Interaction of quercetin and its derivatives with Ca2+-ATPase from sarcoplasmic reticulum: Kinetic and molecular modeling studies.

Sarcoplasmic reticulum Ca2+-ATPases (SERCAs) regulate cellular calcium homeostasis and are targeted for age-related diseases. Among 14 SERCA mRNA splice variants, SERCA1a is specific to adult fast-twitch skeletal muscle. Quercetin derivatives (monochloropivaloylquercetin (CPQ), IC50 = 195.7 µM; 2-chloro-1,4-naphthoquinonequercetin (CHNQ), IC50 = 60.3 µM) were studied for their impact on SERCA1a using molecular modeling and enzyme kinetics. While there were some similarities in kinetic parameters and molecular modeling, the compounds exhibited diverse actions on SERCA1a. Quercetin reduced activity by 48% at 250 µM by binding to the cytosolic ATP-binding pocket with increased ATP affinity. CPQ bound near the Ca2+-binding site, possibly altering the transmembrane domain. CHNQ significantly reduced activity by 94% at 250 µM without binding to substrate sites. It was proposed that CHNQ induced global protein structure changes, inhibiting Ca2+-ATPase activity.

Pycnogenol Cytotoxicity in Pancreatic INS-1E ß cells Induced by Calcium Dysregulation.

Natural standardized flavonoid extract from the bark of Pinus pinaster, Pycnogenol (Pyc), was recently found to decrease intensively the activity of sarcoplasmic reticulum Ca2+ -ATPase of rabbit skeletal muscle (SERCA1). On the basis of this inhibitory effect in a cell-free system and similarities of SERCA1 to its other isoforms, proapoptotic properties of Pyc may be expected in cellular systems. Pycnogenol (40-100 µg/mL) induced a concentration-dependent decrease of the viability of pancreatic INS-1E ß cells associated with induction of apoptosis. In addition, intracellular Ca2+ level increase was found along with reduction of protein expression level of SERCA2b and impairment of insulin secretion by ß cells. These facts indicate that Pyc may induce apoptosis by impairment of calcium homeostasis. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA1) by rutin derivatives.

The effect of lipophilic rutin derivatives (acylated with fatty acid chain length of 16-22) on sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA1 isoform) compared to the parent molecule rutin was evaluated. Rutin derivatives caused concentration dependent decrease of SERCA1 activity (IC50 ~ 23-64 µM) and significant conformational alterations in the transmembrane region of the enzyme. Upon treatment by peroxynitrite, rutin derivatives exerted a hormetic effect, i.e. prevented enzyme activity decrease at low concentrations, while additionally inhibited at high concentrations. Concerning the posttranslational modifications of SERCA1, rutin esters: (i) induced a significant loss of free sulfhydryl groups, (ii) protected the enzyme from protein carbonyl formation, and (iii) prevented SERCA from tyrosine nitration (except R20:4 and R22:1). In silico study revealed a strong affinity of the derivative R20:4 to the transmembrane region of SERCA1, stabilized via hydrogen bonds with Glu90, Glu771, Thr778 and Thr848 residues. Interaction of rutin derivatives with Glu771, a residue involved in Ca(2+) binding, is likely to be responsible for the inhibitory effect of the esters.

Rutin stimulates sarcoplasmic reticulum Ca(2+)-ATPase activity (SERCA1) and protects SERCA1 from peroxynitrite mediated injury.

In this study we analyzed the protective action of the flavonoid rutin on peroxynitrite (ONOO(-)) mediated impairment of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1 isoform), especially related to posttranslational and conformational changes. Rutin concentration dependently protected ONOO(-) induced SERCA1 activity decrease with effective concentration EC50 of 18 ± 1.5 µM. Upon treatment with ONOO(-), this flavonoid also prevented SERCA1 from thiol group oxidation and significantly reduced tyrosine nitration and protein carbonyl formation. In the absence of ONOO(-), rutin (250 and 350 µM) stimulated SERCA1 activity at 2.1 mM [ATP] and 10 µM [Ca(2+)]free. According to changes in the kinetic parameters V max and K m with regard to [ATP], rutin (250 µM) increased the rate of enzyme catalysis and decreased the affinity of SERCA1 to ATP. FITC fluorescence decreased in the presence of rutin (150 and 250 µM), indicating conformational changes in the cytosolic ATP binding region of SERCA1. In silico study confirmed the binding of rutin in the cytosolic region of SERCA1, in the vicinity of the ATP binding site. Residue Glu183 localized within the conserved TGES loop was identified to play a key role in rutin-SERCA1 interaction (H-bond length of 1.7 Å), elucidating the ability of rutin to affect the affinity of SERCA1 to ATP. The binding of rutin in the proximity of Lys515 is likely to cause a decrease in FITC fluorescence.

