Effect of quercetin and its derivatives on peroxynitrite injured sarco/endoplasmic Ca2+-ATPase as a possible therapeutic tool.

L Horakova1, P Zizkova1, J Viskupicova1, D Blaskovic1, I Milackova1, L Ratkovska0,2, M Veverka0,2. 1Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Biochemical Pharmacology, 841 04 Bratislava, Slovakia, 2Bel/Novamann International Ltd., Eurofins, Bel/Novamann International, 940 02 Nove Zamky, Slovakia

The search for mechanisms of oxidative/nitrosative injury involved in human diseases is important in the development new therapeutic approaches. Modulation of Ca 2+ -ATPases by antioxidants may be a therapeutic tool in mitigating complications associated with human diseases.

Peroxynitrite is able to participate not only in physiological regulation but also in pathological effects. Peroxynitrite (25-250 µM) in our experiments, concentration dependently decreased the activity of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) from sarcoplasmic reticulum of white rabbit skeletal muscles. Posttranslational alterations of SERCA as increase of protein carbonyls and nitrotyrosine as well as decrease of cysteine SH groups were observed. Peroxynitrite induced conformational changes in cytosolic region of SERCA as indicated by fluorescein 5´-isothiocyanate (FITC), a competitive inhibitor of the ATP binding site on SERCA. No changes in the transmembrane Ca 2+ binding domain were observed, as measured by N-cyclohexyl-N\'- (4-dimethylamino-1-naphtyl) carbodiimide (NCD-4).

New semisynthetic quercetin derivatives were compared with the standard flavonoid quercetin (Q) with respect to protective effects on SERCA under conditions of peroxynitrite induced oxidative stress. Quercetin and monochloropivaloylquercetin (MPQ,) both in the concentration region of 10-100 µM, significantly prevented the activity of SERCA decreased by peroxynitrite. Chloronaphtochinonequercetin (ChQ) was without any protective effect and even induced additional decrease of SERCA activity. Derivatives of quercetin exerted stronger antiradical activity compared with quercetin as indicated by the DPPH test. In addition their preventive effects against protein carbonyl formation and nitrotyrosine generation were observed. Interestingly, all agents tested decreased free SH groups of cysteines measured by fluorescence probe ThioGlo, probably by their possible prooxidant effects. Quercetin and its derivatives induced conformational changes in cytosolic nucleotide binding site, as well as in transmembrane Ca 2+ binding site.

Our results may at least partially explain the inhibitory effect of ChQ on SERCA by only slight protection against protein carbonyl formation, the most intensive impairment of SH groups and the most intensive conformational alterations in the transmembrane Ca 2+ binding region.

The results point to a dual role of quercetin agents in SERCA activity modulation. Q and MPQ with a protective effect on SERCA could be useful in anti-inflammatory defense and prevention against heart diseases, where protection against cell apoptosis is necesary. On the other hand, the inhibitory effect of ChQ on Ca 2+ -ATPase activity may represent a new therapeutic approach by induction of apoptosis in antitumor treatment.

Supported by The Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic for the Structural Funds of EU, OP R&D of ERDF by realization of the Project „Evaluation of natural substances and their selection for prevention and treatment of lifestyle diseases (ITMS 26240220040) and by VEGA grant 2/0038/11.