SV40 immortalized human corneal epithelial cells express cold sensitive TRPM8 channels

Abstract

Abstract: : Purpose: TRPM8, a non–selective cation sensory transducing channel member of the TRP family, is activated by cold temperatures resulting in rises in intracellular free calcium concentration ([Ca²⁺]_i). These transients are due to Ca²⁺ release from intracellular stores followed by an increase in plasma membrane Ca²⁺ influx through Ca²⁺–permeable ion channels. To date, there has been no study in human corneal epithelial cells (HCEPC) on the expression of TRPM8 channels. We determined whether the cooling compound icilin induces in these cells rises in [Ca²⁺]_i. In addition, human corneal endothelial cells (HCEC) were also tested for such a response. Methods: The effects of icilin were determined on [Ca²⁺]_i in fura2–loaded HCEP and SV40–imortalized human corneal endothelial cells (HCEC) with a single cell fluorescence video imaging system. Results: Extracellular application of icilin (50 µM) reversibly increased [Ca²⁺]_i from 108 ± 3 nM (SEM) to a peak level of 155 ± 17 nM in HCEP after 3 min followed by full recovery (104 ± 14 nM) to the baseline after another 3 min. (n = 8 different coverslips). In contrast, in HCEC, icilin had instead biphasic concentration dependent effects on [Ca²⁺]_i. Whereas a higher concentration (50 µM) reduced [Ca²⁺]_i from 112 ± 5 nM to 79 ± 12 nM followed by a recovery to 102 ± 16 nM (n = 5 different coverslips), a lower concentration of icilin (10 µM) reversibly increased [Ca²⁺]_i from 109 ± 6 nM to 178 ± 7 nM followed by full recovery (114 ± 5 nM) (n = 6 different coverslips). Conclusions: HCEP and HCEC both express functional TRPM8 activity because icilin elicited [Ca²⁺]_i transients. However, in HCEC icilin instead induced also declines in [Ca²⁺]_i. This opposite effect suggests that HCEC also express different temperature sensitive TRP channel isoforms than in HCEP. Our findings implicate an expanded role for these different types of ion channels in somatic and chemical sensory detection. Sensory transduction of cold in HCEP depends on expression of functional TRPM8 channel activity.

Keywords: cornea: epithelium • ion channels • cornea: endothelium