Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 2: Cloning of a 626 bp cDNA portion of a Na+/myo-inositol cotransporter, an osmotic shock protein.
OBJECTIVE
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.
Zhou
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C
Department of Anatomy and Cell Biology, University of North Texas Health Science Center at Fort Worth/North Texas Eye Research Institute 76107.
Agarwal
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N
Cammarata
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PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004848:Epithelium / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D007294:Inositol / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D027981:Symporters; D014882:Water-Electrolyte Balance
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D027981:Symporters; C073884:SLC5A3 protein, human; D007294:Inositol; D004247:DNA