Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells

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. Ca2+ transients induced by either menthol (500 mu mol/1), eucalyptol (3 mmol/l), or icilin (2-60 mu mol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La3+, 100 mu mol/l) as well as the TRPM8 blockers BCTC (10 mu mol/l) and capsazepine (CPZ, 10 mu mol/l) suppressed icilin-induced Ca2+ increases. In and outward currents induced by application of menthol (500 mu mol/l) or icilin (50 mu mol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to approximate to 18 degrees C led to Ca2+ increases that were inhibited by a TRPM8 blocker BCTC (10 mu mol/l). Other thermosensitive TRP pathways whose heterogeneous Ca2+ response patterns are suggestive of other Ca2+ handling pathways were also detected upon strong cooling (a 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. (C) 2013 Elsevier Ltd. All rights reserved.