Inhibition of purified soluble guanylyl cyclase by L-ascorbic acid.
OBJECTIVE
L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide (NO)/cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC).


METHODS
To address this issue we used a highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [alpha-32P]GTP to [32P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a commercially available Clark-type electrode.


RESULTS
Stimulation of sGC by the NO donor 2, 2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate with an IC(50) of approximately 2 microM. Maximal enzyme inhibition ( approximately 70%) was observed at 0.1-1 mM vitamin C. Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1 mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase failed to restore sGC activity.


CONCLUSIONS
Our results suggest that physiological concentrations of L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.
Schrammel
A
A
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, 8010, Graz, Austria. astrid.schrammel@kfunigraz.ac.at
Koesling
D
D
Schmidt
K
K
Mayer
B
B
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors*; D006834:Hydrazines / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D009569:Nitric Oxide / Q000032:analysis; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction
D000975:Antioxidants; D004791:Enzyme Inhibitors; D006834:Hydrazines; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D001205:Ascorbic Acid