Pharmacological Research 48 (2003) 579–584 Effect of thiamine on 3 H-MPP + uptake by Caco-2 cells Conceição Calhau a,b,c,∗ , Fátima Martel a,b , Cˆ andido Hipólito-Reis a , Isabel Azevedo a,b a Department of Biochemistry, University of Porto, 4200-319, Porto, Portugal b Institute of Pharmacology and Therapeutics, Faculty of Medicine (U-38, FCT), University of Porto, 4200-319 Porto, Portugal c Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal Accepted 12 May 2003 Abstract Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP + ) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP + uptake by Caco-2 cells. The present study investigated a putative modulation of MPP + intestinal apical uptake and ecto-ALP activity by thiamine (T + ) and thiamine pyrophosphate (TPP, a T + dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl- 3 H]-4-phenylpyridinium acetate ( 3 H-MPP + ) uptake were evaluated in intact Caco-2 cells. T + and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease 3 H-MPP + apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and 3 H-MPP + uptake were concentration-dependent. The results suggest that the effect of T + and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T + may occur through hEMT, in Caco-2 cells. © 2003 Elsevier Ltd. All rights reserved. Keywords: Caco-2 cells; 1-Methyl-4-phenylpyridinium (MPP + ); Uptake; Ecto-alkaline phosphatase; Thiamine 1. Introduction It was recently described by our group that the model organic cation 1-methyl-4-phenylpyridinium (MPP + ) is efficiently transported by Caco-2 cells in the apical-to- basolateral (absorptive) direction [1], and that absorption of MPP + seems to occur through a Na + -independent trans- porter belonging to the amphiphilic solute facilitator (ASF) family, the extraneuronal monoamine transporter (hEMT) [2,3]. In addition, we recently demonstrated that the MPP + transporter, in Caco-2 cells, is most likely active in the de- phosphorylated state [3] and that ecto-alkaline phosphatase Abbreviations: ALP, alkaline phosphatase; ASF, amphiphilic solute facilitator; hEMT, human extraneuronal monoamine transporter; MPP + , 1-methyl-4-phenylpyridinium; OCT1, organic cation transporter 1; p-NP, p-nitrophenol; p-NPP, p-nitrophenylphosphate; RDA, recommended di- etary allowance; T + , thiamine; TMP, thiamine monophosphate; TPP, thi- amine pyrophosphate; ThTr1, thiamine transporter 1; ThTr2, thiamine transporter 2 ∗ Corresponding author. Tel.: +351-22-5095694; fax: +351-22-5502402. E-mail address: ccalhau@med.up.pt (C. Calhau). (ecto-ALP) seems to be involved in this regulatory pathway [4]. Thiamine (T + ) is required by animal cells to synthesize thiamine pyrophosphate (TPP), the coenzyme of the indis- pensable carbohydrate enzyme transketolase and of the de- hydrogenase complexes for pyruvate, -ketoglutarate, and branched-chain keto acids. T + plasma concentration is reg- ulated both by intestinal and renal mechanisms. Chemically T + is a water-soluble organic cation (quaternary ammonium compound) with a high molecular weight. At concentrations lower than 2 mol l −1 ,T + is transported by intestinal mu- cosa mainly through an active, carrier-mediated system that involves the intracellular phosphorylation and dephosphory- lation of this vitamin [5]. Entry at the luminal side is largely through exchange with H + and very little through enzy- matic transphosphorylation to TMP (by intestinal alkaline phosphatase present in enterocyte apical membrane). Cellu- lar crossing is associated with intracellular enzymatic phos- phorylation to TPP and dephosphorylation of TPP to TMP and T + . For higher concentrations of T + , simple passive diffusion prevails [6]. The expression of the recently cloned thiamine trans- porter, ThTr1, is very high in skeletal muscle, heart and 1043-6618/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00176-2