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

Abstract

The Ca²⁺-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⁻ + Fe³⁺) 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 75 kDa), 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 (50 μM) protected SR against oxidation induced via the Fenton system by preventing SH group oxidation and lipid peroxidation. Pycnogenol^® and EGb761 (40 μg/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 Ca²⁺-ATPase activity. Pycnogenol and EGb761 even enhanced the decrease of the Ca²⁺-ATPase activity induced by the Fenton system, probably by secondary oxidative reactions.

Introduction

In many inflammatory diseases, the phagocytes release HOCl, superoxide and H₂O₂. Release of the latter two agents into a fluid with free iron promotes the production of hydroxyl radical and extensive damage. Free iron is normally not found in most biological fluids (Valko et al., 2005) yet it has been detected for example in synovial fluid (Halliwell and Gutteridge, 1985), which may lead to the production of hydroxyl radical in the presence of H₂O₂, via the Fenton reaction. Instead of a reaction of Fe²⁺ and H₂O₂ producing hydroxyl radical, a ferryl radical, with a valence of +4, has been suggested to occur (Halliwell and Gutteridge, 1990, Meyerstein, 1997). In inflammation, release of ROS by PMNs may damage membranes, thus causing accumulation of intracellular calcium (Ca_i), and chronically increased Ca_i is a final common pathway in cell injury and death (Orrenius et al., 1991, Laporte et al., 2004). Cells have mechanisms for removal of Ca_i, including plasma membrane and sarcoplasmic/endoplasmic (SR/ER) Ca-pump.

Sarcoplasmic Ca²⁺-ATPase (SERCA) is a calcium regulatory protein, preferentially oxidized by ROS (Eu et al., 2000) at critical sites, thus regulating its function in a reversible manner (Bigelow and Squier, 2005). Ca²⁺-ATPase from skeletal muscle represents a single-chain transmembrane protein present in high concentration in sarcoplasmic reticulum (SR) vesicles, with easily measurable function (Starling et al., 1996). The ATPase accounts for approx. 70–80% of the total protein content of the SR, and it can be purified to >95% purity, while still remaining in the native SR membrane (Warren et al., 1974a, Castilho et al., 1996). These facts make the SR Ca²⁺-ATPase a good model for a detailed investigation of the mechanisms involved in oxidative damage Ca²⁺ transport function.

The sarco/endoplasmic reticulum Ca²⁺-ATPases of mammalian tissues can be divided structurally into three main groups (SERCA 1–3) representing the products of different genes. SERCA 1 is found predominantly in fast-twich skeletal muscle. The structure of Ca²⁺-ATPase of sarcoplasmic reticulum was recently reviewed (Sweadner and Donnet, 2001, Martonosi and Pikula, 2003). Combining structural and biochemical information, MacLennan and his colleagues constructed a hypothetical model of the tertiary structure of Ca²⁺-ATPase (MacLennan et al., 1997). The structure was divided into three major parts, designated as the cytoplasmic headpiece, the stalk domain, and the transmembrane domain. Only short loops were assumed to be exposed on the luminal side of the membrane.

More than half of the total mass of the ATPase molecule is exposed on the cytoplasmic surface of the membrane, forming the cytoplasmic headpiece. The headpiece contains six subdomains including the phosphorylation and the nucleotide binding domain. The phosphorylation and nucleotide binding domains form the active site of ATP hydrolysis and are closely related structurally and functionally. The stalk region connects the headpiece to the membrane. High affinity and low affinity binding sites are according to mutagenetic studies located in the transmembrane domain. The transmembrane domain is assumed to contain 10 hydrophobic transmembrane helices that anchor the Ca²⁺-ATPase to the lipid bilayer and form the transmembrane channel for the passage of Ca²⁺ (Toyoshima et al., 2000).

