Pharmacology of [³H]-pyrilamine binding and of the histamine-induced inositol phosphates generation, intracellular Ca²⁺-mobilization and cytokine release from human corneal epithelial cells

Abstract

1. 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.

2. 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.

3. 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 (K_d=3.76±0.78 nM; n=4) and saturable (B_max=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 (K_i=3.6±0.84 nM, n=4) and triprolidine (K_i=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_is>13 μM).

4. 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 (K_i) 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).

5. 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.

6. 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.

7. 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.

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