Measurement of the concentrations of dopamine and its metabolites in PC12 cells and in extracellular fluid by detection of native fluorescence

Kobayashi Yoko

doi:10.34506/bku.24.0_111

Rat pheochromocytoma (PC12) cells synthesize and release neurotransmitters such as dopamine and noradrenaline. Dopamine, which is synthesized from L-tyrosine in cells, is metabolized to dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3MT), and homovanillic acid (HVA). Dopamine is known to show native fluorescence. In this study, we aimed to establish a method to measure the concentrations of dopamine and its metabolites in PC12 cells by using high performance liquid chromatography coupled with a fluorescence detector (HPLC-FL). 　First, several concentration of monoamine standard solutions, including that of dopamine, serotonin, and their metabolites, were separated and detected by HPLC-FL. The concentrations of dopamine, 3MT, and HVA were measured with a fluorescence detector set at an excitation wavelength of 225 nm and emission wavelength of 310 nm. However, the sensitivity of DOPAC detection was low. n-Hexanal (hexanal), a straight aldehyde with a 6-carbon chain length, is known to stimulate dopamine release from PC12 cells. Therefore, the concentrations of dopamine and its metabolites in samples from PC12 cells stimulated by hexanal were measured using HPLC-FL. We observed that PC12 cells released dopamine and that the concentrations of extracellular dopamine and 3MT were elevated after stimulation with hexanal. In future, we will attempt to elucidate the physiological significance of the dopamine released by hexanal by using this method.

Measurement of the concentrations of dopamine and its metabolites in PC12 cells and in extracellular fluid by detection of native fluorescence

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