The Role of P2X7 Purinoceptors in the Pathogenesis and Treatment of Muscular Dystrophies.

Muscular dystrophies are inherited neuromuscular diseases, resulting in progressive disability and often affecting life expectancy. The most severe, common types are Duchenne muscular dystrophy (DMD) and Limb-girdle sarcoglycanopathy, which cause advancing muscle weakness and wasting. These diseases share a common pathomechanism where, due to the loss of the anchoring dystrophin (DMD, dystrophinopathy) or due to mutations in sarcoglycan-encoding genes (LGMDR3 to LGMDR6), the α-sarcoglycan ecto-ATPase activity is lost. This disturbs important purinergic signaling: An acute muscle injury causes the release of large quantities of ATP, which acts as a damage-associated molecular pattern (DAMP). DAMPs trigger inflammation that clears dead tissues and initiates regeneration that eventually restores normal muscle function. However, in DMD and LGMD, the loss of ecto-ATPase activity, that normally curtails this extracellular ATP (eATP)-evoked stimulation, causes exceedingly high eATP levels. Thus, in dystrophic muscles, the acute inflammation becomes chronic and damaging. The very high eATP over-activates P2X7 purinoceptors, not only maintaining the inflammation but also tuning the potentially compensatory P2X7 up-regulation in dystrophic muscle cells into a cell-damaging mechanism exacerbating the pathology. Thus, the P2X7 receptor in dystrophic muscles is a specific therapeutic target. Accordingly, the P2X7 blockade alleviated dystrophic damage in mouse models of dystrophinopathy and sarcoglycanopathy. Therefore, the existing P2X7 blockers should be considered for the treatment of these highly debilitating diseases. This review aims to present the current understanding of the eATP-P2X7 purinoceptor axis in the pathogenesis and treatment of muscular dystrophies.

Palmitate Stimulates Expression of the von Willebrand Factor and Modulates Toll-like Receptors Level and Activity in Human Umbilical Vein Endothelial Cells (HUVECs).

An increased concentration of palmitate in circulation is one of the most harmful factors in obesity. The von Willebrand factor (vWF), a protein involved in haemostasis, is produced and secreted by the vascular endothelium. An increased level of vWF in obese patients is associated with thrombosis and cardiovascular disease. The aim of this study was to investigate a palmitate effect on vWF in endothelial cells and understand the mechanisms of palmitate-activated signalling. Human umbilical vein endothelial cells (HUVECs) incubated in the presence of palmitate, exhibited an increased VWF gene expression, vWF protein maturation, and stimulated vWF secretion. Cardamonin, a Nuclear Factor kappa B (NF-κB) inhibitor, abolished the palmitate effect on VWF expression. The inhibition of Toll-like receptor (TLR) 2 with C29 resulted in the TLR4 overactivation in palmitate-treated cells. Palmitate, in the presence of TLR4 inhibitor TAK-242, leads to a higher expression of TLR6, CD36, and TIRAP. The silencing of TLR4 resulted in an increase in TLR2 level and vice versa. The obtained results indicate a potential mechanism of obesity-induced thrombotic complication caused by fatty acid activation of NF-κB signalling and vWF upregulation and help to identify various compensatory mechanisms related to TLR4 signal transduction.

Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns.

Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene. Although there is evidence for the loss of shorter isoforms having impact in the central nervous system, their role in muscle is unclear. We found that at 8 weeks, the active phase of pathology in dystrophic mice, dystrophin-null mice (mdxßgeo) presented with a mildly exacerbated phenotype but without an earlier onset, increased serum creatine kinase levels, or decreased muscle strength. However, at 12 months, mdxßgeo diaphragm strength was lower, whereas fibrosis increased, compared with mdx. The most striking features of the dystrophin-null phenotype were increased ectopic myofiber calcification and altered macrophage infiltration patterns, particularly the close association of macrophages with calcified fibers. Ectopic calcification had the same temporal pattern of presentation and resolution in mdxßgeo and mdx muscles, despite significant intensity differences across muscle groups. Comparison of the rare dystrophin-null patients against those with mutations affecting full-length dystrophins may provide mechanistic insights for developing more effective treatments for DMD.

Atorvastatin and pravastatin stimulate nitric oxide and reactive oxygen species generation, affect mitochondrial network architecture and elevate nicotinamide N-methyltransferase level in endothelial cells.

Statins belong to the most often prescribed medications, which efficiently normalise hyperlipidaemia and prevent cardiovascular complications in obese and diabetic patients. However, beside expected therapeutic results based on the inhibition of 3-hydroxyl-3-methylglutaryl-CoA reductase, these drugs exert multiple side effects of poorly understood characteristic. In this study, side effects of pravastatin and atorvastatin on EA.hy926 endothelial cell line were investigated. It was found that both statins activate proinflammatory response, elevate nitric oxide and reactive oxygen species (ROS) generation and stimulate antioxidative response in these cells. Moreover, only slight stimulation of the mitochondrial biogenesis and significant changes in the mitochondrial network organisation have been noted. Although biochemical bases behind these effects are not clear, they may partially be explained as an elevation of AMP-activated protein kinase (AMPK) activity and an increased activating phosphorylation of sirtuin 1 (Sirt1), which were observed in statins-treated cells. In addition, both statins increased nicotinamide N-methyltransferase (NNMT) protein level that may explain a reduced fraction of methylated histone H3. Interestingly, a substantial reduction of the total level of histone H3 in cells treated with pravastatin but not atorvastatin was also observed. These results indicate a potential additional biochemical target for statins related to reduced histone H3 methylation due to increased NNMT protein level. Thus, NNMT may directly modify gene activity.

Disrupted Calcium Homeostasis in Duchenne Muscular Dystrophy: A Common Mechanism behind Diverse Consequences.

Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.

Mitochondrial Metabolism behind Region-Specific Resistance to Ischemia-Reperfusion Injury in Gerbil Hippocampus. Role of PKCßII and Phosphate-Activated Glutaminase.

Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCßII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCßII specific inhibitor peptide ßIIV5-3-treated IR. Vehicle or ßIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCßII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 µM) to evaluate the inhibition of GLS1 on neuronal viability. PKCßII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by ßIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCßII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.

Mild palmitate treatment increases mitochondrial mass but does not affect EA.hy926 endothelial cells viability.

A dyslipidaemia-related increase of the concentration of long-chain fatty acids in the plasma is an important pathological factor substantially increasing risk of serious consequences in vascular endothelium. Inflammatory response, atherosclerosis and insulin resistance seem the most severe. Palmitate at excessive concentrations has been shown to have a harmful effect on endothelial cells impairing NO generation, stimulating reactive oxygen species (ROS) formation and affecting their viability. On the other hand we found that palmitate applied for 48 h at 100 µM concentration which is sufficient to induce inflammatory response, increase ROS generation and reduce insulin sensitivity of EA.hy926 cells, unexpectedly also stimulates NO synthesis and increases mitochondrial mass, suggesting a pro-survival rather than anti-survival effect. This finding unveils a potential protective mechanism allowing cells to maintain their energy homeostasis under conditions of a moderate deregulation of lipid metabolism.

Mitochondrial dysfunction in fibroblasts derived from patients with Niemann-Pick type C disease.

Mutations in the NPC1 or NPC2 genes lead to Niemann-Pick type C (NPC) disease, a rare lysosomal storage disorder characterized by progressive neurodegeneration. These mutations result in cholesterol and glycosphingolipid accumulation in the late endosomal/lysosomal compartment. Complications in the storage of cholesterol in NPC1 mutant cells are associated with other anomalies, such as altered distribution of intracellular organelles and properties of the plasma membrane. The pathomechanism of NPC disease is largely unknown. Interestingly, other storage diseases such as Gaucher and Farber diseases are accompanied by severe mitochondrial dysfunction. This prompted us to investigate the effect of absence or dysfunction of the NPC1 protein on mitochondrial properties to confirm or deny a putative relationship between NPC1 mutations and mitochondrial function. This study was performed on primary skin fibroblasts derived from skin biopsies of two NPC patients, carrying mutations in the NPC1 gene. We observed altered organization of mitochondria in NPC1 mutant cells, significant enrichment in mitochondrial cholesterol content, increased respiration, altered composition of the respiratory chain complex, and substantial reduction in cellular ATP level. Thus, a primary lysosomal defect in NPC1 mutant fibroblasts is accompanied by deregulation of the organization and function of the mitochondrial network.

[Mitofusin 2 and mitochondrial dynamics in norm and pathology]. / Mitofuzyna 2 i dynamika mitochondriów w normie i patologii.

