Abstract
S-Nitrosothiols are a group of potent, bioactive compounds that form through the reaction of nitric oxide (NO) with thiols in the presence of oxygen. These compounds are naturally occurring in vivo, stabilize NO and potentiate its biological effects. S-Nitrosoglutathione is the most abundant intracellular S-nitrosothiol, and the kinetics for its formation favors de novosynthesis. In this analysis, we studied the formation of S-nitrosothiols by S-transnitrosation, or exchange of -NO for -H between sulfur atoms; we synthesized S-nitroso-glutathionyl-Sepharose 4B beads (SNO-4B) as a reagent with which to measure S-transnitrosation reactions. We detected a maximum of 1.57 ± 0.24 pmol NO/bead (n = 5) after S-nitrosation of the beads with acidified nitrite. The stability of the S-NO bond was dependent on temperature, but not pH over the 5 to 9 range (except at pH 9 at 37°), with an estimated t1/2 of 30 hr at 22°C and of approximately 2 wk at 4°C. We demonstrated that SNO-4B transfers -NO to glutathione and to cysteine rapidly and in a pH-dependent manner. The initial rate of transfer of -NO from SNO-4B to glutathione at room temperature was 0.53, 3.03 and 5.14 μM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). Under the same conditions, the initial rate of -NO transfer to cysteine was 0.72, 3.71 and 4.69 μM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). There was no appreciable S-transnitrosation between SNO-4B and bovine serum albumin. We further demonstrated that SNO-4B evokes significant vasodilator and platelet inhibitory responses in plasma-free systems and activates platelet soluble guanylyl cyclase. These data suggest a mechanism by which to explain the metabolic fate and distribution of NO among thiol pools in the vasculature, and implicate S-transnitrosation at the cell surface in NO signal transduction.
Footnotes
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Send reprint requests to: Dr. Joseph Loscalzo, Whitaker Cardiovascular Institute, Center for Advanced Biomedical Research, 700 Albany Street, Boston, MA 02118.
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↵1 This work was supported in part by National Institutes of Health Grants HL53919, HL48743, HL55993; by a Merit Review Award from the U.S. Veterans Administration, and by a grant from NitroMed, Inc.
- Abbreviations:
- RSNO
- S-nitrosothiol
- NO
- nitric oxide
- GSNO
- S-nitrosoglutathione
- SNO-4B
- S-nitroso-glutathionyl-Sepharose 4B beads
- SGC
- soluble guanylyl cyclase
- GSH
- glutathione
- SNP
- sodium nitroprusside
- GTP
- guanosine-5′-triphosphate
- cGMP
- cyclic guanosine-3′5′-monophosphate
- trishydroxymethylaminomethane (Tris)
- IBMX, 3-isobutyl-1-methylxanthine
- NEM
- N-ethylmaleimide
- DTNB
- 5,5′-dithiobis(2-nitrobenzoic acid)
- BSA
- bovine serum albumin
- DPTA
- diethylenetriaminepentaacetic acid
- CySNO
- S-nitrosocyseine
- SNO-BSA
- S-nitroso-albumin
- SGC
- soluble guanylyl cyclase
- ACD
- acid-citrate-dextrose
- TCA
- trichloroacetic acid
- DTT
- dithiothreitol
- Received June 24, 1997.
- Accepted October 14, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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