Evidence for δ‐Opioid Binding and GTP‐Regulatory Proteins in 5‐Day‐Old Rat Brain Membranes

Abstract

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.