Human colonic carcinoma Caco-2 cells grown in vitro undergo epithelial differentiation. Electrical measurements showed that they form resistant monolayers of polarized cells. On millipore filters, transepithelial electrical resistance (154 +/- 6.5 omega X cm2) was accompanied by a small potential difference (0.29 +/- 0.02 mV, serosal side positive) and by short-circuit current (1.9 +/- 0.14 microA X cm-2), both of which were ouabain sensitive. Micropuncture of domes formed on plastic supports under standard culture conditions revealed electrical polarity similar to that of filter-grown cells (0.8 +/- 0.2 mV, serosal side positive) combined with a highly negative cytoplasm (-57 +/- 1 mV) and very marked cell asymmetry (76% of total electrical cell resistance was located in the mucosal membrane). These parameters were not affected by the diuretic amiloride nor the hormone aldosterone, suggesting that sodium conductance is very limited in the mucosal membrane. Addition to the mucosal side of the ionophore nystatin or amphotericin B unmasked the possibility of high electrical transport activity. Electrical measurements made it possible to define the epithelial properties of Caco-2 cells, which may resemble those of colonic crypt or fetal cells. These measurements also confirmed that functional differentiation is homogeneous in Caco-2 cells. It is suggested that dome cell micropuncture may be useful in investigating the functional properties of other dome-forming cell lines.
Nine healthy volunteers were studied before, during, and after ingesting a fermented dairy product containing Lactobacillus acidophilus, Bifidobacterium bifidum, and mesophilic cultures (Streptococcus lactis and S cremoris) for 3 wk. Hydrogen and methane productions and fecal beta-galactosidase and beta-glucosidase activities were measured as indicators of fermentation capacity of the colonic flora. Fecal concentrations of nitroreductase, azoreductase, and beta-glucuronidase, which may be implicated in colonic carcinogenesis, were also assessed. Hydrogen and methane productions, fecal beta-galactosidase, beta-glucuronidase, and azoreductase activities did not change over three 3-wk periods whereas fecal beta-glucosidase activity increased (42 +/- 6, 91 +/- 12, and 40 +/- 6 IU/g N, P less than 0.01) and nitroreductase decreased (0.87 +/- 0.13, 0.54 +/- 0.11, and 0.57 +/- 0.08 IU/g N, P less than 0.05).
Quantification and functional characteristics of intact protein uptake, metabolic behavior, and transmission across the intestinal wall were examined using horseradish peroxidase (HRP) transport through adult rabbit jejunum. Transepithelial HRP fluxes were determined with a modified Ussing apparatus. In Ringer solution, no significant difference was found between intact HRP fluxes from mucosa to serosa (JHRPm leads to s) and those from serosa to mucosa (JHRPs leads to m) (3.12 +/- 0.58 and 3.48 +/- 0.45 pmol.h-1.cm-2, respectively). In the presence of 10 mM glucose, slight net secretion was noted. The transport mechanism was shown to be sensitive to metabolic inhibitors, colchicine, and ammonia. Intracellular transfer and catabolism were estimated by measuring transepithelial fluxes of tritiated horseradish peroxidase (J[3H]HRP). Net absorption of 3H equivalent HRP (91 +/- 32 pmol.h-1.cm-2) occurred chiefly in the form of tritiated degraded catabolites of 2-4 kilodaltons. Comparison of the transepithelial fluxes of intact and 3H equivalent HRP made it possible to estimate that 97% of the HRP was degraded while crossing the tissue from mucosa to serosa and 88% while crossing from serosa to mucosa. The saturable absorption observed for JHRPm leads to s became a nonsaturable process for J[3H]HRPm leads to s. These results fit the existence of at least two functional pathways for intestinal protein transport. The main route seems to involve endocytosis, with striking intracellular degradation, possibly during passage through the lysosomal system. The HRP that escapes metabolic degradation is transported by an alternative route requiring the structural and metabolic integrity of the epithelial cells. Although this route only accounts for a small fraction of HRP transport, it may be of immunological importance.
To assess absorption and metabolic effects of enterally delivered glutamine, a total of 10 healthy subjects received perfusions of natural L-glutamine at graded infusion rates (ranging from 0 to 126 mmol/h; n = 2-8 subjects at each rate) along with a nonabsorbable marker (polyethylene glycol) through a double-lumen nasojejunal tube. Perfusions were administered after an overnight fast during three consecutive 1- or 2.5-h periods and in a randomized order. In eight subjects, continuous intravenous infusion of D-[6,6-2H2]glucose, L-[1-13C]leucine, and L-[15N]alanine was simultaneously performed. Glutamine was nearly quantitatively absorbed over the 30-cm study segment; estimated Km and Vmax of glutamine absorption were 2.48 and 2.32 mmol/min over the 30-cm study segment. Enteral glutamine administration induced 1) a dose-dependent increase in plasma glutamine level; 2) a rise in the plasma level and appearance rate (Ra) of alanine (from 191 +/- 42 to 213 +/- 51 mumols.kg-1.h-1, P less than 0.05, for 0 and 46.8-mmol/h glutamine infusion rates, respectively) and in plasma levels of glutamate, citrulline, aspartate, and urea; 3) a decline in plasma free fatty acid and glycerol levels; and 4) no change in leucine or glucose Ra. We conclude that glutamine is efficiently absorbed by human jejunum in vivo and may directly inhibit lipolysis, whereas it neither affects proteolysis nor glucose production in healthy postabsorptive humans.
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