Establishment of low extracellular pH is essential for uptake of the fluorescent anionic dye hydroxypyrenetrisulfonate by suspension-cultured carrot cells

Uptake of the fluorescent dye hydroxypyrenetrisulfonate by carrot (Daucus carota L.) suspension cells only occurs when the external (medium) pH falls to below 4.0. Uptake of the dye was shown to be inhibited by a range of compounds, including the ‘Good’ buffers MES, HEPES, HEPPSO and HEPPS, the growth medium component coconut water (CW) and probenecid, an organic anion translocator inhibitor. Tris(hydroxymethyl)aminomethane stimulated transport. Buffer effects were not correlated with pKa values. Inhibitors of dye uptake (MES, probenecid, CW) also inhibited medium acidification by the cells and Tris stimulated acidification and uptake. Uptake of HPTS correlated strongly with external pH and was restored when external pH was experimentally reduced to below 4.0 even in the presence of inhibitors. This suggests that inhibitors of HPTS uptake at the plasma membrane act primarily by preventing the establishment of a low external pH required for transport. The implications of this on the mechanism of HPTS transport are discussed.