Large-scale preparation of plasma membrane vesicles from PC-12 Pheochromocytoma cells and their use in noradrenaline transport studies

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 μm in diameter) with a mean intravesicular water space of 1.84 μl / mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [³H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31°C). The uptake and accumulation of [³H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.