Protective Role of Magnesium against Oxidative Stress on SO4= Uptake through Band 3 Protein in Human Erythrocytes

n/

Curcumin Protects -SH Groups and Sulphate Transport after Oxidative Damage in Human Erythrocytes

Background/Aims: Erythrocytes, continuously exposed to oxygen pressure and toxic compounds, are sensitive to oxidative stress, namely acting on integral Band 3 protein, with consequences on cell membranes deformability and anion transport efficiency. The aim of the present investigation, conducted on human erythrocytes, is to verify whether curcumin (1 or 10µM), a natural compound with proved antioxidant properties, may counteract Band 3-mediated anion transport alterations due to oxidative stress. Methods: Oxidative conditions were induced by exposure to, alternatively, either 2 mM N-ethylmaleimide (NEM) or pH-modified solutions (6.5 and 8.5). Rate constant for SO4= uptake and -SH groups estimation were measured to verify the effect of oxidative stress on anion transport efficiency and erythrocyte membranes. Results: After the exposure of erythrocytes to, alternatively, NEM or pH-modified solutions, a significant decrease in both rate constant for SO4= uptake and -SH groups was observed, which was prevented by curcumin, with a dose-dependent effect. Conclusions: Our results show that: i) the decreased efficiency of anion transport may be due to changes in Band 3 protein structure caused by cysteine -SH groups oxidation, especially after exposure to NEM and pH 6.5; ii) 10 µM Curcumin is effective in protecting erythrocytes from oxidative stress events at level of cell membrane transport

Sulphate and Chloride-Dependent Potassium Transport in Human Erythrocytes are Affected by Crude Venom from Nematocysts of the Jellyfish Pelagia noctiluca

Background: It has been reported that biologically active compounds extracted from Cnidaria venom may induce damage by oxidative stress. Erythrocytes are constantly exposed to oxidative stresses, which can contribute to sulphydril (SH-) group oxidation and cell membrane deformability accompanied with activation of K-Cl co-transport and inhibition of anion transport. In this regard, Band 3 protein is responsible for mediating the electroneutral exchange of chloride (Cl-) for bicarbonate (HCO3-), particularly in erythrocytes, where it is the most abundant membrane protein. The aim of this study was to elucidate the effect of crude venom extracted from Pelagia noctiluca nematocysts on Band 3 -mediated anion transport in human erythrocytes. Methods: Erythrocytes were tested for SO42- uptake, K+ efflux, glutathione (GSH) levels and concentration of SH- groups. Results: The rate constant of SO42- uptake decreased progressively to 58% of control with increasing venom doses, and showed a 28% decrease after 2 mM NEM treatment. These effects can be explained by oxidative stress, which was reflected by decreased GSH levels in venom-treated erythrocytes. Hence, the decreased efficiency of anion transport may be due to changes in Band 3 structure caused by SH-group oxidation and reduced GSH concentration. In addition, an increased Cl--dependent K+ efflux was observed in venom-treated erythrocytes. Conclusion: Our results suggest that crude venom from Pelagia noctiluca alters cell membrane transport in human erythrocytes

The venom and the toxicity of Pelagia noctiluca (Cnidaria: Scyphozoa). A review of three decades of research in Italian laboratories and future perspectives

Recurrent outbreaks of Pelagia noctiluca and health problems consequent to stings were recorded during the last decades. This phenomenon forced some Italian University laboratories to study this cnidarian. The first studies concerned the distribution, biochemical composition and morphology of nematocysts of Pelagia noctiluca. The discharge mechanism of nematocysts was defined starting from early 1980s when enzymes, cations, anions, and pH were observed to have an influence on this process. Notably, trypsin, extreme pH values, some anions (I–, Cl–, SCN–), and thioglycolate were seen to induce, while La3+ and Gd3+ to prevent, nematocyst discharge. The discharge of both in situ and isolated nematocyst was found to be Ca2+ dependent. Furthermore, Pelagia noctiluca nematocysts were seen to retain their discharging capacity in distilled water. The toxicological evaluations were carried out mainly using the crude venom from Pelagia noctiluca because, unfortunately, to date the composition of venom remains unknown. Hemolytic and cytotoxic properties of crude venom have been evaluated on erythrocytes and cultured guinea-pig fibroblasts, mouse fibroblasts, and cancer (neuroblastoma) cells. The activity of Pelagia noctiluca venom on other cnidarians has been also assessed. The crude venom induced apoptosis by reactive oxygen species generation and decrease in mitochondrial transmembrane potential, loss of mitochondrial integrity, and alteration of cell membrane permeability. A pore-forming action mechanism on mitochondrial membrane with oxidative damage was also suggested. The protective activity of some compounds against envenomations has been also evaluated. Future challenges will concern the attempts to characterize the venom and to perform a wider screening of cytotoxicity induced to normal and cancer cells

Mechanisms of hyposmotic volume regulation in isolated nematocytes of the anthozoan Aiptasia diaphana.

