title: 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: 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.
authors.affiliation: Molecular Pharmacology Unit, Alcon Laboratories, Inc, Fort Worth, Texas 76134-2099, USA.
authors.forename: N A
authors.initials: NA
authors.lastname: Sharif
authors.forename: T K
authors.initials: TK
authors.lastname: Wiernas
authors.forename: B W
authors.initials: BW
authors.lastname: Griffin
authors.forename: T L
authors.initials: TL
authors.lastname: Davis
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology*; D006634:Histamine H1 Antagonists / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007700:Kinetics; D020781:Matrix Metalloproteinase 1; D011738:Pyrilamine / Q000378:metabolism* / Q000494:pharmacology*; D011969:Receptors, Histamine H1 / Q000378:metabolism*; D014316:Tritium
chemical_list: D016207:Cytokines; D006634:Histamine H1 Antagonists; D007295:Inositol Phosphates; D015850:Interleukin-6; D016209:Interleukin-8; D011969:Receptors, Histamine H1; D014316:Tritium; D006632:Histamine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D011738:Pyrilamine; D015232:Dinoprostone; D002118:Calcium