Caterina Morabito

Modulation of redox status and calcium handling by extremely low frequency electromagnetic fields in C2C12 muscle cells: A real-time, single-cell approach.

The biological effects of electric and magnetic fields, which are ubiquitous in modern society, remain poorly understood. Here, we applied a single-cell approach to study the effects of short-term exposure to extremely low frequency electromagnetic fields (ELF-EMFs) on muscle cell differentiation and function using C2C12 cells as an in vitro model of the skeletal muscle phenotype. Our focus was on markers of oxidative stress and calcium (Ca(2+)) handling, two interrelated cellular processes previously shown to be affected by such radiation in other cell models. Collectively, our data reveal that ELF-EMFs (1) induced reactive oxygen species production in myoblasts and myotubes with a concomitant decrease in mitochondrial membrane potential; (2) activated the cellular detoxification system, increasing catalase and glutathione peroxidase activities; and (3) altered intracellular Ca(2+)homeostasis, increasing the spontaneous activity of myotubes and enhancing cellular reactivity to a depolarizing agent (KCl) or an agonist (caffeine) of intracellular store Ca(2+)channels. In conclusion, our data support a possible link between exposure to ELF-EMFs and modification of the cellular redox state, which could, in turn, increase the level of intracellular Ca(2+)and thus modulate the metabolic activity of C2C12 cells.

Effects of acute and chronic low frequency electromagnetic field exposure on PC12 cells during neuronal differentiation.

Background/Aims. The purpose of this study was to provide information about the in vitro neuritogenesis during cell exposure to extremely low frequency electromagnetic fields (ELF-EMFs) of different intensities and durations using pheochromocytoma-derived cell line (PC12 cells) as neuronal model. Methods. Proliferative rates and neuritogenesis were tested by colorimetric assay and morphological analysis, respectively; reactive oxygen species (ROS) levels and intracellular Ca2+ variations monitored using single cell videomicroscopy. Results. The long-lasting ELF-EMF exposure (0.1-1.0 mT) did not appear to significantly affect the biological response (proliferation and neuritogenesis). However, during the acute ELF-EMF exposure (30 min), in undifferentiated PC12 cells, there were increased ROS levels and decreased catalase activity, that, conversely, resulted increased after chronic exposure (7 days) at 1.0 mT. Acute exposure (0.1-1.0 mT) affected the spontaneous intracellular Ca2+ variations in undifferentiated cells, in which basal intracellular Ca2+ resulted increased after chronic exposure. In addition acute exposure affected cell response to a depolarizing agent, while basal membrane potential was not changed. Conclusion. Even if further studies remain necessary to identify the ROS/intracellular Ca2+cross-talking pathway activated by ELF-EMF exposure, we support the hypothesis that ROS and Ca2+ could be the cellular “primum movens” of the ELF-EMF induced effects on biological systems.

Modification of the functional capacity of sarcoplasmic reticulum membranes in patients suffering from chronic fatigue syndrome

In chronic fatigue syndrome, several reported alterations may be related to specific oxidative modifications in muscle. Since sarcoplasmic reticulum membranes are the basic structures involved in excitation-contraction coupling and the thiol groups of Ca(2+) channels of SR terminal cisternae are specific targets for reactive oxygen species, it is possible that excitation-contraction coupling is involved in this pathology. We investigated the possibility that abnormalities in this compartment are involved in the pathogenesis of chronic fatigue syndrome and consequently responsible for characteristic fatigue. The data presented here support this hypothesis and indicate that the sarcolemmal conduction system and some aspects of Ca(2+) transport are negatively influenced in chronic fatigue syndrome. In fact, both deregulation of pump activities (Na(+)/K(+) and Ca(2+)-ATPase) and alteration in the opening status of ryanodine channels may result from increased membrane fluidity involving sarcoplasmic reticulum membranes.

Oxidative-induced membrane damage in diabetes lymphocytes: Effects on intracellular Ca2 + homeostasis

Oxidative stress is linked to several human diseases, including diabetes. However, the intracellular signal transduction pathways regulated by reactive oxygen species (ROS) remain to be established. Deleterious effects of ROS stem from interactions with various ion transport proteins such as ion channels and pumps, primarily altering Ca2 + homeostasis and inducing cell dysfunction. This study characterized the Ca2 + transport system in lymphocytes of patients with type-2 diabetes, evaluating the possible correlation between cell modifications and the existence of specific oxidative stress damage. Lymphocytes from type-2 diabetes patients displayed oxidative stress features (accumulation of some ROS species, membrane peroxidation, increase in protein carbonyls, increase in SOD and Catalase activity) and Ca2 + dyshomeostasis (modified voltage-dependent and inositol 1,4,5-triphosphate-mediated Ca2 + channel activities, decrease in Ca2 + pumps activity). The data support a correlation between oxidative damage and alterations in intracellular Ca2 + homeostasis, possibly due to modification of the ionic control in lymphocytes of type-2 diabetes patients.

Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells

Background Calcimimetics, such as R-568, are thought to activate G protein-linked Ca2+-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects. Methodology/Principal Findings Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels. Conclusions/Significance Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Peripheral Blood Lymphocytes: A Model for Monitoring Physiological Adaptation to High Altitude

Depending on the absolute altitude and the duration of exposure, a high altitude environment induces various cellular effects that are strictly related to changes in oxidative balance. In this study, we used in vitro isolated peripheral blood lymphocytes as biosensors to test the effect of hypobaric hypoxia on seven climbers by measuring the functional activity of these cells. Our data revealed that a 21-day exposure to high altitude (5000 m) (1) increased intracellular Ca(2+) concentration, (2) caused a significant decrease in mitochondrial membrane potential, and (3) despite possible transient increases in intracellular levels of reactive oxygen species, did not significantly change the antioxidant and/or oxidative damage-related status in lymphocytes and serum, assessed by measuring Trolox-equivalent antioxidant capacity, glutathione peroxidase activity, vitamin levels, and oxidatively modified proteins and lipids. Overall, these results suggest that high altitude might cause an impairment in adaptive antioxidant responses. This, in turn, could increase the risk of oxidative-stress-induced cellular damage. In addition, this study corroborates the use of peripheral blood lymphocytes as an easily handled model for monitoring adaptive response to environmental challenge.

Growth Associated Protein 43 Is Expressed in Skeletal Muscle Fibers and Is Localized in Proximity of Mitochondria and Calcium Release Units

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases.