- 1We 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.
- 2The 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.
- 3Specific [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 (Kis>13 μm).
- 4Histamine induced phosphoinositide (PI) hydrolysis 2.7–4.4 fold above basal levels and with a potency of 14.9±4.9 μm (n = 9) and 4.7±0.2 μm (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 μm 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-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8±2.4 μm, n = 3).
- 5Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3±1.5 μm (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.
- 6Histamine (EC50s=1.28–2.77 μm, 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-α, 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 μg ml−1) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells.
- 7It 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.
British Journal of Pharmacology (1998) 125, 1336–1344; doi:10.1038/sj.bjp.0702194