Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells

Abstract

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca(2+) transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5-20 μM) -induced Ca(2+) transients were blocked by capsazepine (CPZ) (10 μM), the TRPV4 activator 4α-PDD (10 μM) -induced Ca(2+) increases were reduced by ruthenium-red (RuR) (20 μM). Different heating (<40°C or >43°C) led to Ca(2+) increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca(2+) transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.

Upregulation of Transient Receptor Potential Vanilloid Type-1 Channel Activity and Ca2+Influx Dysfunction in Human Pterygial Cells

“…In Drosophila, photoreceptors and mammalian photosensitive retinal ganglion cell phototransduction is mediated by G protein-activated phospholipase C (PLC), which generates a force in the lipid-bilayer opening TRP-Canonical channels. 23,24 Transient receptor potential V1 is expressed in human conjunctival epithelial cells (HCjEC), 25 human corneal endothelial cells, 26 corneal fibroblasts, 27 and corneal epithelial cells (HCEC) 28,29 (reviewed in Ref. 30 ).…”

“…Immunohistochemistry was performed with anti-TRPV1 (ACC-030; Alomone Labs, Jerusalem, Israel), TRPM8 (HPA024117; Sigma Aldrich, Munich, Germany), cytokeratin (CK)-13 (sc-57003; Santa Cruz Biotechnology, Heidelberg, Germany), CK-15 (CBL1587417; Millipore, Darmstadt, Germany), and CK-19 (sc-53003; Santa Cruz Biotechnology) antibodies, as previously described in detail. 25,37 Transient receptor potential V1 and -M8 antibody specificity was confirmed based on elimination of antibody reactivity by antibody preadsorption with 1 lg of a respective blocking peptide per lg antibody. Furthermore, control sections were incubated with nonimmune IgG to determine possible nonspecific binding of IgG.…”

“…In addition, an immortalized HCjEC line, was used and cultivated as previously reported. 25,29 Furthermore, an SV40-immortalized human cornea epithelial cell line (HCEC) 35 was cultured according to our previous study. 25 Reverse Transcriptase PCR …”

“…25,40 In brief, the standard intracellular solution contained in mM: 50 CsCl, 10 NaCl, 60 CsF, 20 EGTA, and 10 HEPES-acid at pH of approximately 7.2 and approximately 288 mOsM, which was applied to the inner side of the microchip. The external solution contained in mM: 140 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 5 D-glucose monohydrate, and 10 HEPES (pH » 7.4 and osmolarity » 298 mOsM) was present on the external side of the microchip (resistance of 2.5-3.0 MX).…”

“…All experiments were performed at a constant room temperature (» 208-238C) because experiments performed at higher temperatures led to increased Ca 2þ levels due to increased open probability of thermosensitive-TRPs in ocular surface cells. 25,29 Stock solutions of drugs were prepared in dimethyl sulfoxide (DMSO). Dimethyl sulfoxide concentrations below 0.1% had no detectable effect on intracellular Ca 2þ regulation (data not shown).…”