R E Reeves1, J Sanchez-Torres, M Coca-Prados, P R Cammarata. 1. Department of Anatomy and Cell Biology, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth 76107-2690, USA.
Abstract
PURPOSE: The authors recently established a link between swelling-activated myo-inositol efflux and chloride movement via anion channels in cultured bovine lens epithelial cells (BLECs). To further define this pathway, the relationship between cell volume, myo-inositol movement and mRNA expression of pI(Cln), a proposed chloride channel regulatory protein was investigated. METHODS: To demonstrate the effect of cell volume changes on pIcln transcription, BLECs were exposed to either hypertonic or hypotonic medium conditions. For rapid cellular shrinkage, BLECs were maintained at confluence in physiologic medium (257+/-2 mosm) then transferred to sodium hypertonic medium (473+/-6 mosm) or raffinose hypertonic medium (452+/-2 mosm). For rapid cellular swelling, cells were switched from sodium hypertonic medium to physiologic medium+/-tamoxifen. The expression of pI(Cln) mRNA was determined by Northern blot analysis. RESULTS: Upon cell volume reduction (increasing intracellular osmolality), BLECs upregulate the expression of pI(Cln) mRNA. Contrastly, when cell volume rapidly increases (decreasing intracellular osmolality), BLECs moderately downregulate pIcln mRNA, with expression levels reaching near physiologic control by 24 h. Blockage of swelling-activated chloride movement and osmolyte efflux with either tamoxifen or niflumic acid enhances the downregulation of pIcln mRNA expression. CONCLUSIONS: In cultured BLECs, pI(Cln) transcriptional regulation appears to be responsive to cell volume fluctuations. These data suggest a converse relationship exists between pIcln mRNA expression and changes in cell volume.
PURPOSE: The authors recently established a link between swelling-activated myo-inositol efflux and chloride movement via anion channels in cultured bovine lens epithelial cells (BLECs). To further define this pathway, the relationship between cell volume, myo-inositol movement and mRNA expression of pI(Cln), a proposed chloride channel regulatory protein was investigated. METHODS: To demonstrate the effect of cell volume changes on pIcln transcription, BLECs were exposed to either hypertonic or hypotonic medium conditions. For rapid cellular shrinkage, BLECs were maintained at confluence in physiologic medium (257+/-2 mosm) then transferred to sodium hypertonic medium (473+/-6 mosm) or raffinosehypertonic medium (452+/-2 mosm). For rapid cellular swelling, cells were switched from sodium hypertonic medium to physiologic medium+/-tamoxifen. The expression of pI(Cln) mRNA was determined by Northern blot analysis. RESULTS: Upon cell volume reduction (increasing intracellular osmolality), BLECs upregulate the expression of pI(Cln) mRNA. Contrastly, when cell volume rapidly increases (decreasing intracellular osmolality), BLECs moderately downregulate pIcln mRNA, with expression levels reaching near physiologic control by 24 h. Blockage of swelling-activated chloride movement and osmolyte efflux with either tamoxifen or niflumic acid enhances the downregulation of pIcln mRNA expression. CONCLUSIONS: In cultured BLECs, pI(Cln) transcriptional regulation appears to be responsive to cell volume fluctuations. These data suggest a converse relationship exists between pIcln mRNA expression and changes in cell volume.