Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo

Abstract

The ex vivo tissue concentration of nitrite and nitrate (NO~) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N'~-nitro-L-arginine(L-NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NO. by up to 75% but had no effect on plasma NOR. Selective inhibition of central NOS by intracerebroventricular administration of L-NA methyl ester produced similar decreases in levels of whole-brain NOR. A residual concentration of NO. of 10-15 ,uM remained in all brain regions even after complete inhibition of brain NOS. Brain NO~content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NO. was found to reflect the in vivo efficacy of several different types of NOS inhibitor: L-NA, N~-monomethyl-L-arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate L-arginine increased brain NO. concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NO~is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo. Key Words: Nitric oxide synthase-Nitrite-Nitrate-N~-Nitro-L-arginine-N~-Monomethyl-L-arginine-7-Nitroindazole. oxide; NOS, nitric oxide synthase; NO,, nitrite and nitrate.

Glutathione Depletion, Lipid Peroxidation and Mitochondrial Dysfunction Are Induced by Chronic Stress in Rat Brain

“…(Cortas and Wakid 1990) and NO 2 Ϫ was determined by a colorimetric assay based on the Griess reaction (Green et al 1982) in a Thermomax ® microplate reader (Molecular Devices). The measurement of NO x Ϫ levels has been found to be a reliable technique to determine the synthesizing capacity of NOS in brain (Salter et al 1996).…”

Induction of Cyclooxygenase-2 Accounts for Restraint Stress-Induced Oxidative Status in Rat Brain

“…The [ 14 C]-bound radioactivity was counted using a liquid scintillation counter (Beckman LS-6500) (Salter et al, 1991;Rees et al, 1995 (Cortas and Wakid, 1990) and NO 2 À was determined by a colorimetric assay based on the Griess reaction (Green et al, 1982) in a Thermomax s microplate reader (Molecular Devices). The measurement of NO x À levels has been found to be a reliable technique to determine the synthesizing capacity of NOS in the brain (Salter et al, 1996).…”

Unexpected Effects of Nitric Oxide Synthase Inhibitors on Extracellular Nitrite Levels in the Hippocampus in vivo

“…The evidence to support this assumption is compelling [7][8][9][10][11][12][13][14] , however, in all of these studies NOS inhibitors were administered to sites distinct from those where the measure of NOS activity was made. Salter et al [8,9] applied the drugs by intracerebroventricular injection then determined both NOS activity and tissue nitrite concentration ex vivo. The others administered them intraperitonealy and measured nitrite/nitrate in dialysates collected via a microdialysis probe.…”