In 7.5% gels run under non-reducing conditions, the PMCA appeared as a somewhat diffuse band in the molecular mass range of ~140 kDa, consistent with the presence of four different isoforms and their splice variants in brain neurons [29-31]. donating its free electron to molecular oxygen, thereby generating superoxide free radicals (O2.?) [14]. Superoxide can be further converted to H2O2 by the anti-oxidant enzyme superoxide dismutase (Mn-SOD and Cu/Zn-SOD). The peroxide is usually converted to water and oxygen by glutathione peroxidase and catalase [15, 16]. Exposure of neurons to PQ resulted in nearly instantaneous generation of O2.?, as monitored by the inactivation of aconitase, an enzyme with four iron-sulfur centers, believed to be one of the most sensitive targets of O2.? [17]. Paraquat treatment led to a biphasic response in PMCA activity. Low concentrations of PQ stimulated basal PMCA activity and abolished its sensitivity to CaM, whereas higher concentrations inhibited both basal and CaM-stimulated PMCA activity. Functional changes were accompanied by formation of high molecular excess weight adducts of the PMCA protein. Additionally, evidence for PMCA proteolysis was indicated by the appearance of a faster moving band of PMCA. Proteolysis of the enzyme was prevented by pretreatment with a calpain inhibitor but not with a caspase inhibitor, suggesting a role for the Ca2+-activated protease calpain in the effects of PQ on the activity and structure of PMCA. Our results suggest that PMCA is usually a very sensitive target of oxidative stress in neurons. Alterations in Delcasertib Delcasertib PMCA activity Rabbit Polyclonal to MDM2 (phospho-Ser166) and structure under conditions of prolonged oxidative stress may contribute to altered Ca2+ homeostasis. Materials and Methods Ouabain, thapsigargin, oligomycin, ATP, 1, 2- bis (o-aminophenoxy) ethane-N,N,N,N-tetraacetic acid acetoxy methyl ester (BAPTA-AM), calcein AM, and propidium iodide were purchased from Sigma Chemical Organization (St. Louis, MO). The Protease Inhibitor Cocktail III, bovine brain CaM, calpain inhibitor (carbobenzoxy-valinyl-phenylalaninal) and pan caspase inhibitor (Ac-Asp-Glu-Val-Asp-chloromethylketone) were from Calbiochem (San Diego, CA). Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL). The following sources were used for the various main antibodies: Anti-pan PMCA, PMCA 2, PMCA 4, and anti-SERCA (Affinity Bioreagents Inc., Golden, CO), anti-cytochrome P450 reductase (Stressgen Inc., Victoria, Canada), anti–fodrin (Chemicon, Temecula, CA), anti-calcineurin A and anti-neuronal nitric oxide synthase (nNOS) (Transduction Labs, Lexington, KY), and anti-NCX (Swant laboratories, Bellinzona, Switzerland). Cell Culture Dissociated cortical neuron cultures were established from 18-day aged Sprague Dawley fetuses as explained previously [18-20]. Briefly, pups were delivered by cesarean section while the dam was fully anesthetized with pentobarbital (0.1g/kg), and the brains recovered according to protocols approved by the University or college of Kansas IACUC and in accordance Delcasertib with National Institutes of Health guidelines. The cortical lobes were dissected and cells dissociated by gentle trituration with trypsin. After the final precipitation step, neurons were resuspended in new DMEM/F-12 with 10% fetal calf serum (FCS), and plated at densities ranging from 0.3 – 3 106 cells/dish on 35 mm tissue culture dishes coated with poly-D-lysine. After 24 h to allow cell attachment, the FCS-containing medium was replaced by a defined medium with DMEM/F12 made up of N2 supplements, potassium bicarbonate (15 mM), and 20% glial conditioned medium. Cells were fed twice a week by replacing 1/3 of the medium with new medium. It was estimated that 90% of the cells in the primary cultures were neurons, based on morphological characteristics and confirmation with immunofluorescent staining for glial fibrillary acidic protein and neuron specific enolase. Treatment of Neurons with Paraquat Main cortical neurons were grown in culture for 6 -7 days in a humidified atmosphere of 95% air flow / 5% CO2 at 37C. New stocks of PQ were made in water and sterile-filtered Delcasertib prior to treatment. Following addition of 15 l of a 100-fold concentrated PQ stock answer to 1 1.5 ml of the culture medium, dishes were swirled gently to allow complete mixing of the drug with the medium and incubated at 37C for the indicated periods of time. Determination of Aconitase Activity The activity of aconitase was measured by monitoring the conversion of isocitrate to cis-aconitate, a product that can be measured at 240 nm [21, 22]. Control or PQ-treated neurons were lysed in a buffer made up of 3 mM Tris-HCl, pH 7.5, plus a cocktail of protease inhibitors (5l/ml), and centrifuged at 13,000 for.