title: Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity.
abstract: Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.
authors.affiliation: Department of Biochemistry, Faculty of Medicine (U38-FCT), University of Porto, Porto, Portugal. ccalhau@med.up.pt
authors.forename: C
authors.initials: C
authors.lastname: Calhau
authors.forename: F
authors.initials: F
authors.lastname: Martel
authors.forename: C
authors.initials: C
authors.lastname: Hipólito-Reis
authors.forename: I
authors.initials: I
authors.lastname: Azevedo
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000201:enzymology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000975:Antioxidants / Q000493:pharmacokinetics; D001205:Ascorbic Acid / Q000493:pharmacokinetics; D018938:Caco-2 Cells; D002412:Cations; D002462:Cell Membrane / Q000201:enzymology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D021381:Protein Transport; D014212:Tretinoin / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014638:Vanadates / Q000493:pharmacokinetics
chemical_list: D000975:Antioxidants; D002412:Cations; D014638:Vanadates; D014212:Tretinoin; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid