The effects of sodium nitroprusside (SNP) on dopamine synthesis in a porcine renal epithelial cell line (LLC-PK1) were evaluated. Subsequent studies examined the actions of the degradation products of SNP (cyanide, ferrous ion and nitric oxide) on aromatic amino acid decarboxylase (AAAD) activity in tissue supernatants from LLC-PK1 cells and rat renal cortex. SNP (10–500 µmol/l) significantly inhibited dopamine production in LLC-PK1 cells in a dose-related manner. The activation of guanylate cyclase by nitric oxide was not found to be the mechanism whereby SNP inhibited dopamine synthesis in LLC-PK1 nor did the antioxidant glutathione attenuate the actions of SNP. Ferrous sulfate (0.5 mmol/l) and SNP (0.5 mmol/l) were found to inhibit dopamine synthesis in LLC-PK1 cells and to directly inhibit cytosolic AAAD activity from LLC-PK1 cells. A series of studies were conducted using AAAD from rat renal cortex and confirmed that SNP could directly inhibit the conversion of L-dopa to dopamine by AAAD. Furthermore, potassium ferricyanide (1 mmol/l) and potassium cyanide (1 mmol/l) could produce greater than 80% reductions in AAAD activity. Iron (0.5-1 mmol/l) was found to increase rat kidney AAAD activity. Kinetic analysis revealed that potassium cyanide was a potent (Ki = 40-50 µmol/l) noncompetitive/mixed noncompetitive inhibitor of AAAD. SNP was also found to be a noncompetitive inhibitor of AAAD with a Ki of approximately 300-500 µmol/l. In contrast, ferrous sulfate (0.5 mmol/l) was a competitive inhibitor (Ki = ∼650 µmol/l) that actually increased the Vmax of AAAD. The results of these studies support that cyanide released from SNP can potently inhibit AAAD, although SNP has somewhat more complex interactions with AAAD due to the presence of ferrous ion.
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.
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