Characterization of the Synthesis and Release of Dopamine in LLC-PK₁ Cells

Abstract

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 DA 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-PK₁cells 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.

Tissue-specific Expression of the Nonneuronal Promoter of the Aromatic L-Amino Acid Decarboxylase Gene Is Regulated by Hepatocyte Nuclear Factor 1

“…However, the isolation of the renal epithelial cell line LLC-PK 1 , which is devoid of neural input and tyrosine hydroxylase activity, and does not metabolize dopamine, yet expresses high levels of AADC, has provided a model system which has been used to study renal physiology and the regulation of dopamine synthesis and release (16,23).…”