Pharmacology of [3H]‐pyrilamine binding and of the histamine‐induced inositol phosphates generation, intracellular Ca2+‐mobilization and cytokine release from human corneal epithelial cells
Authors: Sharif N A , Wiernas T K , Griffin B W , Davis T L 
Published: January 2005

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 (Kis>13 μm).
Histamine 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).
Histamine 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.
Histamine (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.
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.

British Journal of Pharmacology (1998) 125, 1336–1344; doi:10.1038/sj.bjp.0702194