Organic Cation Transporter 1 (OCT1/mOct1) Is Localized in the Apical Membrane of Caco-2 Cell Monolayers and Enterocytes

Abstract

Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.

Four Cation-Selective Transporters Contribute to Apical Uptake and Accumulation of Metformin in Caco-2 Cell Monolayers

“…5B). OCT1-knockdown clones [construct 1-clone 43 (1-43), construct 3-clone 21 (3-21), and construct 3-clone 27 (3-27)] with 70% reduction in the OCT1 mRNA expression (corresponding reduction in OCT1 protein expression has been shown previously) (Han et al, 2013) (Fig. 5C) were identified and used as the positive control for the studies with PMAT-knockdown Caco-2 cells.…”

“…The Caco-2 (HTB-37) cell line was obtained from the American Type Culture Collection (Manassas, VA). Human OCT1-knockdown Caco-2 clones were generated in our laboratory previously (Han et al, 2013). SERT transfected human embryonic kidney 293 (HEK 293) cells were provided by Dr. Randy Blakely from Vanderbilt University (Nashville, TN).…”

“…PMAT-knockdown Caco-2 clones were generated using a method similar to that used to generate OCT1-knockdown Caco-2 clones (Han et al, 2013). The PMAT-specific small interfering RNA sequence (sense strand: 59-CAGCUUCAUCACGGACGUG-39; antisense strand: 59-CACGUCCGUGAUGAAGCUG-39) was obtained from published reports (Engel et al, 2004).…”

“…The mRNA expression of human OCT1-3, PMAT, MATE1-2, CHT, and CTL1-5 relative to 18s rRNA in Caco-2 cells and human intestine was determined using quantitative reverse-transcription polymerase chain reaction that was conducted using established methods (Holmes et al, 2006) with minor deviations as described previously (Han et al, 2013).…”

“…Protein concentrations of cell and tissue samples were determined by the bicinchoninic acid assay. Protein from cells (30 mg per lane) and tissues (40 mg per lane) were subjected to Western blot analyses as previously described (Han et al, 2013) using primary antibodies against OCT1, PMAT, SERT, and CHT. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control.…”