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 micron in diameter) with a mean intravesicular water space of 1.84 microliters/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 degrees 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.
D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D009638:Norepinephrine / Q000378:metabolism*; D010673:Pheochromocytoma / Q000378:metabolism / Q000648:ultrastructure*; D051381:Rats; D013696:Temperature; D014882:Water-Electrolyte Balance
D009638:Norepinephrine