Real-time uptake of fluorescent ASP+ via the organic cation transporter 3

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Background The organic cation transporter 3 (OCT3) shows a broad expression pattern in the nervous system and has been detected in neurons and glial cells. Here, OCT3 serves as an additional re-uptake system for neurotransmitters, such as dopamine, serotonin and norepinephrine and therefore OCT3 mediated uptake has been termed “uptake 2”. Methods We measured OCT3 mediated uptake of the fluorescent OCT3 substrate 4-Di-1-ASP (4-(-4(dimethylamino(styryl)-N-methylpyridinium (ASP) in real-time in HEK293 cells stably expressing the human and the mouse isoforms of OCT3. Data obtained on OCT3 mediated uptake of fluorescent ASP were compared to uptake of tritiated 1-methyl-4-phenylpyridinium (MPP). Results We show that ASP is selectively taken up via OCT3 in real time and this allows for sensitive assessment of transport via OCT3. Hence, we analyzed the mode of action of several OCT3 substrates and transport inhibitors, such as the stress hormone corticosterone and decynium-22. All results obtained from ASP uptake studies are comparable with data obtained from uptake studies with tritiated MPP and in line with previously published reports. Finally, we tested if OCT-mediated uptake is sensitive to phosphorylation and found that the protein kinase C inhibitor GF109203X inhibited uptake. Discussion The use of ASP as substrate for OCT3 provides the opportunity to analyze OCT3 mediated transport with a high temporal resolution and is comparable to OCT3 mediated uptake of tritiated MPP. Uptake inhibition by corticosterone was comparable using either ASP or MPP and similar to inhibition of protein kinase C.