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

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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

VRAC Channels and the Cellular Redox Balance

Volume-regulated anion channels (VRAC) are mainly involved in the regulated transport of osmolytes such as ions or small organic compounds across the plasma membrane during anisosmotic cell swelling. However, they also play additional roles in various pathophysiological processes, such as the transport of metabolites and drugs, extracellular signal transduction and anti-cancer drug resistance. These channels are formed by heteromers of LRRC8 proteins, of which LRRC8A is the essential subunit that combines with its paralogs LRRC8B–E to form hexameric complexes. Despite the extensive research devoted to the understanding of VRACs functions, different aspects of these channels are still to be characterized in depth. In this chapter, recent findings concerning the involvement of VRAC channels in the cellular redox balance will be summarized. Also, their relevance as potential targets of antioxidant therapies will be discussed

Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42− uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology

d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42− uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines

Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents

Cellular and Molecular Mechanisms in Oxidative Stress-Related Diseases

The redox equilibrium is important in preserving the correct functionality of vital cellular functions [...

Impact of Scyphozoan Venoms on Human Health and Current First Aid Options for Stings

Cnidaria include the most venomous animals of the world. Among Cnidaria, Scyphozoa (true jellyfish) are ubiquitous, abundant, and often come into accidental contact with humans and, therefore, represent a threat for public health and safety. The venom of Scyphozoa is a complex mixture of bioactive substances—including thermolabile enzymes such as phospholipases, metalloproteinases, and, possibly, pore-forming proteins—and is only partially characterized. Scyphozoan stings may lead to local and systemic reactions via toxic and immunological mechanisms; some of these reactions may represent a medical emergency. However, the adoption of safe and efficacious first aid measures for jellyfish stings is hampered by the diffusion of folk remedies, anecdotal reports, and lack of consensus in the scientific literature. Species-specific differences may hinder the identification of treatments that work for all stings. However, rinsing the sting site with vinegar (5% acetic acid) and the application of heat (hot pack/immersion in hot water) or lidocaine appear to be substantiated by evidence. Controlled clinical trials or reliable models of envenomation are warranted to confirm the efficacy and safety of these approaches and identify possible species-specific exceptions. Knowledge of the precise composition of Scyphozoa venom may open the way to molecule-oriented therapies in the future

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed