Crosstalk among Calcium ATPases: PMCA, SERCA and SPCA in Mental Diseases.

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein-calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.

Hexachloronaphthalene Induces Mitochondrial-Dependent Neurotoxicity via a Mechanism of Enhanced Production of Reactive Oxygen Species.

Hexachloronaphthalene (PCN67) is one of the most toxic among polychlorinated naphthalenes. Despite the known high bioaccumulation and persistence of PCN67 in the environment, it is still unclear to what extent exposure to these substances may interfere with normal neuronal physiology and lead to neurotoxicity. Therefore, the primary goal of this study was to assess the effect of PCN67 in neuronal in vitro models. Neuronal death was assessed upon PCN67 treatment using differentiated PC12 cells and primary hippocampal neurons. At 72 h postexposure, cell viability assays showed an IC50 value of 0.35 µg/ml and dose-dependent damage of neurites and concomitant downregulation of neurofilaments L and M. Moreover, we found that younger primary neurons (DIV4) were much more sensitive to PCN67 toxicity than mature cultures (DIV14). Our comprehensive analysis indicated that the application of PCN67 at the IC50 concentration caused necrosis, which was reflected by an increase in LDH release, HMGB1 protein export to the cytosol, nuclear swelling, and loss of homeostatic control of energy balance. The blockage of mitochondrial calcium uniporter partially rescued the cell viability, loss of mitochondrial membrane potential (ΔΨ m), and the overproduction of reactive oxygen species, suggesting that the underlying mechanism of neurotoxicity involved mitochondrial calcium accumulation. Increased lipid peroxidation as a consequence of oxidative stress was additionally seen for 0.1 µg/ml of PCN67, while this concentration did not affect ΔΨ m and plasma membrane permeability. Our results show for the first time that neuronal mitochondria act as a target for PCN67 and indicate that exposure to this drug may result in neuron loss via mitochondrial-dependent mechanisms.

Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells.

BACKGROUND: Plasma membrane Ca2+-ATPase (PMCA) is the most sensitive cellular calcium detector. It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. In the brain, PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases. These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. METHODS: To mimic the changes in aged neurons we used stable transfected differentiated PC12 cells with downregulated PMCA2 or PMCA3 and analyzed the effect of CCL5 on calcium transients with Fluo-4 reagent. Chemokine receptors were evaluated using Western blot, and IP3 receptors expression level was assessed using qRT-PCR and Western blot. RESULTS: In PMCA-reduced cell lines, CCL5 released more Ca2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer. Also, in these lines we detected altered expression level of CCR5 and IP3 receptors. CONCLUSION: Although modification of PMCAs composition could provide some protection against calcium overload, reduction of PMCA2 appeared to be more detrimental to the cells than deficiency of PMCA3. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons.

The Puzzling Role of Neuron-Specific PMCA Isoforms in the Aging Process.

The aging process is a physiological phenomenon associated with progressive changes in metabolism, genes expression, and cellular resistance to stress. In neurons, one of the hallmarks of senescence is a disturbance of calcium homeostasis that may have far-reaching detrimental consequences on neuronal physiology and function. Among several proteins involved in calcium handling, plasma membrane Ca2+-ATPase (PMCA) is the most sensitive calcium detector controlling calcium homeostasis. PMCA exists in four main isoforms and PMCA2 and PMCA3 are highly expressed in the brain. The overall effects of impaired calcium extrusion due to age-dependent decline of PMCA function seem to accumulate with age, increasing the susceptibility to neurotoxic insults. To analyze the PMCA role in neuronal cells, we have developed stable transfected differentiated PC12 lines with down-regulated PMCA2 or PMCA3 isoforms to mimic age-related changes. The resting Ca2+ increased in both PMCA-deficient lines affecting the expression of several Ca2+-associated proteins, i.e., sarco/endoplasmic Ca2+-ATPase (SERCA), calmodulin, calcineurin, GAP43, CCR5, IP3Rs, and certain types of voltage-gated Ca2+ channels (VGCCs). Functional studies also demonstrated profound changes in intracellular pH regulation and mitochondrial metabolism. Moreover, modification of PMCAs membrane composition triggered some adaptive processes to counterbalance calcium overload, but the reduction of PMCA2 appeared to be more detrimental to the cells than PMCA3.

