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.

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.