Abstract
At the concentrations usually employed as a Ca2+ indicator, arsenazo III undergoes a one-electron reduction by rat liver microsomes to produce an azo anion radical as demonstrated by electron spin resonance spectroscopy. Either NADH or NADPH can serve as a source of reducing equivalents for the production of this free radical by rat liver microsomes. The steady state concentration of the azo anion radical is proportional to the square root of the protein concentration, suggesting that the radical decays through a nonenzymatic second order process. The steady state concentration of the azo anion radical is not altered in the presence of metyrapone or CO, and is decreased in the presence of NADP+ or p-hydroxymercuribenzoate. These observations suggest that the formation of arsenazo III anion radical is mediated through NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Under aerobic conditions, addition of arsenazo III to rat liver microsomes produces an increase in electron flow from NAD(P)H to molecular oxygen, generating both superoxide anion and hydrogen peroxide. The steady state concentration of the azo anion radical, but neither oxygen consumption nor superoxide anion formation, is enhanced by calcium and magnesium, suggesting an enhanced azo anion radical-stabilization by complexation with the metal ions. Accordingly, the arsenazo III anion radical signal is abolished in the presence of paramagnetic metal ions (Fe3+, Gd3+, and Ni2+) and enhanced in the presence of other diamagnetic metal ions (La3+). Antipyrylazo III is less effective than arsenazo III in increasing superoxide anion formation by rat liver microsomes, and gives a much weaker ESR spectrum of an azo anion radical. Murexide is reduced to the monodehydro-5,5'-iminobarbituric acid radical by rat liver microsomes, and its efficiency as a superoxide anion generator is intermediate between arsenazo III and antipyrylazo III.
Highlights
At the concentrations usually employed as a Ca2+ indicator, arsenazo I11 undergoes a one-electron reduction by rat liver microsomes to produce an azo anion radical as demonstrated by electron spin resonance spectroscopy
Arsenazo I11 and antipyrylazo I11 have only recently been recognized as Ca2’ indicators [6,7] and have been applied to the measurements of Ca2+ transport inisolated mitochondria (&lo), sarcoplasmic reticulum [7,11, 12],liver microsomes [13],chromaffin vesito rat liver microsomes produces an increase in elec- cles [14],and for measurements of ionized Caz+ within tron flow from NAD(P)Hto molecular oxygen, gener- single cells [15,16,17]
The steady state concentration of the azo anion radical, diazonaphthol dye closely related to arsenazIo11 and antipyrbut neither oxygen consumption nor superoxide anion ylazo 111, is rapidly reduced by rat liver microsomes to an azo formation, is enhanced bycalcium and magnesium, anion radical whose autoxidation generatessuperoxide anion suggesting an enhanced azo anion radical-stabilization by complexation with themetal ions
Summary
Ide is reduced to the monodehydro-5,5’-iminobarbituric acid radical by rat liver microsomes, and its efficiency as a superoxide anion generator is interme-. CDmale rats (150-200 g, CharlesRiver,Inc.) wereusedin the experiments. Theywere fed standard rat chow and water ad libitum and were not fasted prior to use. The animals were killed and their diate between arsenazo I11 and antipyrylazo 111. Microsomes were prepared as described previously [21]and kept onice until use. ErythrocytseuperoxiddeismutaseN, ADHN, ADP+, NAD’, Metallochromic indicatorsaresubstancesthat undergo NADPH,arsenazo 111 (98% pure), antipyrylazo 111, murexide, pcolor changes when the concentrationof free metal ion in the hydroxymercuribenzoate, and dicumarol were obtained from Sigma
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