Cross talk among PMCA, calcineurin and NFAT transcription factors in control of calmodulin gene expression in differentiating PC12 cells.

Brain aging is characterized by progressive loss of plasma membrane calcium pump (PMCA) and its activator - calmodulin (CaM), but the mechanism of this phenomenon remains unresolved. CaM encoded by three genes Calm1, Calm2, Calm3, works to translate Ca2+ signal into changes in frequently opposite cellular activities. This unique function allows CaM to affect gene expression via stimulation of calcineurin (CaN) and its downstream target - nuclear factor of activated T-cells (NFAT) and to terminate Ca2+ signal by stimulation of its extrusion. PMCA, which exists in four isoforms PMCA1-4, may in turn shape the pattern of Ca2+ transients and control CaN activity by its direct binding. Therefore, the interplay between PMCA, CaM and CaN/NFAT is highly plausible. To verify that, we used differentiated PC12 cells with reduced expression of PMCA2 or PMCA3 to mimic the potential changes in aged brain. Manipulation in PMCAs level decreased CaM protein in PMCA2 or PMCA3-reduced lines that was accompanied by down-regulation of Calm1 and Calm2 in both lines, but Calm3 only in PMCA2-reduced cells. Further studies showed substantially higher NFATc2 nuclear accumulation and increased NFAT transcriptional activity. Blocking of CaN/NFAT signalling resulted in almost full CaM recovery, mainly due to up-regulation of Calm2 and Calm3 genes. Moreover, higher occupancy of Calm2 and Calm3 promoters by NFATc2 and increased expression of these genes in response to NFATc2 silencing were demonstrated in PMCA2 and PMCA3-reduced lines. Our results indicate that decrease in CaM level in response to PMCAs downregulation can be driven by CaN/NFAT pathway.

Malignant melanoma of the lung: case series.

Extracutaneous locations of primary malignant melanoma are rare. In the respiratory system most melanomas present as metastatic tumors. For the diagnosis of primary lung melanoma, strict histopathological and clinical criteria should be met. In this paper we present three cases of malignant melanoma which showed in the respiratory system. The first 2 case studies present primary lung melanomas, while the last one shows late lung metastasis of tumor originated from vaginal mucosa. The treatment of choice for localized disease as well as single metastasis is surgical excision.

Plasma membrane Ca(2+)-ATPase is a novel target for ketamine action.

Ketamine, a high affinity uncompetitive antagonist of voltage-dependent NMDA receptor, has been used for years as a dissociative anesthetic. Although the drug is considered as safe and well-tolerable, it is now evident that it can exert dose-dependent multidirectional effects acting on different cellular targets and pathways. The latest clinical studies also demonstrated its promising antidepressant action. However, the widespread use of this drug in humans is largely limited by a broad range of cognitive adverse effects that resemble some core symptoms of schizophrenia. In line with the hypothesis of unifying role of calcium in schizophrenia symptomology, we used ketamine-induced rat model of experimental psychosis to study the effect of 5-day ketamine treatment (30 mg/kg, ip) on the activity of plasma membrane Ca(2+)-ATPase. Whereas no change in a total amount of the enzyme in cortical synaptosomal membranes was observed, a decrease by ∼50% in hydrolytic activity, as well as lowered phosphointermediate formation were detected. Moreover, ketamine action appeared to be isoform-independent. The experiments on intact Ca(2+)-ATPase purified from vehicle-treated rat cortex revealed dose-dependent inhibition of enzymatic activity. Furthermore, ketamine decreased, but not eliminated, the stimulation by calmodulin. The inhibitory effect, although much weaker, was also evident for truncated form of calcium pump obtained following digestion by trypsin. Our results indicate that plasma membrane Ca(2+)-ATPase is a novel target for ketamine and putative interaction sites may involve central catalytic loop and calmodulin-binding domain.

Regulation of GAP43/calmodulin complex formation via calcineurin-dependent mechanism in differentiated PC12 cells with altered PMCA isoforms composition.

