title: Uptake of (3)H-1-methyl-4-phenylpyridinium ((3)H-MPP(+)) by human intestinal Caco-2 cells is regulated by phosphorylation/dephosphorylation mechanisms.
abstract: Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the intestinal uptake of organic cations by these mechanisms. The intestinal apical uptake of 1-methyl-4-phenylpyridinium (MPP(+)) was studied by incubating Caco-2 cells at 37 degrees for 5 min with 200 nM (3)H-MPP(+). Uptake of (3)H-MPP(+) by Caco-2 cells was not affected by activators of protein kinase G, and was not affected or slightly reduced (by 15-20%) by activators of protein kinase A or protein kinase C. Uptake of (3)H-MPP(+) by Caco-2 cells was reduced in a concentration-dependent manner by non-selective phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine (IBMX), caffeine, teophylline). The IC(50) of IBMX was found to be 119 microM (102-138; n=9). Uptake of (3)H-MPP(+) by Caco-2 cells was not affected by inhibition of protein tyrosine kinase, but it was concentration-dependently reduced in the presence of inhibitors of mitogen-activated protein kinase. Uptake of (3)H-MPP(+) by Caco-2 cells was strongly reduced by Ca(2+)/calmodulin-mediated pathway inhibitors, but it was not dependent on extracellular Ca(2+). Our results suggest that the intestinal apical uptake of MPP(+) is regulated by phosphorylation/dephosphorylation mechanisms, being most probably active in the dephosphorylated state. Moreover, uptake of (3)H-MPP(+) by Caco-2 cells and by the extraneuronal monoamine transporter (EMT) are regulated in a very similar manner, suggesting an important participation of EMT in the intestinal uptake of this compound.
authors: Department of Biochemistry, Faculty of Medicine, 4200-319 Porto, Portugal. fmartel@med.up.pt
authors: Fátima
authors: F
authors: Martel
authors: Elisa
authors: E
authors: Keating
authors: Conceição
authors: C
authors: Calhau
authors: Isabel
authors: I
authors: Azevedo
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism*; D000470:Alkaloids; D053119:Benzophenanthridines; D001692:Biological Transport; D018938:Caco-2 Cells; D002147:Calmodulin / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006540:Herbicides / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D002147:Calmodulin; D004791:Enzyme Inhibitors; D006540:Herbicides; D010617:Phenanthridines; D014316:Tritium; C016299:chelerythrine; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D015655:1-Methyl-4-phenylpyridinium