CHARACTERIZATION OF BRADYKININ RECEPTORS IN GUINEA-PIG GALL-BLADDER

Specific binding of [H-3]bradykinin (BK) to guinea pig gall bladder (G PGB) membranes was protein dependent, rapid (K(on) = 0.067 min-1) with high affinity (K(d) = 0.45 +/- 0.02; n = 3), saturable (B(max) = 546 +/- 56 fmol/mg of protein) and showed no cooperativity (n(H) = 1.19 +/ - 0.08). A BK B2 receptor type was indicated by the rank order of pote ncy for inhibition of binding by B2 antagonists, [(D)Arg-[Hyp3,Thi5,(D )Tic7-Oic8]-bradykinin (HOE140) > (D)Arg (D)Arg-[Hyp3(D)HypE(transprop yl)7-Oic8]-bradykinin (NPC17731) > (D)Arg-[Hyp3,Thi5,(D)Tic7-Tic8]-bra dykinin (NPC16731) > (D)Arg-[Hyp3,(D)Phe7]-bradykinin (NPC567)] and ag onists (BK = kallidin = Tyr(Me)8-BK > Tyr8-BK, > Hyp4-kallidin) as wel l as inactivity of the B1 agonist des(Arg9)-BK. Nonhydrolyzable GTP an alogs (GTP-gamma-S and guanylyl-5'-imido-diphosphate) produced 80% inh ibition of specific binding suggesting receptor coupling to guanine nu cleotide-binding proteins. BK increased polyphosphoinositide hydrolysi s in chopped GPGB in a concentration-dependent manner (0.01-300 muM; E C50 = 414 +/- 171 nM; n = 3-9 tissues/concentration). HOE140 and NPC16 731, inhibited BK-induced polyphosphoinositide hydrolysis but only the latter appeared competitive (pK(b) 8.09 +/- 0.19, n = 3). U73122, an inhibitor of phospholipase C pathway, also inhibited BK-induced turnov er in GPGB (IC50 = 46.9 +/- 17.3 nM). BK produced a concentration-rela ted contraction of isolated strips of GPGB. Indomethacin significantly decreased both the potency and efficacy of BK whereas thiorphan, a ne utral endopeptidase inhibitor, and/or captopril, an angiotensin-conver ting enzyme inhibitor, enhanced potency. NPC567, NPC16731, NPC17731 an d HOE140 were competitive antagonists of BK-induced contraction with p K(b) or pA2 values of: 5.05 +/- 0.20 (n = 12), 7.27 +/- 0.08 (n = 34), 7.75 +/- 0.08 (n = 42) and 8.54 +/- 0.06 (n = 46), respectively. Furt hermore, the potency of HOE140 to inhibit contraction produced by seve ral agonists was not different. The rank order of potency of B2 agonis ts to contract GPGB was independent of the presence or absence of indo methacin. Based on these results 1) B2, but not B1, bradykinin recepto rs appear to exist in GPGB and 2) receptor subtypes were not distingui shable by binding studies, polyphosphoinositide formation or contracti on studies alone but may exist based on differences between the assays -most notably competitive vs. noncompetitive antagonism of HOE140.