C Zhou1, N Agarwal, P R Cammarata. 1. Department of Anatomy and Cell Biology, University of North Texas Health Science Center at Fort Worth/North Texas Eye Research Institute 76107.
Abstract
PURPOSE: Bovine lens epithelial cells (BLECs) accumulate osmotically active organic solutes (i.e., osmolytes) including myo-inositol when exposed to hypertonic stress (osmotic shock). In hypertonic medium, the increase in myo-inositol accumulation is attributed to an elevation in activity of Na+/myo-inositol cotransporter(s). The authors report the nature of the hypertonicity-induced enhancement of myo-inositol uptake in cultured BLECs by amplifying a 626 bp cDNA from lens cell RNA. METHODS: A portion of cDNA encoding a Na+/myo-inositol cotransporter was isolated from cultured BLECs using PCR primers designed from an established myo-inositol transporter from Madin-Darby canine kidney (MDCK) cells. Using the reverse transcription-polymerase chain reaction, a 626 bp PCR product was amplified. Its nucleic acid sequence was determined by the dideoxynucleotide method using Sequenase kit. Na+/Myo-inositol cotransporter mRNA expression in the cultured cells was demonstrated under physiological and hypertonic conditions by northern analysis of poly(A)+ RNA using the lens cell 626 bp cDNA as probe. RESULTS: The BLEC cDNA sequence was 92% identical with the Na+/myo-inositol cotransporter of MDCK cells. Myo-inositol transporter mRNA was demonstrated in cultured BLECs and was significantly induced by hypertonic stress. CONCLUSIONS: These data suggest that cultured bovine lens epithelial cell adaptation to hypertonicity involves intracellular accumulation of small organic osmolytes (i.e., myo-inositol) through elevation of myo-inositol uptake activity resulting from the upregulation of transporter mRNA.
PURPOSE:Bovine lens epithelial cells (BLECs) accumulate osmotically active organic solutes (i.e., osmolytes) including myo-inositol when exposed to hypertonic stress (osmotic shock). In hypertonic medium, the increase in myo-inositol accumulation is attributed to an elevation in activity of Na+/myo-inositol cotransporter(s). The authors report the nature of the hypertonicity-induced enhancement of myo-inositol uptake in cultured BLECs by amplifying a 626 bp cDNA from lens cell RNA. METHODS: A portion of cDNA encoding a Na+/myo-inositol cotransporter was isolated from cultured BLECs using PCR primers designed from an established myo-inositol transporter from Madin-Darby canine kidney (MDCK) cells. Using the reverse transcription-polymerase chain reaction, a 626 bp PCR product was amplified. Its nucleic acid sequence was determined by the dideoxynucleotide method using Sequenase kit. Na+/Myo-inositol cotransporter mRNA expression in the cultured cells was demonstrated under physiological and hypertonic conditions by northern analysis of poly(A)+ RNA using the lens cell 626 bp cDNA as probe. RESULTS: The BLEC cDNA sequence was 92% identical with the Na+/myo-inositol cotransporter of MDCK cells. Myo-inositol transporter mRNA was demonstrated in cultured BLECs and was significantly induced by hypertonic stress. CONCLUSIONS: These data suggest that cultured bovine lens epithelial cell adaptation to hypertonicity involves intracellular accumulation of small organic osmolytes (i.e., myo-inositol) through elevation of myo-inositol uptake activity resulting from the upregulation of transporter mRNA.