Results Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol879.Issue1-2.2000:
S . Paterson et al. Brain Research 879 2000 148 –155
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reaction was started by adding 25 ml of the sample supernatant to an assay tube containing 75 ml of 1 mM
3
NADPH, 0.75 mM CaCl and 150 nM [ H]-
L
-arginine.
2
Following incubation in a water bath for 1 min at 378C, the reaction was stopped by adding 1 ml of 1:1 v v Dowex
1
H O-50W 200–400, 8 cross-linked, Na form and 2
2
ml of 20 mM HEPES buffer containing 2 mM EDTA, pH 5.5 into the tube and transferring the tube onto the ice.
After 10 min, 750 ml of the supernatant were removed and
3
added to 4.25 ml of scintillation fluid in a vial. [ H]- citrulline was quantified by liquid scintillation spectros-
copy, using a Beckman scintillation counter. The counts- per-minute cpm for duplicate samples were averaged and
corrected with respect to the blank control and background radioactivity. NOS activity was expressed as pmol min
mg protein. Protein concentrations in the supernatant were measured based on the Bradford method [18] using a
Bio-Rad protein assay dye reagent concentrate and spec- tramax microplate reader. Ten microlitres of supernatant
were removed from the same tissue sample used for the NOS assay and added to 390 ml of distilled water in a 1.5
ml Eppendorf tube and vortexed. Ten microlitres of standard 0–500 mg ml protein or sample was then added
to 190 ml of reagent that consisted of 40 ml of BioRad reagent and 150 ml of water in a 96 well tray. Protein
formation was then assessed using spectrophotometry at a wavelength of 595 nm. The counts for duplicate samples
were averaged.
2.6. Statistical analysis For SN, YHT and RHT, a single two-factor analysis of
variance ANOVA with repeated measures on time was performed [22]. Factor A represented the drug effect on
SN frequency, YHT or RHT; factor B, the repeated measure, represented time; and the interaction AB repre-
sented the change in the rate of compensation as a result of treatment. As factor B, the repeated measure, was always
significant i.e., since vestibular compensation occurred in all cases, it will not be discussed further. Pairwise
comparisons were conducted, where appropriate, using the
´ Scheffe F-test; linear and exponential regression analyses
were also used on the SN data [22]. The NOS activity data were analysed using one-way ANOVAs and post-hoc
student Newman–Keuls multiple comparison tests [22]. The significance level was set at 0.05 for all comparisons.