Immunocytochemical detection of GLUT2 at the rat intestinal brush-border membrane.

We have proposed a new model of intestinal sugar absorption in which high sugar concentrations promote rapid insertion of the facilitative transporter GLUT2 into the brush-border membrane so that absorptive capacity is precisely regulated to match dietary intake during the assimilation of a meal. However, location of GLUT2 at the brush border by immunocytochemistry has been problematical. We report that control of rapid GLUT2 trafficking and the use of an antibody to a sequence within the large extracellular loop of GLUT2 permits localization of GLUT2 at the brush border. To reveal brush-border GLUT2 fully, it is necessary to digest the sugar chain at the glycosylation site close to the antigenic site. In this way, we have demonstrated by immunocytochemistry PKC-dependent changes in the regulation of brush-border GLUT2 in rat jejunum that correspond to those seen by Western blotting. The functional and immunocytochemical data are now reconciled.

Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine.

Glucose absorption in rat jejunum involves Ca(2+)- and PKC betaII-dependent insertion of GLUT2 into the apical membrane. Ca(2+)-induced rearrangement of the enterocyte cytoskeleton is thought to enhance paracellular flow. We have therefore investigated the relationships between myosin II regulatory light chain phosphorylation (RLC(20)), absorption of glucose, water and calcium, and mannitol clearance. ML-7, an inhibitor of myosin light chain kinase, diminished the phloretin-sensitive apical GLUT2 but not the phloretin-insensitive SGLT1 component of glucose absorption in rat jejunum perfused with 75 mM glucose. Western blotting and immunocytochemistry revealed marked decreases in RLC(20) phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC betaII, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca(2+), or inhibition of unidirectional (45)Ca(2+) absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca(2+), nor clearance of [(14)C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with (45)Ca(2+) absorption or mannitol clearance. We conclude that the Ca(2+) necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Ca(v)1.3, and is dependent on SGLT1. Moreover, terminal web RLC(20) phosphorylation is necessary for apical GLUT2 insertion. The data confirm that glucose absorption by paracellular flow is negligible, and show further that paracellular flow makes no more than a minimal contribution to jejunal Ca(2+) absorption at luminal concentrations prevailing after a meal.