REGULATION OF RIBOFLAVIN INTESTINAL UPTAKE BY PROTEIN-KINASE-A - STUDIES WITH CACO-2 CELLS

Although the mechanism of riboflavin (RF) intestinal uptake has been t he subject of many studies, virtually nothing is known about the cellu lar regulation of the uptake process. In the present study, we investi gated the role of protein kinase A (PKA)- and C (PKC)-mediated pathway s in the regulation of RF intestinal uptake using the confluent Caco-2 monolayers. Treatment of Caco-2 cells with 3-isobutyl-1-methylxanthin e (IBMX), forskolin, cholera toxin, or dibutyryl adenosine 3',5'-cycli c monophosphate caused a significant inhibition in RF uptake. The inhi bitory effect of IBMX was reversible and resulted from a significant d ecrease in the maximal velocity of the RF uptake process with no chang e in its apparent Michaelis constant. The IBMX-induced inhibition in R F uptake was not mediated via inhibition in the synthesis of the RF ca rrier protein or through inhibition in the recruitment of preexisting carrier protein into the plasma membrane. Calyculin A also inhibited R F uptake when added alone and further potentiated the inhibitory effec t of IBMX when added together. Phorbol 12-myristate 13-acetate or chel erythrine, on the other hand, showed no significant effect on RF uptak e. These results demonstrate for the first time that compounds that in crease intracellular cAMP levels downregulate RF intestinal uptake and that this effect is mediated via a decrease in the activity of the RF uptake carrier. It is suggested that a PKA-mediated pathway(s) plays an important role in regulating RF intestinal uptake.