Calcium absorption by Ca(v)1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine

Glucose absorption in rat jejunum involves Ca2+- and PKC II-dependent insertion of GLUT2 into the apical membrane. Ca2+-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 (RLC20), 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 RLC20 phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC II, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca2+, or inhibition of unidirectional 45Ca2+ absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca2+, nor clearance of [14C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with 45Ca2+ absorption or mannitol clearance. We conclude that the Ca2+ necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Cav1.3, and is dependent on SGLT1. Moreover, terminal web RLC20 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 Ca2+ absorption at luminal concentrations prevailing after a meal.