ABSTRACT
The kappa nature of opioid binding sites in a brush border membrane (BBM) fraction from human placenta has been confirmed: these sites display considerably higher apparent affinity (KI = 1.2 nM) for the kappa selective ligand U-50488 than they do for the mu and delta selective ligands [D-Ala2, MePhe4, Glyol5] enkephalin (KI = 1.5-2 microM) and [D-Thr2, Leu5] enkephalyl-Thr (KI = 10-15 microM), respectively. The BBM fraction from human placenta was incubated either with the agonist 3H-etorphine or with the antagonist 3H-diprenorphine and subsequently solubilized with digitonin. The solubilized macromolecular radioactivity was found to behave as a homogeneous entity both in molecular exclusion chromatography (app. rs = 6.1 nm) and in linear sucrose gradients (app. S20.w = 12 S). Two lines of evidence indicated that the placental kappa opioid receptor is capable of interacting with a guanine nucleotide regulatory (G) protein: (i) equilibrium binding of the agonist 3H-etorphine in the BBM fraction was clearly inhibited by 5'-guanylylimidodiphosphate (Gpp(NH)p), especially in the presence of Na+ ions while binding of the antagonist 3H-diprenorphine was significantly less so and (ii) the sedimentation velocity of the kappa opioid receptor was decreased down to about 10 S when the BBM fraction was prelabeled with radioligand in the presence of Gpp(NH)p prior to its solubilization with digitonin. The G protein that mediates the effect of Gpp(NH)p might be neither Gs nor Gi since no adenylate cyclase activity could be demonstrated in the BBM fraction from human placenta.
Subject(s)
GTP-Binding Proteins/metabolism , Placenta/metabolism , Receptors, Opioid/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Adenylyl Cyclases/metabolism , Binding, Competitive , Centrifugation, Density Gradient , Chromatography, Gel , Diprenorphine/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalins/metabolism , Etorphine/metabolism , Female , Guanylyl Imidodiphosphate/pharmacology , Humans , Microvilli/metabolism , Oligopeptides/metabolism , Pregnancy , Pyrrolidines/metabolism , Receptors, Opioid/drug effects , Receptors, Opioid, kappa , Sodium Chloride/pharmacologyABSTRACT
Immunoreactive dynorphin (ir-Dyn) and opiate-like peptides (OLP) were measured in acid (HC1) extracts of human placenta by the use of an antibody to synthetic Dyn-(1-13) and of the displacement of [3H]-naloxone binding to rat brain homogenates, respectively. The placenta contained 57.6 pmoles per g of ir-Dyn and 134.4 pmoles per g of naloxone binding equivalents. After passage of the extract through cartridges of Sep Pak C18, half of the OLP was eluted with ir-Dyn at 35% acetonitrile (ACN), the rest being eluted at 60% ACN. Both fractions obtained from Sep Pak were chromatographed separately on Sephadex G-50, the OLP of the 35% ACN fraction coeluting with the ir-Dyn speak and that of the 60% ACN fraction being eluted at the same volume as synthetic beta-endorphin. Conversely, the fraction of OLP coeluting with synthetic leucine-enkephalin (Leu-Enk) in these two chromatographies was minimal. The Dyn-immunoreactive material was further purified by high pressure liquid chromatography on reverse phase micro-Bondapak C18 columns to give three distinct peaks corresponding to synthetic Dyn-(1-11), Dyn-(1-13) and Dyn-(1-12), respectively. Our results indicate that the human placenta contains several forms of ir-Dyn which account for about half of its endogenous OLP.
Subject(s)
Dynorphins , Placenta/chemistry , Protein Isoforms , Animals , Chromatography, Gel , Dynorphins/isolation & purification , Dynorphins/metabolism , Female , Humans , Narcotics , Pregnancy , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Radioimmunoassay , Rats , Tissue Extracts/chemistry , Tissue Extracts/metabolismABSTRACT
We have previously demonstrated that human placenta contains a homogenous population of kappa binding sites. The selective interaction of dynorphin with these opiate binding sites suggests a possible physiological implication of this endogenous opioid system in the placenta physiology. Ethylketocyclazocine stimulate K+-induced hCG release. This fact favours the hypothesis of a participation of placental opiate receptor on hCG secretion.
Subject(s)
Chorionic Gonadotropin/metabolism , Dynorphins , Endorphins/pharmacology , Peptide Fragments/pharmacology , Placenta/metabolism , Receptors, Opioid/metabolism , Analgesics, Opioid/pharmacology , Cyclazocine/analogs & derivatives , Cyclazocine/pharmacology , Ethylketocyclazocine , Female , Humans , Placenta/drug effects , Pregnancy , Receptors, Opioid/drug effects , Receptors, Opioid, kappaABSTRACT
Opiate binding sites were measured in different placental membrane fractions which were characterized by marker enzyme analysis and electron microscopic examination. The distribution pattern of opiate binding sites in the different fractions closely parallels that of placental alkaline phosphatase. These results clearly show that opiate binding sites are mainly located on the syncytial brush border membrane. The opiate binding sites found on microvillus membrane fraction have the same pharmacological characteristics as the Kappa opiate binding site previously characterized on placental crude membrane fraction.
