EFFECTS OF BRADYKININ ON SIGNAL-TRANSDUCTION, CELL-PROLIFERATION, ANDCYTOKINE, PROSTAGLANDIN E-2 AND COLLAGENASE-1 RELEASE FROM HUMAN CORNEAL EPITHELIAL-CELLS

1 We recently demonstrated the presence of phospholipase C-coupled bra dykinin (BK) B-2-receptors in human primary and SV40 virus-immortalize d corneal epithelial (CEPI) cells. 2 The aims of the present studies w ere to demonstrate the specific binding of [H-3]-BK to CEPI cell membr anes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to variou s physiological and pathological mechanisms in the CEPI cells, includi ng phosphoinositide (PI) turnover, intracellular Ca2+-mobilization ([C a2+](i)), cell proliferation (via [H-3]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloprotein ase-1) and prostaglandin E-2 (PGE(2)). 3 Specific [H-3]-BK binding com prised 83 +/- 2% of the total binding, and was of high affinity (K-d = 1.66 +/- 0.52 nM, n = 5), saturable (B-max = 640 +/- 154 fmol g(-1) w et weight) and reversible. Competition studies yielded the following a ffinity values for BK and a number of BK-related peptides: Hoe-140 (D- Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]BK; icatibant): K-i = 0.17 +/- 0.07 nM; BK: K-i = 1.0 +/- 0.11 nM; [Tyr(8)]-BK; K-i = 12.9 +/- 2.3 nM; [d es-Arg(9)]-BK: K-i > 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 mu M Hoe-140, a selec tive B-2-receptor antagonist, and also inhibited by the selective phos pholipase C (PLC) inhibitor, U73122 (1-(6-((17 beta-3-methoxyestra-1,3 ,5(10)-trien-17-yl)amino) -hexyl)-1 H-pyrrole-2,5-dione) (IC50 = 3.0 /- 1.6 mu M). BK-induced [Ca2+], mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 1 00 nM nifedipine. 5 BK (0.1 nM - 10 mu M) significantly (P < 0.05-0.00 1) stimulated [H-3]-thymidine incorporation into CEPI cellular DNA. Ho wever, while interleukin-1 alpha (IL-1 alpha; 10 ng ml(-1)) potently s timulated the release of IL-6, IL-8 and granulocyte macrophage colony- stimulating factor from CEPI cells, BK (0.1 nM - 10 mu M) was without effect. 6 Whilst phorbol-12-myristate-13-acetate (PMA; 3 mu g ml(-1)) and 10% foetal bovine serum (positive control agents) significantly st imulated the release of both MMP-1 and PGE(2) from CEPI cells, BK (0.1 nM - 10 mu M) was without any significant effect under these conditio ns. 7 In conclusion, these data indicate that the CEPI cells express h igh-affinity [H-3]-BK binding sites representing B-2-subtype BK recept ors coupled to PI turnover and [Ca2+](i) mobilization which appear to stimulate [H-3]-thymidine incorporation into cellular DNA. In contrast , BK failed to elicit the release of PGE(2), 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 prolifer ation.