Kinetic separation and characterization of three sugar transport modes in Caco-2 cells.

The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an "AMG-strict" system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a "DG-strict" system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a "DG/AMG-mixed" system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the "AMG-strict" system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.

The synthesis and accumulation of membrane protein 4.1 in Friend erythroleukemia cells.

The effect of extensive differentiation on the synthesis and accumulation of protein 4.1 were studied on Friend erythroleukemia cells grown in suspension and on fibronectin coated dishes. Whole membranes of Friend erythroleukemia cells (FELC) contained a protein 4.1a and 4.1b doublet of Mr 76 and 74 kDa and two minor bands of Mr 105 and 43 kDa that cross-reacted with anti-human protein 4.1 IgG. These proteins were present even in uninduced cells. The synthesis of protein 4.1 was maximal after 4 days of induction in both suspension culture and in fibronectin-coated dishes whereas the protein 4.1 continued to accumulate until the seventh day. More protein 4.1 accumulated in cells grown on fibronectin-coated dishes, at each stage of differentiation, than in cells grown in suspension. The protein 4.1a/4.1b ratio changed during differentiation. The amounts of protein 4.1b increased progressively after induction until the protein 4.1a/4.1b ratio was similar to that of mouse mature erythrocyte. The protein 4.1a/4.1b ratio appears to be an internal marker of erythroid differentiation.