Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.
Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand. During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded. At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II. Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 4.6 +/- 0.5 nM (mean +/- SE, n = 4). Linear Scatchard plots, Hill coefficients close to unity (0.95-1.06), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity. Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25%. The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor. Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha. This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes.
D001665:Binding Sites; D001920:Bradykinin / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D011464:Epoprostenol / Q000096:biosynthesis; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008297:Male; D018002:Receptors, Bradykinin; D011956:Receptors, Cell Surface / Q000032:analysis*; D012964:Sodium / Q000378:metabolism
D018002:Receptors, Bradykinin; D011956:Receptors, Cell Surface; D012964:Sodium; D011464:Epoprostenol; D008274:Magnesium; D001920:Bradykinin; D002118:Calcium