Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts

Abstract

Background and purpose: Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells.

Experimental approach: Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes.

Key results: [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone.

Conclusions and implications: Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

Figure 1

Bradykinin (BK), MEN16132 and icatibant inhibit [³H]-BK specific binding to human synoviocytes. Cells were incubated for 2 h at 4°C with [³H]-BK (1 nM) and varying concentrations of competing ligands as described in Methods. Data are expressed as mean ± SEM of three independent experiments, each one performed in triplicate.

Figure 2

MEN16132 (A) and icatibant (B) antagonist activity towards BK-induced activation of IP production. Antagonists were added at the indicated concentrations 15 min before the agonist incubation (60 min). C: Schild analysis of data presented in panels A and B. Data are expressed as mean ± SEM of three to four independent experiments, each one performed in triplicate. IP, inositol phosphates.

Figure 3

Bradykinin (BK)-induced release of IL-6 (A) and IL-8 (B) in human synoviocytes. Cells were incubated for 24 h with BK (1 nM–10 µM). Data (basal release = 100%) are expressed as the mean ± SEM of seven independent experiments, each one performed in triplicate. *P < 0.05, **P < 0.01 versus basal values, one-way anova followed by Dunnett's post hoc test. IL, interleukin.

Figure 4

Inhibitory effect of MEN16132 and icatibant on bradykinin (BK)-induced IL-6 (A) or IL-8 (B) release. Human synoviocytes were preincubated for 30 min with the indicated concentrations of antagonists before BK (1 µM, 24 h) stimulation. Data are expressed as % of control response to BK (in the absence of antagonist), and represent the mean ± SEM of five independent experiments, each one performed in triplicate. IL, interleukin.

Figure 5

Effect of indomethacin and nordihydroguaiaretic acid (NDGA) on bradykinin (BK)-induced IL-6 (A) or IL-8 (B) release. Human synoviocytes were preincubated for 60 min with indomethacin (10 µM) or NDGA (10 µM), alone or in combination, before BK (1 µM, 24 h) stimulation. Data are expressed in pg·mL⁻¹, and represent the mean ± SEM of three to five independent experiments, each one performed in triplicate. ^••P < 0.01 versus control basal; *P < 0.05, **P < 0.01 versus the corresponding basal condition; two-way anova followed by Bonferroni's post hoc test. IL, interleukin.