title: Bioactivation of nitroglycerin by ascorbate.
abstract: Bioactivation of nitroglycerin (GTN) into an activator of soluble guanylate cyclase (sGC) is essential for the vasorelaxant effect of the drug. Besides several enzymes that catalyze GTN bioactivation, the reaction with cysteine is the sole nonenzymatic mechanism known so far. Here we show that a reaction with ascorbate results in GTN bioactivation. In the absence of ascorbate, GTN did not affect the activity of purified sGC. However, the enzyme was activated to approximately 20% of maximal NO-stimulated activity by GTN in the presence of 10 mM ascorbate with an EC(50) value of 27.3 +/- 4.9 microM GTN. The EC(50) value of ascorbate was 0.11 +/- 0.011 mM. Activation of sGC was sensitive to oxyhemoglobin, superoxide, and a heme-site enzyme inhibitor. GTN had no effect when ascorbate was replaced by 1000 U of superoxide dismutase per milliliter. Ascorbate is known to reduce inorganic nitrite to NO. In the presence of 10 mM ascorbate, approximately 30 microM nitrite caused the same increase in sGC activity as 0.3 mM GTN. Determination of ascorbate-driven 1,2- and 1,3-glycerol dinitrate formation indicated that a 140 nM concentration of products was generated from 0.3 mM GTN within 10 min, excluding nitrite as a relevant intermediate. Our results suggest that a reaction between GTN and ascorbate or an ascorbate-derived species yields an activator of sGC with NO-like chemical properties. This reaction may contribute to GTN bioactivation in blood vessels under conditions of GTN tolerance and ascorbate supplementation.
authors: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
authors: Alexander
authors: A
authors: Kollau
authors: Matteo
authors: M
authors: Beretta
authors: Antonius C F
authors: AC
authors: Gorren
authors: Michael
authors: M
authors: Russwurm
authors: Doris
authors: D
authors: Koesling
authors: Kurt
authors: K
authors: Schmidt
authors: Bernd
authors: B
authors: Mayer
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D001711:Biotransformation; D002478:Cells, Cultured; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D005996:Nitroglycerin / Q000493:pharmacokinetics*; D051381:Rats
chemical_list: D006834:Hydrazines; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D006162:Guanylate Cyclase; D005996:Nitroglycerin; D001205:Ascorbic Acid