The in vivo binding properties of cerebral mu and
delta opioid receptors were investigated in mice after the intrastriatal injection of [3H][D-Ala2, MePhe4, Gly-ol5]
enkephalin (
DAGO) or [3H][D-Thr2,Leu5]enkephalyl-Thr (
DTLET). Both
peptides exhibited similar diffusion kinetics in the brain and 30-40% of [3H]
DAGO or [3H]
DTLET was shown to be present in the tissue 15 min after injection when maximal binding was observed. The specific binding of both agonists, defined as the fraction of the radioactivity bound to brain membranes which was displaced by 10 nmol of cold
ligand, was reversible, saturable and displayed a pharmacological profile similar to that found in in vitro experiments. At doses producing a similar
analgesic effect in the hot-plate test in mice,
DTLET occupied 64% of delta sites and
DAGO 15% of mu sites. However, because of the residual cross-reactivity of
DTLET for mu sites, it appeared that both
ligands occupied a similar number of
mu receptors at their ED50 values, thus supporting a preferential involvement of mu
opioid binding sites in the supraspinal
pain control. [Met5]
enkephalin inhibited the in vivo binding of both agonists only when the
peptide was protected from degradation by the co-administration of a mixed inhibitor of
enkephalin degrading
enzymes RB38A (N[3(R)(hydroxyaminocarbonyl)-2-benzyl-1-oxopropyl]-
L-phenylalanine). Unlike
thiorphan, 5 nmol RB38A alone was able to inhibit [3H]
DAGO binding by 60%. This result is the first direct demonstration of the existence of an in vivo tonic control of
mu opioid receptor occupation by endogenous
opioid peptides.