Osmosensitive Na+-Dependent Myo-Inositol Transporter (SMIT) Expression in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: To characterize myo-inositol subtype transporter gene expression and its transport activity in human immortalized corneal epithelial cells (HECE). Methods: Confluent HCEC were cultured in hypertonic media (up to 500 mOsm×kg^-1) for as long as 72 h. Sucrose was used to increase medium osmolality. RT-PCR detected myo-inositol transporter gene expression. [³H] myo-inositol uptake characterized transport activity. Myo-inositol transporter RNA amount was semi-quantified with real-time PCR. Cell viability was assayed based on measurements of lactate dehydrogenase (LDH) release. Results: Na⁺ and H⁺-dependent myo-inositol subtype transporter (SMIT and HMIT, respectively) mRNA expression was detected. However, HMIT transport activity was not detected across either the apical or basolateral facing sides whereas SMIT was present on the apical facing surface. SMIT elicited myo-inositol uptake was stimulated following exposure to 450 mOsm×kg^-1 for 24 h. Following culture for 24 h in media whose osmolality varied from 310 to 500 mOsm, myo-inositol uptake/15 min increased up to 25-fold. With the 450 mOsm×kg^-1 medium, uptake maximally increased 10-fold above its isotonic value at 24 h, but after an additional 48 h myoinositol uptake/15 min fell by 50%. Hypertonic exposure increased transporter apparent maximal velocity and the affinity. After 6 h of exposure to 450 mOsm×kg^-1, SMIT expression maximally increased 30% whereas HMIT remained unchanged. Supplementation of the medium with 1000 µM myo-inositol improved HCEC viability 2.3-fold following exposure to 450 mOsm×kg^-1 medium for 48 h. Its intracellular concentration increased up to 60 mM after 24 h in this medium. Conclusions: SMIT is expressed in the apical side of HCEC and is osmosensitive. Increases in its apparent maximal velocity elicited by hyperosmolality are associated with a rise in SMIT gene expression. The mechanisms of increase in its apparent affinity are unknown.

Keywords: cornea: epithelium • gene/expression • ion transporters