UP-REGULATION OF [H-3] DES-ARG(10)-KALLIDIN BINDING TO THE BRADYKININ B-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-Arg(10)-kallidin (-KD) was investigated in smooth muscle cells (SMC) isolated from rabbit mesenteric arteries (RMA). 2 [H-3]-des-Arg(10)-KD specifically bound to interleukin-1 (IL-1)-treated RMA-SMC in a saturable 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 inhibited [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 receptor antagonist [Leu(8)]des-Arg(9)-BK inhibited [H-3]-des-Arg(10)-KD binding with an IC50 of 43 nM as expected for its effect at B-1 receptors. 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 cells did not affect the B-1 agonist response whereas IL-1 beta treatment produced a 7 fold increase in des-Arg(9)-BK-stimulated PGI(2) production. IL-1 beta also stimulated the response to B-2 agonists. 4 Des-Arg(9)-BK-induced PGI(2) secretion in IL-1-primed RMA-SMC was mediated by 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 agonists. 5 IL-1-treated RMA-SMC displayed a 5 fold increase in the number of B-1 receptors without modification of the affinity constant, thus establishing a possible relationship between the receptor density and 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) secretion, This observation suggests that IL-1 but not LPS, in addition to increase in the number of receptors, signals the cell to permit the coupling of B-1 receptors to the PLA(2)/cyclo-oxygenase pathway.