According to Waku et al. (1971), the lipidic content of SR from rabbit skeletal muscle was estimated to be 44.5%, of which 88.8% were phospholipids. In the phospholipids, the choline-phosphoglyceride fraction was predominant (70.8%), followed by the fractions ethanolamine-phosphoglyceride (16.5%), sphingomyelin (6.2%), phosphatidylinositol (2.3%) and phosphatidylserine (1.7%). The neutral lipid fraction contained cholesterol (45.5%), triglycerides (26.4%), cholesterol esters (16.2%) and free fatty acids (11.6%). After isolation, one molecule of Ca²⁺-ATPase is surrounded by approximately 80 lipid molecules of sarcoplasmic reticulum (Shivanna and Rowe, 1997), 30 of them are annular lipids, more tightly bound to Ca²⁺-ATPase protein (Kragh-Hansen et al., 1998a, Lee, 1998).

Phagocyte-derived reactive species are known to contribute to inflammation (Halliwell, 2006) and antioxidants could prove beneficial in the treatment of inflammatory diseases (Kunsch et al., 2005). It was reported that tert-butyl hydroperoxide inhibits Ca-pump in intact RBCs, which can be prevented by antioxidants or free radical scavengers including stobadine (Clinch et al., 1993). Recently we found that HOCl generated in inflammation by myeloperoxidase, inhibited SR Ca²⁺-ATPase at least partially by structural alterations and the enzyme activity decrease was prevented by antioxidants like trolox or standardized extract from leaves of Ginkgo biloba (EGb761) (Strosova et al., 2005). The antioxidants prevented Ca-pump activity decrease also after incubation with the Fenton system including EDTA, which correlated directly with TBARS formation (Rohn et al., 1993). In the present study, oxidation of SR Ca²⁺-ATPase by Fenton reaction (without EDTA) was studied. It is supposed that under these conditions oxidative injury is induced by similar way as in the presence of activated neutrophils. Possible preventive effects of the below mentioned antioxidants were studied.

The pyridoindole derivative stobadine [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido (4,3b)indole] (Sto) (Stolc et al., 1983, Horakova et al., 1994, Horakova and Stolc, 1998) and trolox (Tro), a water soluble vitamin E derivative, were used as simple synthetic drugs with known antioxidant properties. Stobadine is a compound with cardio- and neuroprotective effects based on antioxidant or free radical scavenging mechanisms (Horakova and Stolc, 1998). Stobadine was also effective against chronic hypoxia in maternal, fetal, and new born organs of rats (Navarova et al., 2005). In experimental rheumatoid arthritis a positive effect of stobadine on the survival of rats was observed and also morphological and biochemical parameters of the spleen were improved (Bauerova et al., 2004). The aggregation and cross-linking of eye lens proteins due to free radical-mediated cataractogenesis in diabetes was significantly inhibited by stobadine (Stefek et al., 2005), and the ability of stobadine to attenuate oxidation reactions of lipids in diabetes may account, at least partially, for its anticataract action (Kyselova et al., 2005). The natural products with mutual preventive biological properties, standardized extracts of flavonoids from Pinus pinaster bark Pycnogenol^® (Pyc) (Packer et al., 1999, Rohdewald, 2002) and from leaves of Ginkgo biloba (EGb761) (Diamond et al., 2000, Smith and Luo, 2004) were also studied. EGb761 and Pyc are extracts with multivalent properties, able to up- or down-regulate signaling pathways, gene transcription cellular metabolism, etc. (Smith and Luo, 2004). EGb761 is an extract with antiapoptotic properties which are probably due to its preventive effects to mitochondria (Eckert et al., 2003). A neuroprotective activity (Ahlemeyer and Krieglstein, 2003) was also reported.

In the presented study we showed that inhibition of SERCA induced by Fenton reaction was accompanied by protein oxidation and structural changes of the SERCA protein. This oxidation process might be at least partially blocked by antioxidants but the impaired function of SERCA, probably induced by structural changes, was not involved.