Results of an intensive research performed during last 25 years have revealed that an understanding of biochemical and molecular principles of oxidative phosphorylation has not finished the streak of ground-breaking discoveries of newly identified mitochondrial functions in numerous cellular processes. Among other things it has been shown that mitochondria undergo reversible fission and fusion processes, and may form a complex network which functionally and structurally interacts with the endoplasmic reticulum membranes and probably also other organelles. An organization of mitochondrial network is closely controlled and is of high importance for numerous intracellular processes to occur properly. In this review, mitofusin 2 - one of a few proteins involved in a maintenance of an appropriate mitochondrial architecture, and in the consequence in the regulation of mitochondrial metabolism and calcium signalling, the controlling of the mitochondrial DNA level, and the regulation of cell proliferation and differentiation is the focus. Mutations within mitofusin 2-encoding gene are a cause of Charcot-Marie-Tooh 2A - type neuropathies while an affected expression of this protein seems to be related to neoplasia, type 2 diabetes, or vascular hyperplasia. Numerous experimental data confirm pleiotropic effects of mitofisin 2 in animal cells.

Store-operated calcium entry contributes to abnormal Ca²âº signalling in dystrophic mdx mouse myoblasts.

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca(2+) homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca(2+) concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

Hyperglycaemia modifies energy metabolism and reactive oxygen species formation in endothelial cells in vitro.

There is significant evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetic vascular complications through many metabolic and structural derangements. However, despite the advanced knowledge on the crucial role of ROS in cardiovascular damage, their intracellular source in endothelial cells exposed to high concentrations of glucose has not been precisely defined. Moreover, the molecular mechanism of action of elevated glucose on mitochondria has not been fully elucidated. The main aim of this study was to describe changes in the mitochondrial metabolism of human umbilical vein endothelial cells (HUVECs) treated with high glucose concentrations and to indicate the actual source of ROS in these cells. HUVECs exposed to 30 mM glucose exhibited an increased content of vascular adhesive molecule-1 (VCAM-1) and an excessive ROS production. Faster oxygen consumption and increased abundance of selected respiratory complexes coexist with slightly declined mitochondrial membrane potential and substantially elevated amount of uncoupling protein-2 (UCP2). Inhibition of NADPH oxidase (NOX) and modification of mitochondrial ROS generation with a mitochondrial uncoupler or respiratory chain inhibitors allowed concluding that the major source of ROS in HUVECs exposed to hyperglycaemic conditions is NOX. The mitochondrial respiratory chain seems not to participate in this phenomenon.

Potassium channel openers prevent palmitate-induced insulin resistance in C2C12 myotubes.

Insulin resistance (IR) of muscle cells is an early symptom of type 2 diabetes. It often results from excessive lipid accumulation in muscle fibers which under in vitro experimental conditions may be induced by incubation of muscle cells with palmitate. IR is manifested as a reduced response of cells to insulin expressed by lowered Akt kinase phosphorylation and decreased insulin-dependent glucose uptake. Stimulation of mitochondrial oxidative metabolism by mild dissipation of the mitochondrial potential is thought to increase fatty acid utilization and thereby prevent insulin resistance. Here it is shown that nicorandil and NS1619, which are openers of two different mitochondrial potassium channels, protect C2C12 myotubes from palmitate-induced insulin resistance. Preincubation of myotubes with 5-hydroxydecanoate abolishes the protective effect of nicorandil. The efficient concentrations of both openers are far below those commonly applied for cytoprotection. This is probably why their effects on the mitochondrial energy metabolism are small. These data suggest that opening of mitochondrial potassium channels could be a promising approach in prevention and therapy of insulin resistance related to dyslipidemia and obesity.

Purinergic receptors in skeletal muscles in health and in muscular dystrophy.

The P2 purinergic (nucleotide) receptor super-family comprises of two families of protein. The P2X, which are channel-forming ionotropic receptors and the P2Y metabotropic receptors activating G protein-mediated signalling pathways. Members of both groups have been identified in skeletal muscle cells at different stages of differentiation. It is well documented that sequential expression and down-regulation of particular P2 receptors on the surface of sarcolemma is closely associated with muscle maturation during embryogenesis and postnatal growth. P2 receptors are also involved in muscle regeneration following injury. Moreover, enhanced expression of specific purinergic receptors together with increased availability of extracellular ATP in dystrophic muscles are important elements of the dys- trophic pathophysiology considerably increasing severity.

Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the "calcium signalling toolkit" proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue.

INTRODUCTION: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. RESULTS: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. CONCLUSION: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment.

Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.

[Calcium homeostasis in the animal cell--an outline]. / Homeostaza wapnia w komórce zwierzecej--w zarysie.

Calcium ions are universal and versatile intracellular signalling molecule which is involved in regulation of many cellular functions in all living cells throughout all animal species. It results from unique properties of Ca2+ in comparison to other two- and monovalent cations commonly present inside and outside cells. On the other hand an excessive increase of intracellular Ca2+ accumulation may exert toxic effect leading to cell death. Therefore calcium content in particular cellular compartment must be precisely regulated. All cells have a complex set of proteins which allow them to remove, store or take up Ca2+ in very controlled manner. This article gives a concise survey of mechanisms involved cellular calcium homeostasis and signalling.

[Store-operated calcium entry--are all elements of the system already identified?]. / Pojemnosciowy naplyw jonów wapnia--czy wszystkie elementy maszynerii zostaly zidentyfikowane?

Store-operated Ca2+ entry (SOCE) is an ubiquitous mechanism leading to a transient increase of Ca2+ concentration in the cytoplasm ([Ca2+]c) of a leaving cell followed by refill of the internal stores with calcium. Discovery of STIM1 and STIM2 proteins located in the endoplasmic reticulum (ER) and playing a role of sensors of calcium, led to our understanding how the calcium signal from ER is propagated to calcium release-activated calcium channels (CRAC) located in the plasma membrane, resulting in their activation, flow of calcium into a cytoplasm and activation of calcium-dependent signaling. In light of controversies existing in identification of CRAC channels (such as Oral, TRPC and others), as well as identification of mechanisms of calcium entry that are independent of the presence of calcium in the internal calcium stores, in this review we discuss the newest theories about SOCE, proteins that are engaged in this mechanism as well as pathologies related to impaired SOCE.

Lipopolysaccharide affects energy metabolism and elevates nicotinamide N-methyltransferase level in human aortic endothelial cells (HAEC).

This study aimed to investigate the putative role of nicotinamide N-methyltransferase in the metabolic response of human aortic endothelial cells. This enzyme catalyses S-adenosylmethionine-mediated methylation of nicotinamide to methylnicotinamide. This reaction is accompanied by the reduction of the intracellular nicotinamide and S-adenosylmethionine content. This may affect NAD+ synthesis and various processes of methylation, including epigenetic modifications of chromatin. Particularly high activity of nicotinamide N-methyltransferase is detected in liver, many neoplasms as well as in various cells in stressful conditions. The elevated nicotinamide N-methyltransferase content was also found in endothelial cells treated with statins. Although the exogenous methylnicotinamide has been postulated to induce a vasodilatory response, the specific metabolic role of nicotinamide N-methyltransferase in vascular endothelium is still unclear. Treatment of endothelial cells with bacterial lipopolysaccharide evokes several metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Among the spectrum of biochemical changes substantially elevated protein level of nicotinamide N-methyltransferase was particularly intriguing. Here it has been shown that silencing of the nicotinamide N-methyltransferase gene influences several changes which are observed in cells treated with lipopolysaccharide. They include altered energy metabolism and rearrangement of the mitochondrial network. A complete explanation of the mechanisms behind the protective consequences of the nicotinamide N-methyltransferase deficiency in cells treated with lipopolysaccharide needs further investigation.

Interaction of plasma membrane Ca(2+)-ATPase isoform 4 with calcineurin A: implications for catecholamine secretion by PC12 cells.

PMCA1-4 isoforms have been recently recognised as regulators of various signalling pathways in mammalian cells. PMCAs were found to interact with calcineurin A in an isoform specific manner. In this study we focus on the interaction of calcineurin A with PMCA4 and its effect on catecholamine secretion in PC12 cells with reduced PMCA2 or PMCA3 content. Reduction of synthesis of PMCA2 or PMCA3 led to upregulation of PMCA4 manifested by preferential interaction of PMCA4 with calcineurin A. On the other hand, we observed a significant reduction of dopamine secretion, which did not correspond with an increased [Ca(2+)](c). This result indicates that the interaction of PMCA4 with calcineurin A plays a regulatory role in the signalling during catecholamine secretion.