Characterization of the synthesis and release of dopamine in LLC-PK1 cells

The ability of a renal epithelial cell line of porcine origin (LLC-PK1) to synthesize dopamine (DA) from L-dopa and release DA into the culture media was studied. L-Dopa was rapidly taken up by LLC-PK1 cells and converted to DA which was then released into the media. L-Dopa uptake and conversion to DA was competitively inhibited by other aromatic amino acids (Tyr and Phe). Studies were conducted to assess L-dopa uptake, DA synthesis and DA release in LLC-PK1 cells in response to changes in the ionic composition of the Krebs-Ringer bicarbonate (KRB) buffer media and to various drugs known to alter renal transport function. Sodium chloride addition to KRB media resulted in enhanced DNA release into the media that was significantly attenuated by both quinine (1 mM) and benzamil (0.1 mM). Other sodium salts (sodium acetate and sodium phosphate) also enhanced DA release from LLC-PK1 cells. Salts containing chloride (LiCl, NH4Cl, choline chloride) stimulated a greater efflux of DA from LLC-PK1 cells than sodium salts at equimolar concentrations. Osmotic stimuli (sucrose, mannitol and dextran, 50-200 mM) also elicited increased DA release from LLC-PK1 cells into the media but not to the same extent as inorganic salts. DA release (secretion) from LLC-PK1 cells was strongly inhibited (25-50%) by drugs known to be substrates for the renal cation transport system. These studies have characterized extensively the factors that govern and regulate the synthesis of DA from L-dopa and the transport system responsible for the secretion (release) of intracellular DA into the media.