Novel quercetin derivatives in treatment of peroxynitrite-oxidized SERCA1.

Sarco/endoplasmic reticulum calcium ATP-ase (SERCA) is regulated by low concentrations of peroxynitrite and inhibited by high levels, as indicated in human diseases. We studied quercetin (Q) and its novel derivatives monochloropivaloylquercetin (MPQ) and chloronaphthoquinonequercetin (CHQ) as agents with expected preventive properties against peroxynitrite-induced SERCA impairment. Q and MPQ protected the SERCA1 against peroxynitrite induced activity decrease, while CHQ potentiated the inhibitory effect of peroxynitrite. Quercetin derivatives were found to be weaker antioxidants compared with Q, as indicated by their ability to scavenge peroxynitrite and prevent of SERCA1 carbonylation, both decreasing in the order (Q > MPQ > CHQ). Quantum-chemical values of theoretical parameter E HOMO also indicated lower antioxidant capacities for MPQ and CHQ. Prooxidant properties estimated by calculations of frontier molecular orbitals (E LUMO) correlated with experimentally determined SH-group decrease induced by the compounds studied. Both methods showed a decrease of prooxidant properties as follows: CHQ > MPQ > Q. In addition, experimentally measured half-wave potentials indicated stronger prooxidant properties of quercetin derivatives as compared to Q. More expressive alterations of conformation in the transmembrane region of SERCA1 induced by quercetin derivatives, as compared with Q, may at least partially correlate with their higher lipophilicities. The protective effects of Q and MPQ on different isoforms of SERCA activity may be useful in prevention and treatment of inflammation or muscle diseases. The inhibitory effect of CHQ on SERCA isoforms may be beneficial in therapeutic approaches aimed at anti-tumor treatment.

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle modified by peroxynitrite.

OBJECTIVE: Effect of peroxynitrite on SERCA1 activity was studied in correlation with enzyme carbonylation. Kinetic parameters and location of peroxynitrite effect on SERCA1 were determined. METHODS: Carbonyls were determined by immunoblotting. FITC, NCD-4 and Trp fluorescence were used to indicate changes in cytosolic and transmembrane regions of SERCA1. RESULTS: Peroxynitrite-concentration-dependent decrease of SERCA1 activity was associated with elevation of protein carbonyls. 4-HNE was not involved in carbonylation of SERCA1. Increased FITC fluorescence in the presence of peroxynitrite correlated with the decrease of the enzyme affinity to ATP. DISCUSSION AND CONCLUSION: Peroxynitrite-induced SERCA1 carbonylation that was not accompanied with the formation of 4-HNE-SERCA1 adducts is indicative of direct oxidation of SERCA1. As assessed by FITC fluorescence and decreased affinity of the enzyme to ATP, peroxynitrite impairment was found to occur in the cytosolic ATP-binding region of SERCA1.

Effects of novel quercetin derivatives on sarco/endoplasmic reticulum Ca2+-ATPase activity.

OBJECTIVES: We examined effect of novel quercetin derivatives on sarcoplasmic reticulum (SR) Ca-ATPase activity isolated from skeletal muscles and their potential to prevent injury of SERCA induced by peroxynitrite that is elevated in multiple pathological processes. METHODS: SR was isolated by ultracentrifugation, ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Sulfhydryl and carbonyl groups content was determined to test oxidation of SERCA. Conformational changes in ATP and calcium binding site were assessed using specific fluorescent labels. RESULTS: Di(diacetylcafeoyl)-mono-(monoacetylcafeoyl) quercetin (DACQ) restored and diquercetin significantly decreased activity of SERCA in the presence of peroxynitrite. Diquercetin significantly decreased SERCA activity in absence of peroxynitrite. All tested quercetin derivatives decreased thiol group content of SR and caused change in SERCA conformation. Significant decrease of protein carbonyls was observed in SERCA treated with di(diacetylcafeoyl)-mono-(monoacetylcafeoyl) quercetin in the presence of peroxynitrite. CONCLUSION: DACQ protected SERCA in SR against formation of carbonyls in vitro and protected activity of the pump against inhibition caused by peroxynitrite. However, none tested quercetin derivative did protect SERCA against conformational changes and sulfhydryl group oxidation. Diquercetin inhibited SERCA at relatively low concentrations in the presence of peroxynitrite. Diquercetin and DACQ may prove to be beneficial in treatment of cancer and inflammatory diseases, respectively.

Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

Phenolic Compounds from Morus nigra Regulate Viability and Apoptosis of Pancreatic ß-Cells Possibly via SERCA Activity.

The ability of phenolic compounds from Morus nigra to modulate sarco-endoplasmic Ca2+-ATPase (SERCA1) activity was analyzed. Enzyme activity decrease correlated with the binding energy of agents to SERCA1. Results from theoretical and experimental approaches were coherent in identifying binding sites to SERCA1. Albanol A inhibited SERCA1 by immersion in the luminal gate at the site of Ca2+ release. Kuwanon U exerted an inhibitory effect by preventing ATP binding in the cytosolic region of SERCA1, and this was associated with conformational alterations. On the basis of similarities of SERCA isoforms, the viability of beta-cells containing SERCA2b was analyzed. Both correlation of viability and negative correlation of SERCA2b expression with SERCA1 activity were found for agents with the highest binding energy to SERCA1. The compounds studied may regulate viability and apoptosis of pancreatic beta-cells via modulation of SERCA activity. Novel pharmacological interventions in diabetes may be realized via compounds restoring ER calcium levels.

Modulating effect of flavonoids and their derivatives on sarcoplasmic reticulum Ca2+-ATPase oxidized by hypochloric acid and peroxynitrite.

OBJECTIVES: Effect of rutin and its lipophilic derivatives on Ca2+-ATPase of sarcoplasmic reticulum (SERCA) oxidized by hypochloric acid and peroxynitrite was investigated to examine the role of flavonoids in SERCA activity modulation. METHODS: Ca2+-ATPase activity was measured spectrophotometrically at 37 degrees C using NADH-coupled enzyme pyruvate kinase/lactate dehydrogenase assay. SERCA was oxidized by HOCl (3 min) or ONOO- (30 s) after previous treatment with flavonoids (2 min) at 37 degrees C. Lipophilic rutin derivatives were prepared by lipase-catalyzed esterification of flavonoids with fatty acids. RESULTS: Both hypochloric acid (HOCl) and peroxynitrite (ONOO-) decreased ATPase activity concentration-dependently with IC50 of 50+/-10 micromol/l and 150+/-15 micromol/l, respectively. Rutin was found to have a protective effect on SERCA activity in both oxidation systems in the concentration range 5 - 250 micromol/l. Lipophilic rutin derivatives (rutin oleate, rutin linoleate, rutin linolenate) exerted inhibitory effect on ATPase activity both in the presence and absence of oxidants. CONCLUSION: The results suggest that selective lipophilization of the flavonoid skeleton may represent a useful tool for SERCA activity modulation.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox.

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Quercetin based derivatives as sirtuin inhibitors.

Polyphenols synthesized by plants and fungi have various pharmacological effects. The ability of polyphenols to modulate sirtuins has gained considerable interest due to the role of sirtuins in aging, insulin sensitivity, lipid metabolism, inflammation, and cancer. In particular, sirtuin 6 (SIRT6) has gained importance in regulating a variety of cellular processes, including genomic stability and glucose metabolism. On the other hand, quercetin has been demonstrated to modulate sirtuins and to protect against several chronic diseases. In this study, two quercetin derivatives, diquercetin and 2-chloro-1,4-naphtoquinone-quercetin, were identified as promising SIRT6 inhibitors with IC50 values of 130 µM and 55 µM, respectively. 2-Chloro-1,4-naphtoquinone-quercetin also showed potent inhibition against SIRT2, with an IC50 value of 14 µM. Diquercetin increased the Km value of NAD+, whereas 2-chloro-1,4-naphthoquinone-quercetin increased the Km value of the acetylated substrate. Molecular docking studies suggest that diquercetin prefers the binding site of the nicotinamide (NAM) moiety, whereas 2-chloro-1,4-naphtoquinone-quercetin prefers to dock into the substrate binding site. Overall, the results of in vitro studies and molecular modeling indicate that diquercetin competes with nicotinamide adenine dinucleotide (NAD+), whereas 2-chloro-1,4-naphthoquinone-quercetin competes with the acetylated substrate in the catalytic site of SIRT6. Natural polyphenolic compounds targeting sirtuins show promise as a new approach in the search for novel and effective treatments for age-related diseases.

