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Characterisation and comparison of temporal release profiles of nitric oxide generating donors.

Bradley SA, Steinert JR - J. Neurosci. Methods (2015)

Bottom Line: We found that donors such as NOC-5 and PAPA-NONOate decayed substantially within days, whereas SNP and GSNO showed greater stability releasing consistent levels of NO over days.In all donors tested, the amount of released NO differs between frozen and unfrozen stocks.Fluorescent and amperometric approaches to measure NO concentrations yield a wide range of levels.

View Article: PubMed Central - PubMed

Affiliation: MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LE1 9HN, UK.

No MeSH data available.


Related in: MedlinePlus

Temporal release of NO from SNAP. (A) Graph depicting the varying NO release profiles from the same concentration of SNAP (5 μM) at three consecutive time points: 0–30 min, 30 min–1 h and 1 h+, note the signal saturation at recordings 0–30 min. (B) Average release profiles of NO yield from fresh SNAP stock at 5, 10 and 20 μM in the presence of the copper chelator EDTA. (C) Box and whisker plots displaying the NO concentration range at plateau after 30 min recording time from fresh SNAP stock (1 h+) in PBS with no EDTA at 5, 10 and 20 μM. (D) Box and whisker plots demonstrating the area under the curve from fresh SNAP stock (1 h+) with no EDTA at 5, 10 and 20 μM.
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fig0030: Temporal release of NO from SNAP. (A) Graph depicting the varying NO release profiles from the same concentration of SNAP (5 μM) at three consecutive time points: 0–30 min, 30 min–1 h and 1 h+, note the signal saturation at recordings 0–30 min. (B) Average release profiles of NO yield from fresh SNAP stock at 5, 10 and 20 μM in the presence of the copper chelator EDTA. (C) Box and whisker plots displaying the NO concentration range at plateau after 30 min recording time from fresh SNAP stock (1 h+) in PBS with no EDTA at 5, 10 and 20 μM. (D) Box and whisker plots demonstrating the area under the curve from fresh SNAP stock (1 h+) with no EDTA at 5, 10 and 20 μM.

Mentions: SNAP is an S-nitrosothiol that is obtained through the S-nitrosation of tertiary thiols. SNAP releases NO by catalytic activity of various physical, chemical and enzymatic factors that include temperature, light, transition metals, thiols and superoxide. Here we characterised the release of NO from fresh stock over a one day period only due to the presence of an uncontrolled variability in NO release following longer time periods of storage. Fresh stock that was diluted in PBS immediately after it was made up has a different release profile and saturated the used sensitivity range in comparison to release tested 30 min later. The release profile changed once more and settles when recorded >1 h after making stock solutions (Fig. 6A). This characteristic was unique to SNAP and not seen in any other donor. As copper is a potent catalyst of SNAP decomposition and copper is present as a contaminant in varying levels in water used to prepare buffer solutions (Dicks and Williams, 1996), we next sought to chelate any free copper ions using EDTA in both the fresh stock solution (40 μM) and recording PBS solutions (200 nM). Addition of EDTA resulted in a dramatic reduction in NO release from SNAP at all concentrations due to removal of catalysing transition metal ions (Mcaninly et al., 1993) (5 μM: 21 ± 9 nM, 10 μM: 29 ± 15 nM and 20 μM: 27 ± 6 nM NO) (Fig. 6B). However, when copper ions were re-introduced to the PBS solution (100 μM CuSO4) the NO release from 20 μM SNAP rose rapidly to NO concentrations that were above the measurable constraints of the probe (not shown). As plateau levels of NO release were not reached during the 30 min recording period (after >1 h of making fresh SNAP stock) the NO concentration given is the final value at the end of the 30 min recording period in PBS with no EDTA (300 ± 9 nM at 5 μM, 374 ± 12 nM NO at 10 μM and 841 ± 158 nM NO at 20 μM donor, Fig. 6C). The total amount of NO released (AUC) following >1 h after making up the stock increased with donor concentration as expected (Fig. 6D). These results indicate the importance of controlling copper levels if using SNAP as an NO donor.


