Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.
Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)
Tousignant
C
C
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Qué., Canada.
Guillemette
G
G
Barabé
J
J
Rhaleb
N E
NE
Regoli
D
D
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D006168:Guinea Pigs; D007082:Ileum / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007601:Jugular Veins / Q000187:drug effects / Q000502:physiology; D007609:Kallidin / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008297:Male; D009130:Muscle, Smooth / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011817:Rabbits; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D007457:Iodine Radioisotopes; D008024:Ligands; D010455:Peptides; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C026201:bradykinin, Tyr-; D007609:Kallidin; D001920:Bradykinin