Transcytosis and coenzymatic conversion of [(57)Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in caco-2 cells.
We have examined the intracellular route, coenzyme conversion and transcytosis rate of [(57) Co]-labeled cobalamin (Cbl) in function of its presentation to the apical side of Caco-2 cells, either free or bound to intrinsic factor (IF). The free-presented Cbl was progressively bound to endogenous transcobalamin II (TCII) which may stem, in part, from a basolateral to apical passage. Its transcytosis was TCII-mediated as it was abolished when antibodies to TCII were added to the apical medium. The apparent permeability coefficient (P(app)) was estimated at 20.8+/-3.6, 103.5+/-17.7, 0.9+/-0.3 x 10(-5) cm/h for TCII-Cbl, IF-Cbl and haptocorrin-Cbl, respectively. Chloroquine inhibited the transcytosis rate of both TCII and IF-bound Cbl in a dose-dependent manner. Approximately 80% of apical Cbl, bound to either exogenous IF or endogenous TCII, was transported to the basolateral side as intact cyano[(57)Co]Cbl whereas the remainder was converted into Ado-Cbl and CH(3)-Cbl within the cells, as shown by HPLC analyses of a 1,000-g pellet and a 12,000-g supernatant. Coenzymatic conversion was virtually abolished by chloroquine. In conclusion, we suggest that apically presented free Cbl is internalized via TCII-dependent transport. The apically internalized CN-Cbl, bound to either IF or TCII, is processed via an acidic vesicle and part of it is converted to coenzymes, whereas bulk of CN-Cbl is transcytosed intact.
Pons
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Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, Equipe INSERM 00-14, Faculté de Médecine, Université Henri Poincaré, B.P. 184, 54 505 Vandoeuvre-lès-Nancy Cedex, France.
Guy
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M
Lambert
D
D
Hatier
R
R
Guéant
J
J
D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D016764:Cell Polarity / Q000502:physiology*; D002738:Chloroquine / Q000494:pharmacology; D003037:Cobalt Radioisotopes; D003078:Colchicine / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007437:Intrinsic Factor / Q000378:metabolism*; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D003037:Cobalt Radioisotopes; D014155:Transcobalamins; D002738:Chloroquine; D007437:Intrinsic Factor; D004492:Edetic Acid; D014805:Vitamin B 12; D003078:Colchicine