Evidence for delta-opioid binding and GTP-regulatory proteins in 5-day-old rat brain membranes.
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.
Szücs
M
M
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Coscia
C J
CJ
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D012984:Software
D004745:Enkephalins; D006150:Guanine Nucleotides; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D006165:Guanylyl Imidodiphosphate; D020881:Enkephalin, D-Penicillamine (2,5)-; D019204:GTP-Binding Proteins