ-opioid receptor densities could be rate-limiting steps in the G-protein activation by μ-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological μ-receptor reserve. Furthermore, little change in δ₁-, D₂- or κ₁-opioid receptor-G-protein complex appears to accompany μ-opioid receptor gene deletions in this region.