CHARACTERIZATION OF BRADYKININ RECEPTORS IN GUINEA-PIG GALL-BLADDER

Specific binding of [H-3]bradykinin (BK) to guinea pig gall bladder (GPGB) 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 potency for inhibition of binding by B2 antagonists, [(D)Arg-[Hyp3,Thi5,(D)Tic7-Oic8]-bradykinin (HOE140) > (D)Arg (D)Arg-[Hyp3(D)HypE(transpropyl)7-Oic8]-bradykinin (NPC17731) > (D)Arg-[Hyp3,Thi5,(D)Tic7-Tic8]-bradykinin (NPC16731) > (D)Arg-[Hyp3,(D)Phe7]-bradykinin (NPC567)] and agonists (BK = kallidin = Tyr(Me)8-BK > Tyr8-BK, > Hyp4-kallidin) as well as inactivity of the B1 agonist des(Arg9)-BK. Nonhydrolyzable GTP analogs (GTP-gamma-S and guanylyl-5'-imido-diphosphate) produced 80% inhibition of specific binding suggesting receptor coupling to guanine nucleotide-binding proteins. BK increased polyphosphoinositide hydrolysis in chopped GPGB in a concentration-dependent manner (0.01-300 muM; EC50 = 414 +/- 171 nM; n = 3-9 tissues/concentration). HOE140 and NPC16731, 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 turnover in GPGB (IC50 = 46.9 +/- 17.3 nM). BK produced a concentration-related contraction of isolated strips of GPGB. Indomethacin significantly decreased both the potency and efficacy of BK whereas thiorphan, a neutral endopeptidase inhibitor, and/or captopril, an angiotensin-converting enzyme inhibitor, enhanced potency. NPC567, NPC16731, NPC17731 and HOE140 were competitive antagonists of BK-induced contraction with pK(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. Furthermore, the potency of HOE140 to inhibit contraction produced by several agonists was not different. The rank order of potency of B2 agonists to contract GPGB was independent of the presence or absence of indomethacin. Based on these results 1) B2, but not B1, bradykinin receptors appear to exist in GPGB and 2) receptor subtypes were not distinguishable by binding studies, polyphosphoinositide formation or contraction studies alone but may exist based on differences between the assays-most notably competitive vs. noncompetitive antagonism of HOE140.