Involvement of TRPC Cation Channels in 15(S)–HETE– Regulated Mucin Secretion by Human Corneal Epithelial Cells.

Abstract

Abstract: : Purpose: 15(S)–HETE stimulates release of ocular surface mucins by a mechanism dependent on external Ca²⁺. This study was undertaken to investigate the role of transient receptor potential cation channels (TRPCs) and voltage–activated Ca²⁺ channels (VACCs) in 15(S)–HETE–induced MUC1 mucin secretion by human corneal epithelial cells. Methods: MUC1 secretion was measured by quantitative immunoassay of conditioned medium from CEPI–17 Cl4 cells, a human corneal epithelial cell line. Molecular composition of secretion products was analyzed by Western blotting. Changes in intracellular Ca²⁺ levels were monitored by fura–2 fluorescence microscopy. Expression of TRPCs of the TRPC3/6/7 subfamily was determined by RT–PCR. Results: 15(S)–HETE (0.1–10 µM) dose–dependently induced release of a soluble form of MUC1 that lacked the C–terminal cytoplasmic domain of the transmembrane form. The 15(S)–HETE response was mimicked by the diacylglycerol analogue oleoyl acetyl glycerol (OAG), but not by the phorbol esters PMA or phorbol dibutyrate. 15(S)–HETE–stimulated MUC1 release was unaffected by staurosporine, indicating the response to be PKC–independent. 15(S)–HETE–stimulated MUC1 secretion was suppressed by the TRPC antagonists 2–APB, SK&F 96365 and MRS 1845, whereas L–type VACC antagonists nitrendipine and verapamil were without effect. 15(S)–HETE did not mobilize [Ca²⁺]_i as measured by fura–2 fluorescence. The pharmacological profile of inhibitors suggested the involvement of receptor–activated TRPCs of the TRPC3/6/7 subfamily. Expression of TRPC3 and TRPC6 transcripts in CEPI 17 cells was confirmed by RT–PCR. Conclusions: The results suggest that 15(S)–HETE utilizes a unique mechanism – namely, activation of cation channels of the TRPC3/6/7 subfamily – to stimulate Ca²⁺–dependent secretion of MUC1 mucin by corneal epithelial cells. The precise nature of 15(S)–HETE – TRPC interactions and the downstream steps in stimulus–secretion coupling await further study.

Keywords: cornea: tears/tear film/dry eye • cornea: surface mucins • cornea: epithelium