intrinsic-factor and leupeptin

Intracellular events within enterocytes following receptor-mediated endocytosis of intrinsic factor-cobalamin (IF-Cbl) are poorly understood. We have examined the fate of IF and Cbl in filter-grown Caco-2 cells which express both IF receptors and transcobalamin II and which transcytose Cbl. Uptake of IF-Cbl from the apical surface leads to the intracellular accumulation of Cbl in a process that reaches an equilibrium between internalization and secretion only after a 20-h continuous incubation. Transcytosed Cbl is detectable in the basolateral medium 4 h after the onset of endocytosis. Cbl is released from the basolateral surface with the same kinetics irrespective of from which cell surface endocytosis of IF-Cbl took place. Following uptake, internalized IF is degraded with a half-time of 4 h. Leupeptin causes a partial block in the proteolysis of IF, an intracellular accumulation of Cbl bound to IF, and a decrease in transcytosis of Cbl. Finally, an analysis of intracellular Cbl during transcytosis shows that free Cbl is present within cells during transcytosis.

Topics: Biological Transport; Cell Line; Cell Polarity; Cytoplasm; Endocytosis; Humans; Intestinal Mucosa; Intrinsic Factor; Leupeptins; Vitamin B 12

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.

Topics: Animals; Chloroquine; Chromatography, Liquid; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Intrinsic Factor; Kidney Cortex; Leupeptins; Male; Microvilli; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Vitamin B 12

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.

Topics: Ammonium Chloride; Animals; Autoradiography; Biological Transport; Cells, Cultured; Chromatography, Gel; Cobalt Radioisotopes; Electrophoresis, Polyacrylamide Gel; Epithelium; Intrinsic Factor; Iodine Radioisotopes; Kidney; Leupeptins; Molecular Weight; Opossums; Rats; Vitamin B 12

To ascertain the mechanism of release of cobalamin (Cbl) from intrinsic factor (IF) and subsequent formation of transcobalamin II (TC-II)-Cbl complex, we studied the intracellular distribution of 57Co-labeled Cbl after its uptake in suckling and adult rats. The amount of Cbl bound to IF, to the IF-Cbl receptor via IF, and to TC-II was determined by immunoprecipitation with monospecific antisera raised to these proteins. IF-Cbl receptor activity was found to be very low in suckling rats up to 12 days after birth. Oral administration of leupeptin in amounts known to alter protein turnover had no effect on the release of Cbl from IF nor did it inhibit the formation of the TC-II-Cbl complex in either adult or suckling animals. However, oral administration of chloroquine resulted in a transient increase in the intestinal concentration of Cbl in both adult and suckling rats and in total inhibition of Cbl released from IF in adults rats. Chloroquine prevented completely the transfer of Cbl to TC-II in adult rats and inhibited the transfer by 50% in suckling rats. These data demonstrate that in adult mucosa utilizing receptor-mediated endocytosis, Cbl is transferred from IF to TC-II. This transfer does not require the IF-Cbl receptor, as it occurs in suckling rats. Finally, transfer of Cbl to TC-II is decreased by a drug that alters vesicular pH. Because Cbl can be released at acid pH from IF, it is proposed that release of Cbl from IF and its transfer to TC-II occurs in an acidic vesicle.

Topics: Administration, Oral; Animals; Animals, Suckling; Chloroquine; Gastric Mucosa; Immune Sera; Intestinal Mucosa; Intestines; Intrinsic Factor; Leupeptins; Male; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Transcobalamins; Vitamin B 12