Acute and chronic stress-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate |
| |
Authors: | Bagchi D. Carryl O.R. Tran M.X. Bagchi M. Garg A. Milnes M.M. Williams C.B. Balmoori J. Bagchi D.J. Mitra S. Stohs S.J. |
| |
Affiliation: | (1) Departments of Medicine and Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA |
| |
Abstract: | To determine the role of translocation vs. activation of Glut1 in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation, we measured the abundance of myc-tagged Glut1 in plasma membrane of stably transfected Clone 9 cells, a rat liver cell line expressing only the Glut1 isoform. The myc epitope-tag is located between Ile56 and Pro57 in the putative first extracellular loop of Glut1. Under basal conditions, transfected cells expressed ~3 fold higher levels of Glut1 and exhibited a ~3 fold higher rate of glucose transport than non-transfected cells. To delineate the mechanism mediating the stimulation of glucose transport by a azide we employed two strategies: (1) mild cell surface biotinylation followed by isolation of plasma membranes and quantitation of Glut1 sites in Western blots employing anti-Glut1 and anti-myc antibodies, and (2) quantitative immunofluorescence of myc epitopes in plasma membrane sheets. The rate of glucose transport increased 2.9 ± 0.5 fold in transfected cells exposed to 5 mM azide for 1 h. Exposure to azide, however, resulted in no significant increase in Glut1 content of plasma membranes using anti-Glut1 or anti-myc antibodies in Western blots (1.0 ± 0.1 and 0.9 ± 0.2 fold, respectively; azide/control), and was associated with no detectable increase in immunofluorescence using either anti-Glut1 or anti-myc antibodies (p > 0.1 for both measurements). Treatment of cells with cobalt chloride (employed as a positive control) resulted in marked increases in glucose transport, cell and plasma membrane Glut1 content, and immunofluorescence of plasma membrane sheets (8-10 fold increase in each parameter). We conclude that the stimulation of glucose transport by azide results mainly from activation of Glut1 transporters pre-existing in the plasma membrane. |
| |
Keywords: | azide cobalt chloride activation of Glut1 biotinylation transfection immunogluorescence |
本文献已被 SpringerLink 等数据库收录! |
|