Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase-1 release from human corneal epithelial cells.

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B2-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells. 2. The aims of the present studies were to demonstrate the specific binding of [3H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+-mobilization ([Ca2+]i), cell proliferation (via [3H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E2 (PGE2). 3. Specific [3H]-BK binding comprised 83 +/- 2% of the total binding, and was of high affinity (Kd = 1.66 +/- 0.52 nM, n = 5), saturable (Bmax = 640 +/- 154 fmol g(-1) wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]BK; icatibant): Ki = 0.17 +/- 0.07 nM; BK: Ki = 1.0 +/- 0.11 nM; [Tyr8]-BK: Ki = 12.9 +/- 2.3 nM; [des-Arg9]-BK: Ki > 9,200 nM (all n = 3-5)). 4. BK potently stimulated PI turnover (EC50 = 2.3 +/- 0.3 nM; n = 7) and [Ca2+]i mobilization (EC50 = 8-20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM-10 microM Hoe-140, a selective B2-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione) (IC50 = 3.0 +/- 1.6 microM). BK-induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine. 5. BK (0.1 nM-10 microM) significantly (P<0.05-0.001) stimulated [3H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1alpha (IL-1alpha; 10 ng ml(-1)) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM-10 microM) was without effect. 6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 microg ml(-1)) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE2 from CEPI cells, BK (0.1 nM-10 microM) was without any significant effect under these conditions. 7. In conclusion, these data indicate that the CEPI cells express high-affinity [3H]-BK binding sites representing B2-subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.