UP-REGULATION OF [H-3] DES-ARG(10)-KALLIDIN BINDING TO THE BRADYKININB-1 RECEPTOR BY INTERLEUKIN-1-BETA IN ISOLATED SMOOTH-MUSCLE CELLS - CORRELATION WITH B-1 AGONIST-INDUCED PGI(2) PRODUCTION

1 Binding of the specific bradykinin B-1 receptor agonist, [H-3]-des-A rg(10)-kallidin (-KD) was investigated in smooth muscle cells (SMC) is olated from rabbit mesenteric arteries (RMA). 2 [H-3]-des-Arg(10)-KD s pecifically bound to interleukin-1 (IL-1)-treated RMA-SMC in a saturab le fashion with an equilibrium dissociation constant (K-D) of 0.3-0.5 nM. The number of binding sites per cell was 20,000-35,000. Kinins inh ibited [H-3]-des-Arg(10)KD binding to RMA-SMC with an order of potency very similar to that observed in typical B-1 specific bioassays: des- Arg(9)-bradykinin (BK)approximate to KD>>BK. Furthermore, the B-1 rece ptor antagonist [Leu(8)]des-Arg(9)-BK inhibited [H-3]-des-Arg(10)-KD b inding with an IC50 of 43 nM as expected for its effect at B-1 recepto rs. The B-2 receptor antagonists, NPC 567 and Hoe 140 only affected [H -3]-des-Arg(10)-KD binding at very high concentrations (IC50 = 0.8 mu M and IC50> 10 mu M, respectively). 3 Des-Arg(9)-BK (B-1 agonist) and [Hyp(3)]Tyr(Me)(8)-BK (B-2 agonist) did not induce prostacyclin (PGI(2 )) production by RMA-SMC. Lipopolysaccharide (LPS) treatment of the ce lls did not affect the B-1 agonist response whereas IL-1 beta treatmen t produced a 7 fold increase in des-Arg(9)-BK-stimulated PGI(2) produc tion. IL-1 beta also stimulated the response to B-2 agonists. 4 Des-Ar g(9)-BK-induced PGI(2) secretion in IL-1-primed RMA-SMC was mediated b y B-1 receptors since it was inhibited by [Leu(8)]des-Arg(9)-BK (IC50 = 56-73 nM) but not by Hoe 140. High concentrations of NPC 567 (IC50 = 2.4 mu M) were required to inhibit PGI(2) production induced by B-1 a gonists. 5 IL-1-treated RMA-SMC displayed a 5 fold increase in the num ber of B-1 receptors without modification of the affinity constant, th us establishing a possible relationship between the receptor density a nd the IL-1-primed B-1 response. 6 LPS treatment of the cells induced a 4 fold increase in B-1 receptor number without modifying PGI(2) secr etion, This observation suggests that IL-1 but not LPS, in addition to increase in the number of receptors, signals the cell to permit the c oupling of B-1 receptors to the PLA(2)/cyclo-oxygenase pathway.