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  • Although we showed that RBL H Sc cells took up

    2021-11-25

    Although we showed that RBL-2H3 Sc98 Canrenone took up histamine from the microenvironment, it was unclear which transporter mediated its passage across the plasma membrane. Other biogenic amines have selective transporters that belong to the neurotransmitter sodium symporter family, including SERT for serotonin, dopamine transporter for dopamine, and norepinephrine transporter for noradrenaline (Rudnick, 2006). However, a histamine-selective transporter in mast cells has not been reported. Recently, uptake-2 transporters, including OCT1, OCT2, OCT3, and PMAT, have been shown to transport some biogenic amines such as histamine, serotonin, and catecholamines. The expression of these transporters in human, rat, and mouse tissues has already been reported along with their substrate specificities (Grundemann et al., 1999, Jonker and Schinkel, 2004, Engel and Wang, 2005, Duan and Wang, 2010, Koepsell, 2013, Duan et al., 2015). To ascertain which protein could potentially act as a histamine transporter in mast cells, we analyzed the ability of drugs, which are known selective inhibitors of each transporter, to suppress histamine uptake in RBL-2H3 Sc98 cells. Uptake-2 transporters have common biogenic amine substrates. We evaluated the uptake of serotonin, dopamine, adrenaline, and noradrenaline by RBL-2H3 Sc98 cells, and found that they did not take up dopamine, adrenaline, or noradrenaline (data not shown). These results suggest that neuronal transporters DAT and NET are not present in these cells. In contrast, serotonin was taken up into the cells, which might have been facilitated by SERT or uptake-2 transporters. However, when both histamine and serotonin were added to the system, the uptake of amines was suppressed, suggesting that histamine and serotonin may compete for the same transporter. Because suppression of histamine uptake was greater than that of serotonin, the transporter appears to have a higher affinity for serotonin than histamine. Previously, it was reported that histamine is transported via OCT2 and OCT3, while serotonin is transported by OCT1 and OCT2 (Grundemann et al., 1999, Jonker and Schinkel, 2004). Schneider et al. (2005) showed that histamine is taken up into murine basophils by OCT3, and revealed D-22 as a potent selective inhibitor of OCT3 (Duan et al., 2015). Because basophils and mast cells have properties that are similar to those of cytoplasmic granule-containing cells, OCT3 is capable of mediating histamine transport into RBL-2H3 mast cells. Therefore, we examined whether D-22 inhibits histamine uptake in RBL-2H3 Sc98 cells. Using D-22 at a single concentration of 0.01 μM and total contact time of 45 min, histamine uptake was suppressed by approximately 30%. Other reported experimental methods, such as radioisotope labeling and fluorescence imaging, have demonstrated that D-22 is an effective inhibitor after only a short incubation period of 5–15 min (Schomig et al., 1993, Gasser et al., 2006, Inyushin et al., 2010). Other inhibitors of each selected transporter, namely desipramine, cimetidine, corticosterone, and lopinavir, were used to block OCT1, OCT2, OCT3, and PMAT, respectively (Duan et al., 2015). Among these inhibitors, lopinavir suppressed histamine uptake the most in RBL-2H3 Sc98 cells. Desipramine slightly inhibited histamine uptake, whereas cimetidine and corticosterone did not show any inhibitory activities. There is no evidence that indicates inhibitory activities of lopinavir and desipramine against VMAT2. Some reported substrates and inhibitors of VMAT2 include reserpine, tetrabenazine, 3-amino-2-phenylpropene, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridinium, and their respective derivative compounds (Wimalasena, 2011) with essentially different structures from lopinavir and desipramine. Therefore, further studies are required to investigate the effects of lopinavir and desipramine on VMAT2. However, using the RBL-2H3 cell line for this purpose will be challenging because there are both plasma and vesicle membrane transporters. Using reserpinized RBL-2H3 cells, the results suggested that the changes in histamine transport by lopinavir and desipramine were due to their activities towards only the plasma membrane transporter in RBL-2H3 cells and not in combination with vesicle membrane transporters.