Characterisation and comparison of temporal release profiles of nitric oxide generating donors.

Bradley SA, Steinert JR - J. Neurosci. Methods (2015)

Temporal release of NO from SNAP. (A) Graph depicting the varying NO release profiles from the same concentration of SNAP (5 μM) at three consecutive time points: 0–30 min, 30 min–1 h and 1 h+, note the signal saturation at recordings 0–30 min. (B) Average release profiles of NO yield from fresh SNAP stock at 5, 10 and 20 μM in the presence of the copper chelator EDTA. (C) Box and whisker plots displaying the NO concentration range at plateau after 30 min recording time from fresh SNAP stock (1 h+) in PBS with no EDTA at 5, 10 and 20 μM. (D) Box and whisker plots demonstrating the area under the curve from fresh SNAP stock (1 h+) with no EDTA at 5, 10 and 20 μM.
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Related In: Results  -  Collection

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fig0030: Temporal release of NO from SNAP. (A) Graph depicting the varying NO release profiles from the same concentration of SNAP (5 μM) at three consecutive time points: 0–30 min, 30 min–1 h and 1 h+, note the signal saturation at recordings 0–30 min. (B) Average release profiles of NO yield from fresh SNAP stock at 5, 10 and 20 μM in the presence of the copper chelator EDTA. (C) Box and whisker plots displaying the NO concentration range at plateau after 30 min recording time from fresh SNAP stock (1 h+) in PBS with no EDTA at 5, 10 and 20 μM. (D) Box and whisker plots demonstrating the area under the curve from fresh SNAP stock (1 h+) with no EDTA at 5, 10 and 20 μM.
Mentions: SNAP is an S-nitrosothiol that is obtained through the S-nitrosation of tertiary thiols. SNAP releases NO by catalytic activity of various physical, chemical and enzymatic factors that include temperature, light, transition metals, thiols and superoxide. Here we characterised the release of NO from fresh stock over a one day period only due to the presence of an uncontrolled variability in NO release following longer time periods of storage. Fresh stock that was diluted in PBS immediately after it was made up has a different release profile and saturated the used sensitivity range in comparison to release tested 30 min later. The release profile changed once more and settles when recorded >1 h after making stock solutions (Fig. 6A). This characteristic was unique to SNAP and not seen in any other donor. As copper is a potent catalyst of SNAP decomposition and copper is present as a contaminant in varying levels in water used to prepare buffer solutions (Dicks and Williams, 1996), we next sought to chelate any free copper ions using EDTA in both the fresh stock solution (40 μM) and recording PBS solutions (200 nM). Addition of EDTA resulted in a dramatic reduction in NO release from SNAP at all concentrations due to removal of catalysing transition metal ions (Mcaninly et al., 1993) (5 μM: 21 ± 9 nM, 10 μM: 29 ± 15 nM and 20 μM: 27 ± 6 nM NO) (Fig. 6B). However, when copper ions were re-introduced to the PBS solution (100 μM CuSO4) the NO release from 20 μM SNAP rose rapidly to NO concentrations that were above the measurable constraints of the probe (not shown). As plateau levels of NO release were not reached during the 30 min recording period (after >1 h of making fresh SNAP stock) the NO concentration given is the final value at the end of the 30 min recording period in PBS with no EDTA (300 ± 9 nM at 5 μM, 374 ± 12 nM NO at 10 μM and 841 ± 158 nM NO at 20 μM donor, Fig. 6C). The total amount of NO released (AUC) following >1 h after making up the stock increased with donor concentration as expected (Fig. 6D). These results indicate the importance of controlling copper levels if using SNAP as an NO donor.

Bottom Line: We found that donors such as NOC-5 and PAPA-NONOate decayed substantially within days, whereas SNP and GSNO showed greater stability releasing consistent levels of NO over days.In all donors tested, the amount of released NO differs between frozen and unfrozen stocks.Fluorescent and amperometric approaches to measure NO concentrations yield a wide range of levels.

View Article: PubMed Central - PubMed

Affiliation: MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LE1 9HN, UK.

No MeSH data available.


Related in: MedlinePlus