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

Abstract

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.

Figures

Figure 1. The effect of MLCK inhibition on glucose absorption across the in vivo luminally perfused rat jejunum

Rat jejunum was perfused in vivo with 75 mm glucose as control (▪) and 5 μm ML-7 (□) as described in the Methods section. At 40 min, the perfusate reservoir was switched (arrow) to an identical one containing 1 mm phloretin. Values are the mean rate of glucose absorption from 6 experiments. Error bars represent s.e.m.

Figure 2. Effect of ML-7 on the protein levels of GLUT2, SGLT1 and PKC βII in the brush-border membrane

Vesicles were prepared from the jejunum of rats perfused for 30 min with either 75 mm glucose (Glu) as control or 75 mm glucose and 5 μm ML-7 (ML-7). A, vesicle protein (20 μg) was separated on 10% SDS-PAGE gels, transblotted onto PVDF and Western blotted with GLUT2, SGLT1 and PKC βII antibodies. B, expression of protein levels relative to 75 mm glucose control determined from three separate preparations using two rats each. ****P < 0.0001 by unpaired Student's t test.

Figure 3. Apical GLUT2-like immunoreactivity in response to 75 mM glucose is abolished by ML-7

Frozen sections of rat jejunum were prepared after perfusion in vivo of the lumen with 20 mm glucose (A), 75 mm glucose (B) or 75 mm glucose and 5 μm ML-7 (C). Sections of 7 μm were probed with anti-GLUT2 antibody. The secondary antibody was a FITC-conjugated goat anti-rabbit IgG. The peptide control (D) was a section of jejunum treated with 75 mm glucose and antibody that had been pre-absorbed with excess antigenic peptide. All sections are at × 40 magnification and were taken at the same settings with a Zeiss LSM 510 confocal microscope; scale bars, 50 μm. E, relative pixel intensity values are expressed relative to the 75 mm glucose as control ± s.e.m. where n=3. ****P < 0.0001 by unpaired Student's t test.

Figure 4. The effect of glucose, ML-7, phloridzin, calmidazolium, nifedipine and luminal Ca2+ on phosphorylation of myosin II RLC20

Rat jejunum was perfused for 30 min with either 75 mm glucose (Glu) as control, 75 mm mannitol (Man), 75 mm glucose and 5 μm ML-7 (ML-7), 75 mm glucose and 0.2 mm phloridzin (Phl), 75 mm glucose and 20 μm calmidazolium (Cal), 75 mm glucose and 10 μm nifedipine (Nif) or 75 mm glucose in a perfusate deplete of Ca2+ (Ca2+-deplete). A–C, protein (20 μg) was separated on 12% SDS-PAGE gels, transblotted onto PVDF membrane and Western blotted with anti-pan and anti-phospho(Ser19) myosin II RLC20 antibodies. D, expression of the ratio of phospho(Ser19) to pan signal determined from the band intensity of three separate preparations using two rats each. ****P < 0.0001 by unpaired Student's t test.

Figure 5. Immunolocalization of phospho(Ser19) RLC20 and pan RLC20 of myosin II in rat jejunum

Frozen sections of rat jejunum were prepared after perfusion with 75 mm mannitol (A), 75 mm glucose (B), 75 mm glucose and 5 μm ML-7 (C) or 75 mm glucose and 10 μm nifedipine (D). Sections 7 μm thick were probed with anti-pan and anti-phospho(Ser19) myosin II RLC20 antibody. Binding was visualized with a secondary goat or rabbit antibody conjugated to FITC; myosin II was located in the terminal web (TW) and lamina propria (LP). The peptide controls (E) were sections from jejunum perfused with 75 mm glucose and incubated with primary antibodies that had been pre-absorbed with excess antigenic peptides. All sections are at × 40 magnification and were taken at the same settings with a Zeiss LSM 510 confocal microscope; scale bars, 50 μm. F, differential interference contrast (DIC) image at 75 mm glucose with anti-phospho(Ser19) antibody confirming that phosphorylated myosin II (green) is located in the terminal web. G, pixel intensity values are expressed as the ratio of anti-phospho(Ser19) to pan relative to the 75 mm glucose as control as determined from three separate preparations ± s.e.m. where n=3. ****P < 0.0001 by unpaired Student's t test.

Figure 6. The effect of ML-7 and nifedipine on unidirectional 45Ca2+ absorption

Rat jejunum was perfused in vivo with 75 mm glucose and 1.25 mm Ca2+ (45Ca2+ 0.35 kBq ml−1). At 40 min, the perfusate was switched (arrow) to an identical one containing either 5 μm ML-7 (▪) or 10 μm nifedipine (□). Values are presented as the average rate of absorption from 5 experiments. Error bars represent s.e.m.

Figure 7. The effect of luminal Ca2+ on apical GLUT2, SGLT1 and PKC βII protein levels

Apical membrane vesicles were prepared from the jejunum of rats following perfusion for 30 min with 75 mm glucose (Glu) as control; 75 mm glucose and 10 μm nifedipine (Nif) or 75 mm glucose and a perfusate without luminal Ca2+ (Ca2+-deplete). A, protein (20 μg) was separated using 10% SDS-PAGE gels, transblotted onto PVDF membrane and Western blotted for GLUT2, SGLT1 and PKC βII. B, expression of protein levels relative to 75 mm glucose control determined from three separate preparations using two rats each. ***P < 0.001 and ****P < 0.0001 by unpaired Student's t test.

Figure 8

Activation of Cav1.3 by glucose transport through SGLT1 links dietary Ca2+ to terminal web myosin II RLC20 phosphorylation, cytoskeletal rearrangement and apical GLUT2 insertion.