Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase.
NO synthase (NOS) is a unique P-450-type enzyme containing both a reductase and a heme domain on a single polypeptide. We show that ebselen [Ebs, 2-phenyl-1,2-benzisoselenazol-3-(2H) one], a nontoxic selenoorganic compound known to break a cysteine thiolate/Fe bond of some of P-450 enzymes, is a relatively selective inhibitor of endothelial isoform of NOS. In rings of rabbit aorta, Ebs irreversibly blocked both the basal as well as acetylcholine- or calcium ionophore A23187-stimulated release of nitric oxide with an IC50 of 6 microM. In homogenates of bovine aortic endothelial cells, Ebs inhibited the activity of NOS, assayed by monitoring conversion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline, with an IC50 of 8.5 microM. The inhibitory action of Ebs was prevented by glutathione, N-acetyl-L-cysteine or dithiothreitol (30-500 microM). The prevention by thiols of Ebs-induced inhibition of NOS suggests that these are competing with a thiol group of NOS that is essential for the catalytic activity of the enzyme. The consequence of the presence of thiols is the "trapping" of Ebs in the form of inactive selenyl sulfides. Consistent with the proposed mechanism of action of Ebs is lack of activity of diselenide of Ebs, which also demonstrates that the action of Ebs is independent of its glutathione peroxidase-like activity. In comparison to endothelial preparations, IC50 values of Ebs for inhibition of soluble isoforms of NOS present in homogenates of porcine cerebellum and of spleens obtained from lipopolysaccharide-treated rats were more than 30-fold higher.
Zembowicz
A
A
Department of Pharmacology, Copernicus Academy of Medicine, Kraków, Poland.
Hatchett
R J
RJ
Radziszewski
W
W
Gryglewski
R J
RJ
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D001393:Azoles / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology; D002417:Cattle; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D004789:Enzyme Activation; D005260:Female; D005979:Glutathione Peroxidase / Q000378:metabolism; D066298:In Vitro Techniques; D054833:Isoindoles; D008297:Male; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D019001:Nitric Oxide Synthase; D016566:Organoselenium Compounds / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013552:Swine
D001393:Azoles; D001549:Benzamides; D054833:Isoindoles; D016566:Organoselenium Compounds; D013438:Sulfhydryl Compounds; C067619:bis(2-(N-phenylcarboxamido)phenyl)diselenide; C042986:ebselen; D005979:Glutathione Peroxidase; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases