Original ContributionsHydroxyguanidines inhibit peroxynitrite-induced oxidation
Introduction
Nitric oxide (NO), a free radical produced by nitric oxide synthase (NOS) isoenzymes, has many essential physiological functions. In addition, NO has been implicated in many pathophysiological processes. Some of the cytotoxic effects of NO are mediated via its rapid reaction with superoxide anions (O2−) to form peroxynitrite (PN) [1], [2], a reactive oxidant species that is generally considered to be more toxic than either NO or superoxide anion alone [3], [4]. The cytotoxic processes triggered by PN include initiation of lipid peroxidation [5], inhibition of mitochondrial respiration [6], [7], inhibition of membrane pumps [8], depletion of glutathione [9] and damage to DNA [10] with subsequent activation of poly (ADP-ribose) synthetase and concomitant cellular energy depletion [11], [12]. The biological activity, toxicity and decomposition of PN is very much dependent on the cellular or chemical environment (concentration of proteins, thiols, glucose, carbon dioxide, and the ratio of NO and superoxide) [1], [2], [4]. Compounds that scavenge PN may provide an alternative to inhibition of NO production for protection against NO/PN mediated cytotoxicity. Indeed, this strategy may be preferable under conditions where peroxynitrite is formed from NO produced by the constitutive isoforms of NOS [4], [13].
We have recently reported that the iNOS inhibitors, mercaptoalkylguanidines, are also effective as direct inhibitors of peroxynitrite [13]. In the present study, we investigate the PN scavenging capability of hydroxyguanidines (OHGs). OHGs, including NG-hydroxy-l-arginine (OH-arg), a product of the oxidation of l-arginine by NOS, can react with NOx species in vitro [14], [15]. The results presented here provide evidence for the rapid reaction of OHGs with PN and associated protection against PN-induced cellular damage.
Section snippets
Measurement of peroxynitrite-induced oxidation of dihydrorhodamine 123
The peroxynitrite-dependent oxidation of dihydrorhodamine 123 (DHR) to rhodamine 123, was measured as described [16], [17]. Briefly, peroxynitrite (5 μM final concentration) was added to phosphate-buffered saline containing DHR (10 μM), in the absence or presence of test compounds (1 μM–3 mM). After a 10 min incubation at 22°C, the fluorescence of rhodamine 123 was measured using a Model LS50B fluorimeter (Perkin-Elmer, Norwalk, CT) at excitation and emission wavelengths of 500 nm and 536 nm,
Hydroxyguanidines reduce the peroxynitrite-induced oxidation of dihydrorhodamine 123
As previously reported [16], [17], peroxynitrite induced a significant oxidation of DHR to rhodamine 123. This oxidation was dose-dependently inhibited by hydroxyguanidine and a range of substituted OHGs with EC50 values ranging from 0.7 to 16 μM (Fig. 1, Table 1). These values compare with values for previously studied thiols of 6 to 21 μM (compounds 1–6, Table 1). By contrast, the guanidines tested (34, 35) were without effect, except for aminoguanidines (32, 33) which had modest effects.
Discussion
The present results demonstrate that hydroxyguanidines (OHGs) are potent scavengers of peroxynitrite and substantially reduce peroxynitrite-induced cell damage.
Although we acknowledge that the order of protection of the various OHGs tested in the DHR assay may be different if a different assay system would have been used in the current study (e.g., cytochrome C oxidation assay, as in ref. [13]), we noted that those compounds that offered the best protection against oxidation of DHR also offered
Acknowledgements
This work was supported by a grant from the National Institutes of Health (R29GM54773). We are grateful for the expert technical assistance of Steve Fox (N2O measurement), Michael O’Connor (DHR, respiration cell damage and blots) and Hank Fales (electrospray-MS). Thanks also to Drs. Hank Fales, Joe Hrabie, Larry Keefer, and Aloka Srinivassen for helpful discussions).
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Present address: Inotek Corporation, 3130 Highland Avenue, Cincinnati, OH 45219, USA; Tel: (513) 475-6655; Fax: (513) 221-8079; E-Mail: [email protected].
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Present address: Inotek Corporation, 3130 Highland Avenue, Cincinnati, OH 45219, USA; Tel: (513) 475-6655; Fax: (513) 221-8079; E-Mail: [email protected].