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S-nitrosothiol detection in isolated perfused rat heart.
Sandra Ghelardoni1, Sabina Frascarelli, Simonetta Ronca-Testoni
1Dipartimento di Scienze dell'Uomo e dell'Ambiente, University of Pisa, Pisa, Italy.
Molecular and Cellular Biochemistry
|October 28, 2003
Summary
This study developed a method to detect S-nitrosothiols in rat heart membranes. S-nitrosothiol formation was observed with nitric oxide donors but not after ischemia or reperfusion.
Area of Science:
- Cardiovascular physiology
- Biochemistry
- Nitric oxide signaling
Background:
- Nitric oxide (NO) plays a key role in cardiovascular function.
- NO's cardiovascular effects may involve S-nitrosothiol formation.
- S-nitrosothiols are formed by NO reacting with protein sulfhydryl groups.
Purpose of the Study:
- To develop and validate a method for detecting S-nitrosothiols in rat heart membranes.
- To investigate S-nitrosothiol levels under various conditions, including NO donor infusion, ischemia, and reperfusion.
Main Methods:
- Crude membrane preparations from isolated perfused rat hearts were used.
- Hearts were treated with NO donors (SIN-1, isosorbide dinitrate) or subjected to ischemia-reperfusion.
- S-nitrosothiol concentration was measured fluorometrically using 2,3-diaminonaphthylene.
Main Results:
- The assay demonstrated sensitivity of 1-2 pmol/mg protein using S-nitrosoalbumin.
- S-nitrosothiols were undetectable in control, ischemic, or ischemic-reperfused hearts.
- Significant S-nitrosothiol formation (26.4 ± 7.4 and 19.9 ± 5.6 pmol/mg protein) was detected after SIN-1 and isosorbide dinitrate infusion, respectively.
Conclusions:
- A reliable method for S-nitrosothiol detection in rat heart membranes was established.
- Nitric oxide donors induce S-nitrosothiol formation in rat heart membranes.
- S-nitrosothiol levels remain below detection limits under baseline, ischemic, or reperfusion conditions.