Inhibition of aromatic L-amino acid decarboxylase under physiological conditions: optimization of 3-hydroxybenzylhydrazine concentration to prevent concurrent inhibition of monoamine oxidase.
The activity of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) is commonly studied indirectly by quantifying the formation of the product, 3,4-dihydroxyphenylalanine (DOPA), after inhibition of aromatic L-amino acid decarboxylase (AAAD; EC 4.1.1.28), the enzyme which metabolizes DOPA. This study was done to determine if the concentration of the hydrazine derivative 3-hydroxybenzylhydrazine (NSD-1015), a drug frequently used in vitro to inhibit AAAD, could be adjusted such that it would inhibit that enzyme, but would not simultaneously inhibit a second, potentially important enzyme, monoamine oxidase (MAO; EC 1.4.3.4). MAO catalyzes the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG) from dopamine (DA) and norepinephrine (NE), respectively. Five concentrations of NSD-1015 in superfusate (0.01 to 20 microM) were tested in strips of canine portal vein superfused and stimulated in vitro. DOPA, DA, NE, and DOPEG in superfusate and in the veins after superfusion were quantified by HPLC with electrochemical detection. The efficacy of NSD-1015 in inhibiting AAAD and MAO was determined by examining the levels of DOPA and DOPEG, respectively. NSD-1015, only when applied at 0.1 microM, resulted in the marked augmentation of total DOPA levels, but did not affect levels of DOPEG, which suggests that this concentration of the drug inhibits AAAD, but does not inhibit MAO. Therefore, it is concluded that, of the concentrations of NSD-1015 tested, 0.1 microM is the optimum concentration to use in this preparation for studies designed to examine TH activity by measuring DOPA after the inhibition of AAAD.
Hunter
L W
LW
Department of Anesthesiology, Mayo Clinic, Rochester, MN.
Rorie
D K
DK
Tyce
G M
GM
D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D004285:Dogs; D004298:Dopamine / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D006834:Hydrazines / Q000494:pharmacology*; D066298:In Vitro Techniques; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives / Q000378:metabolism; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology*; D011169:Portal Vein; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D006834:Hydrazines; D008996:Monoamine Oxidase Inhibitors; D015102:3,4-Dihydroxyphenylacetic Acid; D008734:Methoxyhydroxyphenylglycol; C004297:3-hydroxybenzylhydrazine; D014446:Tyrosine 3-Monooxygenase; C010117:3,4-dihydroxyphenylglycol; D004298:Dopamine