The mu-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in mu-opioid receptor knockout mice.

There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Characterization of endomorphin-1 and -2 on [35S]GTPgammaS binding in the mouse spinal cord.

In the present study, G-protein activation by newly-isolated opioid peptides, endomorphin-1 and -2, was examined in the mouse spinal cord by monitoring the binding of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). Both endomorphin-1 and -2 increased [35S]GTPgammaS binding to mouse spinal cord membranes in a concentration-dependent and saturable manner and reached a maximal stimulation of 57.3+/-5.0 and 60.2+/-3.2%, respectively, at 10 microM. In contrast, the synthetic selective micro-opioid receptor agonist [D-Ala2,NHPhe4,Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced 103.4+/-5.4% of the maximal stimulation. The receptor specificity of endomorphin-stimulated [35S]GTPgammaS binding was verified by co-incubating membranes with endomorphins in the presence of specific micro-(beta-funaltrexamine and D-Phe-Cys-D-Tyr-Om-Thr-Pen-Thr-NH2 (CTOP)), delta-(naltrindole) or K-(nor-binaltorphimine) opioid receptor antagonists. Co-incubation with either beta-funaltrexamine or CTOP blocked both endomorphin-1- and-2-stimulated [35S]GTPgammaS binding in a concentration-dependent manner, whereas neither naltrindole nor nor-binaltorphimine had any effect on the [35S]GTPgammaS binding stimulated by either endomorphin-1 or -2. The data presented indicate that either endomorphin-1 or -2 activate G-proteins by specific stimulation of micro-opioid receptors, and may act as partial agonists with moderate catalytic efficacies in the mouse spinal cord.

Differential antinociceptive effects of endomorphin-1 and endomorphin-2 in the mouse.

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous mu-opioid receptor ligands. We determined the antinociceptive effects of these two ligands at the supraspinal level in mice with the tail-flick and hot-plate responses. The i.c.v. injection of endomorphin-1 and -2 inhibited the tail-flick and hot-plate responses in a dose-dependent manner. The endomorphin-1 was found to be 3.3- and 2.4-fold more potent than endomorphin-2 in inhibiting the tail-flick and hot-plate responses, respectively. The antinociception induced by endomorphin-1 was blocked by i.c.v. pretreatment with the mu-opioid receptor antagonist beta-funaltrexamine but not by the kappa-opioid receptor antagonist nor-binaltorphimine, the delta(1)-opioid antagonist 7-benzylidene naltrexamine, or the delta(2)-opioid receptor antagonist naltriben. In contrast, the antinociception induced by endomorphin-2 was blocked by i.c.v. pretreatment with beta-funaltrexamine or nor-binaltorphimine but not by 7-benzylidene naltrexamine or naltriben. The inhibition of the tail-flick response induced by endomorphin-2 was blocked by pretreatment with an antiserum against dynorphin A(1-17) but not by antisera against Met-enkephalin, Leu-enkephalin, or beta-endorphin. None of these antisera reduced the endomorphin-1-induced tail-flick inhibition. We propose that endomorphin-1 produces antinociception by stimulating one type of mu-opioid receptor, whereas endomorphin-2 initially stimulates different mu-opioid receptors, which subsequently induce the release of dynorphins that act on kappa-opioid receptors to produce antinociception.