benzofurans and dynorphin-(1-8)

A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and > 500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in that < or = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.

Topics: Animals; Benzofurans; COS Cells; Diprenorphine; Dynorphins; Kinetics; Mutation; Narcotic Antagonists; Peptide Fragments; Pyrrolidines; Radioligand Assay; Receptors, Opioid, kappa; Receptors, Opioid, mu; Recombinant Fusion Proteins; Tritium

In the present study, we examined the peptidergic content of lumbar spinoreticular tract neurons in the colchicine-treated rat. This was accomplished by combining the retrograde transport of the fluorescent dye True Blue with the immunocytochemical labeling of neurons containing cholecystokinin-8, dynorphin A1-8, somatostatin, substance P or vasoactive intestinal polypeptide. After True Blue injections into the caudal bulbar reticular formation, separate populations of retrogradely labeled cells were identified as containing cholecystokinin-like, dynorphin-like, substance P-like or vasoactive intestinal polypeptide-like immunoreactivity. Retrogradely labeled somatostatin-like neurons were not identified in any of the animals examined. Each population of double-labeled cells showed a different distribution in the lumbar spinal cord. The highest yield of double-labeling occurred for cholecystokinin, with 16% of all intrinsic cholecystokinin-like neurons containing True Blue. These double labeled neurons were found predominantly at the border between lamina VII and the central canal region. About 11% of intrinsic vasoactive intestinal polypeptide-like neurons in the lumbar spinal cord were retrogradely labeled from the bulbar reticular formation. These neurons were found mostly in the lateral spinal nucleus, with only a few double-labeled cells located deep in the gray matter. Dynorphin-like double-labeled neurons were localized predominantly near the central canal; a smaller population was also seen in the lateral spinal nucleus. It was found that double-labeled dynorphin-like neurons made up 8% of all intrinsic dynorphin-like neurons. Retrogradely-labeled substance P-like neurons were rare; the few double-labeled neurons were found in the lateral spinal nucleus and lateral lamina V. These findings suggest a significant role for spinal cord peptides in long ascending systems beyond their involvement in local circuit physiology.

Topics: Afferent Pathways; Animals; Benzofurans; Dynorphins; Fluorescent Dyes; Immunohistochemistry; Male; Neuropeptides; Peptide Fragments; Rats; Rats, Inbred Strains; Reticular Formation; Sincalide; Somatostatin; Spinal Cord; Substance P