Several lines of evidence suggest the contribution of age-related decline in plasma membrane calcium pump (PMCA) to the onset of neurodegenerative diseases. From four PMCA isoforms, PMCA2, and PMCA3 respond to a rapid removal of Ca(2+) and are expressed predominantly in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in differentiated PC12 cells accelerated cell differentiation, but increased apoptosis in PMCA2-deficient line. We also demonstrated that altered expression of voltage-dependent calcium channels correlated with their higher contribution to Ca(2+) influx, which varied between PMCA-reduced lines. Here, we propose a mechanism unique for differentiated PC12 cells by which PMCA2 and PMCA3 regulate pGAP43/GAP43 ratio and the interaction between GAP43 and calmodulin (CaM). Although down-regulation of PMCA2 or PMCA3 altered the content of GAP43/pGAP43, of paramount importance for the regulatory mechanism is a disruption of isoform-specific inhibitory PMCA/calcineurin interaction. In result, higher endogenous calcineurin (CaN) activity leads to hypophosphorylation of GAP43 in PMCA2- or PMCA3-deficient lines and intensification of GAP43/CaM complex formation, thus potentially limiting the availability of free CaM. In overall, our results indicate that both "fast" PMCA isoforms could actively regulate the local CaN function and CaN-downstream processes. In connection with our previous observations, we also suggest a negative feedback of cooperative action of CaM, GAP43, and CaN on P/Q and L-type channels activity. PMCAs- and CaN-dependent mechanism presented here, may signify a protective action against calcium overload in neuronal cells during aging, as well a potential way for decreasing neuronal cells vulnerability to neurodegenerative insults.

Silencing of plasma membrane Ca2+-ATPase isoforms 2 and 3 impairs energy metabolism in differentiating PC12 cells.

A close link between Ca(2+), ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca(2+) may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca(2+) in cytosol. In differentiation process plasma membrane Ca(2+) ATPase (PMCA) is considered as one of the major players for Ca(2+) homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca(2+)]c resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.

Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations.

Plasma membrane Ca(2+)-ATPase (PMCA) by extruding Ca(2+) outside the cell, actively participates in the regulation of intracellular Ca(2+) concentration. Acting as Ca(2+)/H(+) counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca(2+) overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca(2+) clearance and partially attenuated cellular acidification during KCl-stimulated Ca(2+) influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca(2+) overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca(2+)-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.

Gene expression pattern in PC12 cells with reduced PMCA2 or PMCA3 isoform: selective up-regulation of calmodulin and neuromodulin.

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.

Functional characteristic of PC12 cells with reduced microsomal glutathione transferase 1.

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.

Adaptation of microsomal glutathione transferase 1 in PC12 cells with modified PMCA isoforms composition.

Microsomal glutathione transferase 1 (MGST1) is an integral homo-trimeric membrane protein with transferase and peroxidase activities. With glutathione as a co-substrate, it scavenges toxic compounds and may exert anti-apoptotic effect. We examined the effect of suppression of plasma membrane Ca(2+)-ATPase isoforms--PMCA2 or PMCA3 on MGST1 in PC12 cells. GSH level was significantly higher in PMCA2-reduced line, but similar GSSG/GSH ratios in all cell lines suggested an efficient protection or absence of oxidative stress. The ATP concentration decreased in both modified lines, although in PMCA2-suppressed cells the decrease was higher. Total GSTs activity in postmitochondrial fraction increased by 30% in the cells with reduced PMCA3. After treatment with MGST1 activator N-ethylmaleimide (NEM), the activity increased in both transfected lines by 30-40%. Real-time PCR also showed a higher mRNA expression of MGST1 in these lines. Staining with antibody recognizing all cytosolic and membrane-bound GSTs revealed the difference in oligomeric forms of GSTs, and specific anti-MGST1 antibody showed the presence of MGST1 hexamers in the transfected cells. Formation of similar hexamers was detected in the control line after treatment with peroxynitrite. Modification of MGST1 under reduced PMCAs amount may represent an adaptive mechanism that offers protection against the cytotoxicity mediated by increased Ca2+.

Calmodulin effects on steroids-regulated plasma membrane calcium pump activity.

It is now generally accepted that non-genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca(2+) is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca(2+) increase is ATP-consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non-excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca(2+) uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10(-9) to 10(-6) M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca(2+) uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30-40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca(2+) extrusion in membranes incubated with 17-beta-estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure.