Mechanism of sodium nitroprusside-mediated inhibition of aromatic amino acid decarboxylase activity.
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 mumol/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 mumol/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 mumol/l. In contrast, ferrous sulfate (0.5 mmol/l) was a competitive inhibitor (Ki = approximately 650 mumol/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.
Dawson
R
R
Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville 32610, USA.
Felheim
R
R
Nguyen
S
S
D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D002478:Cells, Cultured / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives; D003676:Deferoxamine / Q000494:pharmacology; D004298:Dopamine / Q000096:biosynthesis*; D005296:Ferrous Compounds / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology*; D011190:Potassium Cyanide / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D013552:Swine
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D005296:Ferrous Compounds; D009599:Nitroprusside; D011732:Pyridoxal Phosphate; D005978:Glutathione; D006152:Cyclic GMP; D003676:Deferoxamine; D011190:Potassium Cyanide; D004298:Dopamine