Shreyasi Sarkar - January 22

Characterization of trace amine transport properties across human intestinal epithelial cells

Trace amines (TA), typified by 2-phenylethylamine (PEA) and p-tyramine (TYR), are a family of endogenous compounds involved in the regulation of various physiological processes through selective activation of trace amine associated receptor 1 (TAAR1). Unlike other G protein-coupled receptors, TAAR1 has an intracellular localization, which requires that TA cross cell membranes to access the receptor. We have previously demonstrated that TA readily diffuse across synthetic lipid bilayers. Further, in neurons membrane passage also involves a transporter exhibiting the pharmacological profile of Organic Cation Transporter 2 (OCT2; Slc22A2). Highly selective inhibitors of individual OCT are not known and gene knockdown is difficult in ex vivo neuronal preparations, making validation of OCT2 as the TYR transporter difficult in neurons. Since TA are also present in commonly consumed foods, and are also produced by the intestinal microbiota by decarboxylation of dietary amino acids, the Caco-2 intestinal epithelial cell line was selected as a relevant model for further validation studies. In culture Caco-2 cells differentiate into polarized epithelial cells which form monolayers that are morphologically and functionally consistent with the in vivo situation containing distinct apical and basolateral sides. Caco-2 cells were grown for 23 days and monolayer integrity was confirmed by lucifer yellow exclusion and trans epithelial electrical resistance measurement and the passage of 100 nM TYR from apical and basolateral compartments determined. Results so far indicate that TYR passage across the apical membrane occurs by a diffusion mediated process which could involve a facilitated diffusion transporter such as OCT2. The presence of OCT2 in Caco-2 cells has been confirmed by western blot. In contrast, transport across the basolateral membrane shows characteristics of active transport. Current studies are focusing on the identification of the transporter on the apical side using inhibitors of different selectivity towards OCT family members. Should one or more inhibitors alter TYR passage across apical membranes, the effect of selective knock-down of the implicated transporter will be examined to validate its role as a TYR transporter. Future studies will further characterize the active transport process on the basolateral membrane. Thus, there appears to be different TYR transport processes occurring on the apical and basolateral membranes of human intestinal epithelial cells, with facilitated diffusion across the apical side and active transport across the basolateral side.

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