• knockout mice;
  • endomorphins;
  • G-proteins;
  • homologous recombination;
  • opioid peptides;
  • μ-opioid receptors;
  • signal transduction;
  • spinal cord
  • 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 non-hydrolyzable analogue of GTP, guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS).

  • 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.

  • 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.

  • No differences in the [35S]GTPγS binding stimulated by specific δ1- ([D-Pen2,5]enkephalin), δ2- ([D-Ala2]deltorphin II) or κ1- (U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes.

  • 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-, δ2- or κ1-opioid receptor-G-protein complex appears to accompany μ-opioid receptor gene deletions in this region.

British Journal of Pharmacology (1999) 126, 451–456; doi:10.1038/sj.bjp.0702330