Summary. Hamster intrinsic factor (IF) preparations markedly enhanced the uptake of 57cobalt-labeled cyanocobalamin (B_2-57Co) by brush borders and microvillous membranes isolated from villous absorptive cells obtained from the distal but not the proximal half of hamster intestine. A similar effect was observed with rat and rabbit IF preparations, but IF preparations obtained from man, dog, and hog were ineffective. After -fractionation of hamster IF preparations by gel filtration or ion exchange chromatography, the extent to which each fraction enhanced B_2-57Co uptake by brush borders correlated closely with the vitamin B12 binding capacity of the fraction. IFmediated attachment of B12-57Co to brush borders occurred rapidly, was not diminished by removal of glucose or oxygen from the incubation medium, and was not significantly altered when incubation temperatures were reduced from 370 C to 70 C. Marked reduction in uptake occurred, however, in the absence of divalent cations.IF enhanced B12-57Co uptake by brush borders isolated from the proximal half of the intestine when these proximal brush borders were preincubated with supernatant fluid obtained after centrifugation of homogenates of distal intestinal mucosa at 28,500 g. The factor in this supernate responsible for the effect on proximal brush borders was shown to be particulate in nature upon centrifugation at speeds of 54,500 g or greater. The resultant pellet contained ribosomes and membranous fragments.Prolonged incubation of brush borders with crude saline extracts of hamster gastric mucosa resulted in decreased uptake of B_2-57Co and marked lysis of brush borders with concomitant release of tissue nitrogen. Neither lysis of brush borders nor decreased uptake of B12-57Co with prolonged incubation was observed when hamster IF was partially purified. Furthermore, uptake of 12-57Co by brush borders increased with increasing purity of the IF preparation used.These results demonstrate IF-mediated attachment of B,2-57Co to brush borders and microvillous membranes of hamster intestinal cells and provide further support for the presence of a specific receptor for IF-bound vitamin
A B S T R A C T Elucidation of mechanisms involved inthe control of colonic production of mucus requires direct examination of glycoprotein synthesis and secretion by colonic mucosa. In the past, the limited viability of intestinal mucosa in vitro has hampered such investigations. When maintained in an organ culture system, mucosal biopsies of rabbit colon and human rectum remained viable for 24 h as documented by morphologic appearance and a steady rate of protein synthesis and secretion. These biopsies also incorporated 'C-labeled glucosamine into tissue. glycoproteins and secreted labeled glycoproteins at a steady rate for 24 h. Glucosamine was predominantly incorporated into macromolecules that were ultimately secreted, in contrast to leucine, which was predominantly incorporated into tissue macromolecules. When studied by autoradiography, cultured rabbit colonic biopsies synthesized and secreted glycoproteins in vitro at cellular sites and over a time-course similar to those previously described for the intestine of intact animals. Acetylcholine consistently stimulated secretion of labeled glycoproteins but did not alter glycoprotein synthesis. In contrast, cycloheximide inhibited glycoprotein synthesis but had no effect on the secretion of newly synthesized glycoproteins. Rectal biopsies from patients with active ulcerative colitis incorporated increased amounts of [1'C]
glucosamine into glycoproteinsThis work was presented in part at
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