Subject(s)
Cocaine , Opioid-Related Disorders/drug therapy , Receptors, Opioid/drug effects , Substance-Related Disorders/drug therapy , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Cyclic AMP/biosynthesis , Electric Stimulation , Guinea Pigs , Humans , Ileum/metabolism , In Vitro Techniques , Male , Mice , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Rats , Receptors, Dopamine/drug effects , Receptors, Dopamine/metabolism , Receptors, Opioid/metabolism , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolismABSTRACT
Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more than 52 million different hexapeptides. The five compounds with the highest affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affinity for ORL1 in the nanomolar range, similar to the recently discovered endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The activity of these compounds was investigated in three different assays: stimulation of [35S]GTPgammaS binding and inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with ORL1, and inhibition of electrically induced contractions in the mouse vas deferens. In each assay, the five hexapeptides acted as partial agonists. The EC50 values for stimulation of [35S]GTPgammaS binding and inhibition of cAMP accumulation were in the range of that for N/OFQ, but maximal effects ranged from 70 to 90% of N/OFQ in the cAMP assay, and 30 to 60% of N/OFQ in the GTPgammaS assay. The positive hexapeptides identified were found to have minimal structural similarity to N/OFQ. The peptides are positively charged, which could enable them to bind to the negatively charged second extracellular loop thought to be a likely binding site for N/OFQ.
Subject(s)
Oligopeptides/metabolism , Receptors, Opioid/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cyclic AMP/biosynthesis , Male , Mice , Molecular Sequence Data , Oligopeptides/pharmacology , Rats , Vas Deferens/drug effects , Nociceptin ReceptorABSTRACT
The presence of kappa-opioid receptor subtypes has been clearly established in guinea pig brain. Using [3H]bremazocine in the presence of reversible blockers of mu, delta and kappa 1 receptors, two additional binding sites can be determined in guinea pig brain membranes. The site with higher affinity for the opioid ligands represents kappa 2, while the other site has low affinity and is poorly characterized. The kappa 2 site has high affinity for ethylketocyclazocine and other benzomorphans, as well as for the dynorphin gene products tested. The dynorphin analogs have no appreciable affinity for the low affinity site, so this site should not be called a kappa receptor. With an appropriate membrane preparation, kappa 2 binding can also be demonstrated in the guinea pig ileum. Binding affinities for selected ligands at kappa 2 in guinea pig ileum membranes are very similar to affinities found in brain membranes.
Subject(s)
Brain Chemistry/physiology , Ileum/metabolism , Receptors, Opioid, kappa/metabolism , Analgesics/metabolism , Animals , Benzomorphans/metabolism , Brain Chemistry/drug effects , Dynorphins/analogs & derivatives , Dynorphins/pharmacology , Ethylketocyclazocine/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Guinea Pigs , Ileum/drug effects , In Vitro Techniques , Kinetics , Ligands , Male , Membranes/metabolism , Myenteric Plexus/drug effects , Myenteric Plexus/metabolism , Receptors, Opioid, kappa/drug effectsABSTRACT
An endopeptidase isolated from bovine kidney displays high affinity and selectivity for the Ser-Phe bond located in the C-terminal region of atrial peptides. Enzymatic activity converts APIII and APII to the less active peptide API. This peptidase is inhibited by both metal chelators and sulfhydryl-reactive agents, suggesting both a tightly bound metal and a cysteine residue are important for enzymatic activity. This enzyme may be important for the processing and/or degradation of atrial peptides.