Limited degradation of oxidized calmodulin by proteasome: formation of peptides.

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H2O2 concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.

Protective effect of antioxidants against sarcoplasmic reticulum (SR) oxidation by Fenton reaction, however without prevention of Ca-pump activity.

The Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle was oxidized by Fe2+/H2O2/ascorbic acid (AA), a system which generates HO(.) radicals according to the Fenton reaction: (Fe2(+)+H2O2-->HO(.)+OH(-)+Fe(3+)) under conditions similar to the pathological state of inflammation. Under these conditions, when hydroxyl-radicals and/or ferryl-radicals are generated, a 50% decrease of the SERCA activity was observed, a significant decrease of SH groups and an increase of protein carbonyl groups and lipid peroxidation were identified. Two new bands, time dependent in density, appeared in the SERCA protein electrophoresis after incubation with the Fenton system (at approximately 50 and 75kDa), probably due to structural changes as supported also by trypsin digestion. Immunoblotting of DNPH derivatized protein bound carbonyls detected a time dependent increase after incubation of SERCA with the Fenton system. Trolox and the pyridoindole stobadine (50microM) protected SR against oxidation induced via the Fenton system by preventing SH group oxidation and lipid peroxidation. Pycnogenol((R)) and EGb761 (40microg/ml) protected SERCA in addition against protein bound carbonyl formation. In spite of the antioxidant effects, trolox and stobadine were not able to prevent a decrease in the SERCA Ca(2+)-ATPase activity. Pycnogenol and EGb761 even enhanced the decrease of the Ca(2+)-ATPase activity induced by the Fenton system, probably by secondary oxidative reactions.

Oxidative impairment of plasma and skeletal muscle sarcoplasmic reticulum in rats with adjuvant arthritis - effects of pyridoindole antioxidants.

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA). METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations. RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants. CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence.

Dysfunction of SERCA pumps as novel mechanism of methylglyoxal cytotoxicity.

A novel pathway of methylglyoxal (MGX)-induced apoptosis via sarcoplasmic reticulum Ca2+-ATPase (SERCA) is presented. Interaction of SERCA1 with MGX was investigated by molecular docking and experimentally in a cell-free system. MGX concentration- and time-dependently decreased SERCA1 activity. A significant increase of sarcoplasmic reticulum (SR) carbonylation was found in the concentration range of 1-10 mM caused by MGX and a decrease of thiol groups at the concentrations of 5 and 40 mM. Affinities of SERCA1 to ATP and Ca2+ were not influenced by MGX, however decreases of Vmax related to both binding sites were observed. Molecular docking indicated binding of MGX at the cytosolic region of SERCA1, inducing conformational changes in the cytosolic-transmembrane interface. This interaction resulted in conformational changes in the cytosolic region (FITC fluorescence decrease) as well as in the transmembrane region of SERCA1 (Trp fluorescence decrease) without direct binding to the cytosolic ATP or transmembrane Ca2+ binding sites. Regarding the MGX inhibitory effect in a cell-free system and similarities of SERCA1 to its other isoforms, proapoptotic properties of MGX may be expected in cellular systems. At cellular level, MGX induced a decrease of SERCA2b expression in the pancreatic INS-1E ß-cell line. This was accompanied by cell viability decrease, increase in apoptosis, impaired insulin secretion and elevation of basal intracellular Ca2+ levels. Decreased expression of SERCA2b may contribute to induction of apoptosis of pancreatic ß-cells.

Novel quercetin derivatives: From redox properties to promising treatment of oxidative stress related diseases.

