title: Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells.
abstract: Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca(2+) transients induced by either menthol (500 μmol/l), eucalyptol (3 mmol/l), or icilin (2-60 μmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La(3+), 100 μmol/l) as well as the TRPM8 blockers BCTC (10 μmol/l) and capsazepine (CPZ, 10 μmol/l) suppressed icilin-induced Ca(2+) increases. In and outward currents induced by application of menthol (500 μmol/l) or icilin (50 μmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca(2+) increases that were inhibited by a TRPM8 blocker BCTC (10 μmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca(2+) response patterns are suggestive of other Ca(2+) handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype.
authors.affiliation: Charité - Universitätsmedizin Berlin, Campus Virchow-Clinic, Department of Ophthalmology, Augustenburger Platz 1, 13353 Berlin, Germany. Electronic address: stefan.mergler@charite.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mergler
authors.forename: Charlotte
authors.initials: C
authors.lastname: Mertens
authors.forename: Monika
authors.initials: M
authors.lastname: Valtink
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
authors.forename: Violeta Castelo
authors.initials: VC
authors.lastname: Székely
authors.forename: Nefeli
authors.initials: N
authors.lastname: Slavi
authors.forename: Fabian
authors.initials: F
authors.lastname: Garreis
authors.forename: Suzette
authors.initials: S
authors.lastname: Abdelmessih
authors.forename: Ersal
authors.initials: E
authors.lastname: Türker
authors.forename: Gabriele
authors.initials: G
authors.lastname: Fels
authors.forename: Uwe
authors.initials: U
authors.lastname: Pleyer
mesh_terms: D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006358:Hot Temperature; D006801:Humans; D018408:Patch-Clamp Techniques; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D050053:TRPM Cation Channels / Q000096:biosynthesis / Q000235:genetics*; D013697:Thermosensing / Q000235:genetics*
chemical_list: D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D012313:RNA; D002118:Calcium
keywords: BCTC; CAP; CPZ; HCEC-12; N-(4-tert.butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide; TRPA; TRPM; capsaicin; capsazepine; human corneal endothelium; immortalized human corneal endothelial cell line; intracellular Ca(2+); planar patch-clamp technique; transient receptor potential ankyrin; transient receptor potential melastatin; transient receptor potential melastatin 8 channel