Characterization of MPP⁺ secretion across human intestinal Caco‐2 cell monolayers: role of P‐glycoprotein and a novel Na⁺‐dependent organic cation transport mechanism

Abstract

-upon-Tyne, NE2 4HH 1 In the kidney, a number of transport proteins involved in the secretion of permanently charged organic cations have recently been cloned. To evaluate the possible similarities between intestine and kidney in the handling of organic cations we investigated the transport of 1-methyl-4-phenylpyridinium (MPP + ) across monolayers of intestinal Caco-2 cells. MPP + is a prototypic substrate of the cloned organic cation transporters hOCT1 and hOCT2. 2 In Caco-2 cell monolayers, the basolateral to apical¯ux of MPP + was signi®cantly greater than the apical to basolateral¯ux, consistent with net secretion of MPP + . 3 Net secretion of MPP + was abolished by addition of either 10 mM cyclosporin A or 100 mM verapamil to the apical membrane. In contrast, secretion of MPP + was una ected by addition of either TEA (2 mM) or decynium-22 (2 mM) to either apical or basolateral membranes. These results suggest that MPP + secretion is mediated primarily by P-glycoprotein located at the apical membrane. We found no evidence of a role for hOCT1 or hOCT2 in the secretion of MPP + . 4 In addition to net secretion of MPP + , we found evidence of a Na + -dependent MPP + uptake mechanism at the apical membrane of Caco-2 cells. 5 Na + -dependent MPP + uptake was sensitive to inhibition by the organic cations; decynium-22 (2 mM), TEA (2 mM) and cimetidine (5 mM) but not by carnitine, guanidine or proline. 6 These results suggest that net secretion of MPP + across the apical membrane of Caco-2 cells is a function of the relative contributions of MPP + secretion mediated by P-glycoprotein and MPP + absorption mediated by a novel Na + -dependent transport mechanism.

Differential Substrate and Inhibitory Activities of Ranitidine and Famotidine toward Human Organic Cation Transporter 1 (hOCT1; SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3)

“…Zhang et al (1999) reported intestinal expression of hOCT1 as determined by reverse-transcriptase-polymerase chain reaction, suggesting that this isoform is present in the human intestine. A number of studies have also reported expression of hOCT1, hOCT2, and hOCT3 in the Caco-2 cell model of intestinal epithelium (Zhang et al, 1999;Bleasby et al, 2000;Martel et al, 2001;Hayer-Zillgen et al, 2002). Functional studies suggest a lack of saturable OCT-mediated uptake of TEA along the basolateral membrane of Caco-2 cells (Lee et al, 2002).…”

“…hOCT1 mRNA expression has been reported in human small intestine (Zhang et al, 1999), and mRNA expression of hOCT1, hOCT2, and hOCT3 has been demonstrated in the Caco-2 cell model of intestinal epithelium (Bleasby et al, 2000;Martel et al, 2001;Hayer-Zillgen et al, 2002). The function of these transporters in the intestine or Caco-2 cells, however, is unclear.…”

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

“…OCTN2 is unique in that it transports carnitine with a high affinity (K m Ϸ 4.3 M) in an Na ϩ -dependent manner, and organic cations in an Na ϩ -independent manner. Recently, Bleasby et al (2) reported that 1-methyl-4-phenylpyridinium absorption by Caco-2 cells is mediated by an Na ϩ -dependent transport mechanism. This raises the possibility of OCTN2 in these cells, because 1-methyl-4-phenylpyridinium can inhibit carnitine uptake via OCTN2 (55,56).…”

Transport Mechanisms of Nicotine across the Human Intestinal Epithelial Cell Line Caco-2

“…Previously, the putative involvements of ASF transporters such as OCT1 or OCT2 were reported to mediate the uptake of organic cations by Caco-2 cells (Bleasby et al, 2000;Martel et al, 2001). However, nicotine uptake by Caco-2 cells was not inhibited by either tetraethylammonium, cimetidine, or NMN, all of which were known to be typical substrates for organic cation transport systems, OCT1 and OCT2 (Urakami et al, 1998(Urakami et al, , 2001).…”

“…Multiple mechanisms appear to be involved in organic cation transport in both intestinal brushborder membrane vesicles and Caco-2 cells. The involvement of P-glycoprotein (Hsing et al, 1992;Hunter et al, 1993a,b) and some members of the amphiphilic solute facilitator (ASF) family such as OCT1, OCT2, or extraneuronal monoamine transporter (EMT) (Bleasby et al, 2000;Martel et al, 2001) have been reported. We previously demonstrated that diphenhydramine, an antihistamine, was transported across Caco-2 cell monolayers by H ϩ -coupled specific transport systems that exist in both the apical and basolateral membranes (Mizuuchi et al, 1999(Mizuuchi et al, , 2000aKatsura et al, 2000).…”