Intracellular Ca2+ in neuronal cells is an essential regulatory ion responsible for excitability, synaptic plasticity, and neurite outgrowth. Plasma membrane calcium ATPase (PMCA) is the most sensitive enzyme in decreasing of the Ca2+ concentration. The diverse PMCA isoforms composition in the membranes suggests their specific function in the cell, and whereas PMCA1 and 4 appear to be ubiquitous, PMCA2 and 3 are characteristic isoforms for excitable cells. The aim of our study was to elucidate if and how the elimination of neuron-specific isoforms affects the pattern of cell growth and development. We have obtained stable-transfected PC12 cell lines with a suppressed expression of PMCA2, PMCA3, or both neuron-specific isoforms. The modified profile of PMCA generated considerable changes in morphology of examined PC12 lines, suggesting the activation of a differentiation process to pseudoneuronal phenotype. Experiments with Fura-2/AM-loaded cells revealed an increased cytosolic Ca2+ concentration in the cell lines with blocked PMCA2 isoform. The suppression of PMCA2 concomitantly altered expression of sarco/endoplasmic Ca2+-ATPase 2 isoform (SERCA2) at the protein level. Comparative flow cytometry analysis, using Annexin V/PI conjugate, showed the difference in the mean percentage of apoptotic cells in modified PC12 lines. Our data suggest that specific PMCA isoforms presence can regulate the intact cell development; however, it may involve multiple unidentified yet signaling pathways.
Background: Magnesium is required for the proper activity of many metabolic pathways in every cell type. Mg deficiency gives rise to preterm birth and low body weight that are associated with pathological circumstances, including disturbed ions homeostasis and insufficient antioxidant protection. Antenatal MgSO4 treatment has been reported to exhibit a protective effect on the developing fetus; however, the molecular mechanism of this protection remains not fully understood. Objectives: Since Mg ions very rapid cross the placenta, our study tested the hypothesis whether prenatal exposure to MgSO4 may modify the expression and activity of erythrocyte plasma membrane calcium pump (PMCA), and total erythrocyte glutathione (GSH) concentration in preterm newborns. Methods: Immunocharacteristics of erythrocyte PMCA in control and MgSO4-treated newborns were done using general and isoform-specific antibodies. The hydrolytic activity of PMCA was determined with and without calmodulin. Total GSH erythrocyte levels were analyzed by a quantitative assay using Ellman reagent. All assays were done after delivery and 24 h later. Results: Significant differences were present in PMCA amount, isoform composition, basal activity and sensitivity for stimulation by calmodulin in both groups of neonates. The total GSH content altered during examined time, but only in the control group. Conclusions: Prenatal treatment with MgSO4 may exert a significant influence on calcium homeostasis and nonenzymatic antioxidant reserve in the erythrocytes of preterm newborns.
Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.
Calcium ions are essential to proper neurotransmission. Impairment in cytosolic Ca(2+) concentration and Ca(2+) signaling disturbs neuronal activity, leading to pathological consequences. In cells, a high-affinity plasma membrane calcium pump (PMCA) keeps free Ca(2+) in the nanomolar range. Among four genes encoding the enzyme, PMCA2 and 3 are primary in excitable cells. To elucidate the relationship between PMCAs' composition and susceptibility for neurosteroid regulation, we obtained PC12 cells with suppressed neuron-specific isoforms and analyzed the effect of selected steroids on Ca(2+) uptake. Our results indicate that hormones affected Ca(2+) transport activity and that this effect depended on both PMCA isoform composition and steroid structure.
Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.
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