Characterization of trace amine transport properties across human intestinal epithelial cells

Sarkar, Shreyasi (2023) Characterization of trace amine transport properties across human intestinal epithelial cells. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Trace amines (TA), typified by p-tyramine (TYR) and 2-phenylethylamine (2-PE), function by activating trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor. TAAR1 is intracellularly localized and plays important physiological functions. Thus, it is important to know the mechanisms by which TA cellular levels can be controlled. TA can readily diffuse across synthetic lipid bilayers and Organic Cation Transporter 2 (OCT2; Slc22A2) was shown to transport TYR in synaptosomes. Whether a similar transporter regulates diet-derived TYR uptake across intestinal epithelial cells is unknown. In this study, I selected the Caco-2 human intestinal cell line to characterize TA transport properties across a Caco-2 monolayer. On studying TYR and 2-PE transport pattern, their transport across the apical membrane was shown to be mediated by a facilitated diffusion transporter which for TYR was decynium-22 (P = 0.0092) and pentamidine sensitive (P = 0.001), but not atropine (ATR) sensitive, suggesting it is OCT2. Conversely, across the basolateral membrane, Na⁺-dependent (P = 0.0174) and ATR sensitive (P = 0.020) active TYR transport was observed (P < 0.0001), with a trend (P = 0.0714) towards active 2-PE transport also observed. Kinetic parameters for the TYR active transporter were Kₜ = 33.1 nM and Vmax = 43.0 nM/second. Protein purification by coupling TYR to a N-hydroxysuccinimide activated column led to isolation of 124 common TYR and ATR binding proteins from Caco-2 cells, although none of them were Na+-dependent transporters. Finally, TYR transport was modelled by developing ordinary differential equations in MATLAB using kinetic parameters of known TYR transport processes and compared to experimental data. Known TYR kinetics did not recapitulate experimental observations, suggesting the presence of additional transporters. Further simulations indicated that asymmetry in apical OCT2 transport (Kₜ_OCT2_apicaltocell = 110.4 nM, Kₜ_OCT2_celltoapical = 1227.9 nM), and an additional non-OCT2, symmetric, basolateral facilitated diffusion transporter (Vmax = 6.0 nM/s, Kₜ = 628.3 nM) were required to recapitulate experimental parameters. In conclusion, OCT2 transports TYR across the apical Caco-2 membrane, while as yet unidentified Na⁺-dependent active transporter and symmetric facilitated diffusion transporter are responsible for TYR transport across Caco-2 basolateral membranes.

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