The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from aconti

An approach to high frequency monitoring of lakes: the SAILING and PITAGORA projects

Recent developments in sensor technology allow highresolution monitoring of lakes and reservoirs from sensor based systems such as buoys and platforms. The data collected by these systems may be provided to end-users by web-based technology and in this way be readily available for both advanced users (e.g. researchers) and citizens. With respect to traditional monitoring, based on discrete samples, high frequency data may resolve diel or short-term events and detect horizontal and vertical patterns in large deep lakes. Starting in 2014, we assembled a multi-parameter measuring system, adaptable to specific needs and implementable with different type of sensors. This system was initially tested on a sailing boat, in the framework of the SAILING project (Sensor-based Assessment on In Lake processes and water quality - Scientific INvestigation and Growing environmental awareness): two monitoring campaigns were performed, on Lake Maggiore (during 2014) and Orta (during 2015), with the aim of measuring basic limnological parameters (water temperature, pH, conductivity, dissolved oxygen) in surface water with high temporal and spatial frequency. Successively, through the PITAGORA project (Platform of interoperable technology for the acquisition, management and organization of environmental data), a similar measuring system was installed on a couple of buoys, deployed in both the two lakes. In this paper we describe the approach we used in the development of these sensor based systems and present some preliminary results obtained from the analysis of high frequency data. Results highlight the usefulness of these data in detecting processes that occur on very short time scales, such as those driven by rapid changes in meteorological condition. These activities have been performed in the framework of the EU COST Action NETLAKE (Networking Lake Observatories in Europe), aiming to build a network of sites and individuals to support the development and deployment of sensor-based systems in lakes and reservoirs

Evidence for aquaporin-mediated water transport in nematocytes of the jellyfish Pelagia noctiluca.

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K+, Cl- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca2+ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl2 (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO3 (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival

Effect of cadmium on anion exchange capability through Band 3 protein in human erythrocytes

The efficiency of Band 3 protein, mediating HCO3-/Cl- exchange across erythrocytes membrane, is reduced by oxidative stress. The aim of the present study was to verify whether Band 3 protein efficiency is compromised by treatment with Cadmium (Cd2+), an extremely toxic heavy metal known to interfere with antioxidant enzymes, energy metabolism, gene expression and cell membranes. To this end, the rate constant for SO4= uptake through Band 3 protein (accounting for velocity of anion exchange) was measured along with membrane –SH groups, Malonyldialdehyde (MDA) and Band 3 protein expression levels in Cd2+ -treated human erythrocytes (300 µM, 1 mM). Our results show that Cd2+ reduced the rate constant for SO4= uptake, with a significant increase in MDA levels at both concentrations and with a reduction in –SH groups observed after 1 mM Cd2+ treatment, whereas Band 3 protein expression levels were unchanged in both conditions. In conclusion: i) Cd2+ reduces Band 3 protein efficiency via different mechanisms depending on metal concentration and with unchanged expression levels; ii) the assessment of Band 3 protein anion exchange capability is a good tool to assay the impact of heavy metals on cell homeostasis and, possibly, useful for diagnosis and monitoring of devalopment of Cd2+ toxicity-related pathologies

oxidative stress affects responsiveness to hypotonicity of renal cells

Oxidative stress plays a critical role in the pathophysiology of several kidney diseases and is the consequence of alterations like ischemic events. The regulatory volume decrease (RVD) is an homeostatic response essential to many cells, including renal cells, to counteract changes in the osmolarity of the external medium. The aim of the present work is to verify whether oxidative stress affects RVD in a model of renal cells (human embryonic kidney cells, HEK 293 Phoenix). To accomplish this aim, the experimental procedure consisted in: i) cell culture preparation and treatment with 200 μM H2O2; and ii) measurement of cell volume changes in isotonic conditions or following hypotonic stress. H2O2 added to the extracellular isotonic solution induced a significant reduction in cell volume, and added to the extracellular hypotonic solution dramatically impaired the expected osmotic cell swelling. Pre-incubation of cells in an extracellular isotonic solution containing H2O2 prevented cell from swelling after hypotonic stress application. In conclusion, H2O2 leads to cell shrinkage in isotonic conditions, inhibits the hypotonicity-induced cell swelling and consequently prevents RVD, hypothetically due to an activation of transport pathways determining ion loss and, in turn, water efflux. Cell shrinkage in isotonic conditions is a hallmark of apoptosis and is known as the apoptotic volume decrease

regulatory volume decrease in isolated nematocytes is affected by crude venom from the jellyfish pelagia noctiluca

Crude venom from nematocysts of the Scyphozoan Pelagia noctiluca possesses hemolytic and cytotoxic power on cultured cells and elicits local and systemic inflammation reactions in vivo. The ability of regulating their volume after exposure to an anisosmotic solution is a fundamental feature common to cells from vertebrates and invertebrates, including Cnidarians. The aim of the present work i s to assay whether crude venom from Pelagia noctiluca may affect the regulatory volume decrease (RVD) of nematocytes isolated from the Anthozoan Aiptasia mutabilis, here employed as a cell model. For this purpose, nematocytes were isolated by 605 mM NaSCN plus 0.01 mM Ca2+ application on acontia of Aiptasia mutabilis, while crude venom was obtained by sonication of a population of, respectively, 10, 25 and 50 nematocysts/µL (n/µL). Isolated nematocytes were pre-treated for 30 min with crude venom, submitted to hypotonic stress and their osmotic response and RVD were measured optically. Our results show that, after exposure to crude venom, nematocytes were morphologically intact, as shown by the Trypan blue exclusion test, but did not exhibit RVD. This effect was dose-dependent and reversed by the ionopho re gramicidin. The last observation suggests an inhibitory effect of venom on cell membrane ion transport mechanisms involved in RVD. Further studies are needed to verify this hypothesis and ascertain if a similar effect could be observed in human cells