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 ef fect 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 (f rom the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [alpha-P-32]GTP to [P-32]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a com mercially available Clark-type electrode. Results: Stimulation of sGC by th e NO donor 2,2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate wi th an IC50 of similar to 2 mu M. Maximal enzyme inhibition (similar to 70%) was observed at 0.1-1 mM vitamin C. Stimulation of sGC by the NO-independe nt activator protoporphyrin-M 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 b y vitamin C. Consumption of NO by ascorbate was prevented by reduced glutat hione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide di smutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase fai led to restore sGC activity. Conclusions: Our results suggest that physiolo gical concentrations of L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC. (C) 2000 Elsevier Science B .V. All rights reserved.