A set of O-substituted quercetin derivatives was prepared with the aim to optimize bioavailability and redox properties of quercetin, a known agent with multiple health beneficial effects. Electron-acceptor/-donor properties of the agents were evaluated theoretically by quantum chemical calculations and by experimental methods in cell-free model systems (2,2-diphenyl-1-picrylhydrazyl (DPPH) test, the ferric reducing ability of plasma (FRAP), peroxynitrite scavenging, protein-thiol oxidation) and in cellular systems of fibroblasts, microglials and cancer lines. The order of individual antioxidant effects varied dependently on the system used. In cellular systems, quercetin derivatives were shown to be better antioxidants compared to quercetin. Monochloropivaloylquercetin (CPQ), monoacetylferuloylquercetin (MAFQ) and chloronaphthoquinonequercetin (CHNQ) showed a prominent inhibitory effect on the key enzymes involved in diabetic complications, aldose reductase and α-glucosidase, suggesting their promising therapeutic application. In the cellular models of BHNF-3 fibroblasts, microglial cell line BV-2, colorectal cancer cell lines HCT-116 and HT-29, CHNQ and CPQ were studied for their cytotoxic, antiproliferative and antiinflammatory properties. In the rat model, CHNQ attenuated colon inflammation induced by acetic acid. In summary, our studies revealed CPQ and CHNQ as potential remedies of chronic age-related metabolic or inflammatory diseases, including diabetes and neurodegenerations. Furthermore, CHNQ represents a novel promising agent exerting its anticancer effect through induction of oxidative stress-dependent cell death.

Ferritin oxidation and proteasomal degradation: protection by antioxidants.

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H2O2 induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation. Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.

Effect of some antioxidants on sarcoplasmic reticulum Ca2+-ATPase activity from rabbit skeletal muscle.

OBJECTIVES: Effects of phenolic antioxidants, synthetized (trolox, pyridoindole stobadine) and plant extracts (EGb 761 and Pycnogenol) were investigated on the activity of Ca(2+)-ATPase from sarcoplasmic reticulum (SR) of rabbit skeletal muscle to examine their potency to modulate the activity of this calcium level regulating enzyme. METHODS: SR vesicles and pure Ca(2+)-ATPase were isolated by ultracentrifugation. Ca(2+)-ATPase activity was measured spectrophotometrically by enzyme-coupled assay. RESULTS: Pycnogenol (Pyc) significantly decreased the activity of Ca(2+)-ATPase incorporated into SR vesicles as well as the activity of purified Ca(2+)-ATPase, the latter with respect to both enzyme substrates, Ca(2+) and ATP. Trolox, stobadine and EGb 761 did not influence significantly the activity SR- vesicle incorporated or pure Ca(2+)-ATPase, the latter with respect to either substrate, in spite of alterations of kinetic parameters in some cases. CONCLUSIONS: The decrease of SR Ca(2+)-ATPase activity induced by Pyc may be associated with its proapoptotic and anticancerogenic properties.

Antioxidants prevented oxidative injury of SR induced by Fe2+/H2O2/ascorbate system but failed to prevent Ca2+-ATPase activity decrease.

Dysfunction of sarcoplasmic reticulum (SR) Ca2+-ATPase induced by oxidative stress may be a contributing factor to the development of serious age related diseases. Incubation of sarcoplasmic reticulum (SR) vesicles of rabbit skeletal muscles with Fe2+/H2O2/ascorbate decreased the SH group content of SR approximately to 35% and Ca2+-ATPase activity to 50% of control not oxidized sample. Protein carbonyls increased twofold, lipid peroxidation was also significantly elevated. The antioxidant effects of trolox, the pyridoindole derivative stobadine and of the standardized extracts from bark of Pinus Pinaster PycnogenolR (Pyc) and from leaves of Ginkgo biloba (EGb 761) were studied on oxidatively injured SR. All antioxidants exerted preventive effects against the oxidized lipids and protein SH groups of SR vesicles. Trolox and stobadine did not influence protein carbonyl formation, while flavonoid extracts prevented carbonyl generation, probably by binding to protein. The preventive effects of the antioxidants studied on lipids and protein SH groups were however not associated with protection of Ca2+-ATPase activity. Stobadine and trolox exerted no effect on enzyme activity, Pyc and EGb 761 enhanced the inhibitory effect of Ca2+-ATPase activity in oxidatively injured SR. Concluding, under the conditions of oxidative stress induced by Fe2+/H2O2/ascorbate against SR of rabbit skeletal muscle, the agents studied demonstrated antioxidant effects yet failed to protect Ca2+-ATPase activity.