The kappa-opioid receptor from human placenta: hydrodynamic characteristics and evidence for its association with a G protein.
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.
Porthé
G
G
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique 205, Toulouse, France.
Francés
B
B
Verrier
B
B
Cros
J
J
Meunier
J C
JC
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D000262:Adenylyl Cyclases / Q000378:metabolism; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004174:Diprenorphine / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D005048:Etorphine / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D008871:Microvilli / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011759:Pyrrolidines / Q000378:metabolism; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D017473:Receptors, Opioid, kappa; D012965:Sodium Chloride / Q000494:pharmacology
D004745:Enkephalins; D009842:Oligopeptides; D011759:Pyrrolidines; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004174:Diprenorphine; D006165:Guanylyl Imidodiphosphate; D005048:Etorphine; D012965:Sodium Chloride; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; C037653:deltakephalin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases