Membrane Potential Generation Research Articles

Synergistic neurological threat from Сu and wood smoke particulate matter

Trace metal Cu and carbonaceous airborn particulate matter (PM) are dangerous neuropollutants. Here, the ability of Cu2+ to modulate the neurotoxicity caused by water-suspended wood smoke PM preparations (SPs) and vice versa was examined using presynaptic rat cortex nerve terminals. Interaction of Cu2+ and SPs, changes of particle size and surface properties were shown in the presence of Cu2+ using microscopy, DLS, and IR spectroscopy. In nerve terminals, Cu2+ and SPs per se elevated the ambient levels of excitatory and inhibitory neurotransmitters L-[14C]glutamate and [3H]GABA, respectively. During combined application, Cu2+ significantly enhanced a SPs-induced increase in the ambient levels of both neurotransmitters, thereby demonstrating a cumulative synergistic effect and significant interference in the neurotoxic threat associated with Cu2+and SPs. In fluorimetric measurements, Cu2+ and SPs also demonstrated cumulative synergistic effects on the membrane potential, mitochondrial potential, synaptic vesicle acidification and ROS generation. Therefore, synergistic effects of Cu2+ and SPs on the most crucial presynaptic characteristics and neurohazard of multiple pollutants through excitatory/inhibitory imbalance, disruption of the membrane and mitochondrial potential, vesicle acidification and ROS generation were revealed. Increased expansion and burden of neuropathology may result from underestimation of synergistic interference of the neurotoxic effects of Cu2+ and carbonaceous smoke PM.

A Glucose Oxidase-Curcumin Composite Nanoreactor for Multimodal Synergistic Cancer Therapy.

Glucose oxidase (GOx) selectively oxidizes β-d-glucose into gluconic acid and hydrogen peroxide; thus, it has emerged as a promising anticancer agent by tumor starvation and oxidative therapy. Here, we developed a nanoscale platform or "nanoreactor" that incorporates GOx and the bioactive natural product curcumin (CUR) to achieve a multimodal anticancer nanocomposite. The composite nanoreactor was formed by loading CUR in biodegradable polymeric nanoparticles (NPs) of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL). Prime-coating of the NPs with an iron(III)-tannic acid complex enabled facile immobilization of GOx on the NP surface. The NPs were monodisperse with a hydrodynamic diameter of 122 nm and a partially negative surface charge. The NPs were also associated with an excellent CUR loading efficiency and sustained release up to 96 h, which was accelerated by surface-immobilized GOx and followed supercase II transport. Viability assays were conducted on two model cancer cell lines, MCF-7 and MDA-MB-231 cells, as well as human dermal fibroblasts as a representative normal cell line. The assays revealed significantly improved potency of CUR in the composite nanoreactor, with up to 6000- and 1280-fold increase in MCF-7 and MDA-MB-231 cells, respectively, and lower toxicity toward normal cells. The NPs were also able to promote intracellular reactive oxygen species (ROS) generation and dissipation of the mitochondrial membrane potential, providing important clues on the mechanism of action of the nanoreactor. Further investigation of caspase-3 activity revealed that the nanoreactor had no effect or inhibited caspase-3 levels, signifying a caspase-independent mechanism of inducing apoptosis. Our findings present a promising nanocarrier platform that combines therapeutic agents with distinct mechanisms of action acting in synergy for more effective cancer therapy.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

BackgroundHydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown.ResultsWe report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes.ConclusionsThe hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

Nr1h4 and Thrb ameliorate ER stress and provide protection in the MPTP mouse model of Parkinson's.

Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the differential expression of all the nuclear receptors (NRs) in the presence of classical ER stress inducers. Activation of Nr1h4 and Thrb by their cognate ligands (GW4064 and T3) ameliorates the tunicamycin (TM)-induced expression of ER stress genes. A combination of both ligands is effective in mitigating cell death induced by TM. Further exploration of their protective effects in the Parkinson's disease (PD) model shows that they reduce MPP+-induced dissipation of mitochondrial membrane potential and ROS generation in an in vitro PD model in neuronal cells. Furthermore, the generation of an experimental murine PD model reveals that simultaneous treatment of GW4064 and T3 protects mice from ER stress, dopaminergic cell death, and functional deficits in the MPTP mouse model of PD. Thus, activation of Nr1h4 and Thrb by their respective ligands plays an indispensable role in ER stress amelioration and mounts protective effects in the MPTP mouse model of PD.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Cassia fistula L. bark fraction modulated GSK3β/ p53 expression for mitochondrial mediated apoptosis in HeLa cells

In Ayurveda, Cassia fistula L. commonly known as Aragvadha (disease killer) is highly valuable medicinal plant. In the present study, hexane fraction (CaMH), chloroform fraction (CaMC), ethyl acetate fraction (CaME) and methanol fraction (CaMM) isolated from C. fistula bark were explored for their cytotoxic potential against cervical carcinoma (HeLa), breast carcinoma (MCF-7) and osteosarcoma (MG-63) cell lines using MTT assay. Our results unveiled that C. fistula bark fractions were selectively cytotoxic to carcinoma cells and among the tested fractions CaMC fraction had highest selectivity index (SI) with a value of 9.77 in HeLa cells. CaMC fraction actuated apoptosis in HeLa cells, as evident from chromatin condensation, DNA fragmentation, externalization of phosphatidylserine, altered mitochondrial membrane potential and ROS generation. Moreover, CaMC fraction upregulated the expression of GSK3β and p53 which modulated the Bax/ Bcl-2 ratio and initiated the release of cytochrome c from mitochondria. The expression of caspase 3/9 was also found to be upregulated which ultimately induced apoptosis. We also found that CaMC induced cell cycle arrest at G0/G1 phase and significantly inhibited the migration in HeLa cells. GC–MS analysis showed Oleic Acid, Piperine, Isopiperine and Tris(2,4-di‑tert-butylphenyl) phosphate as major constituents which could be attributed to anticancer activity of CaMC. The present study affirms the potential of CaMC fraction in modulating the aberrant signaling pathway for the induction of apoptosis in HeLa cells.

H2S Protects from Rotenone-Induced Ferroptosis by Stabilizing Fe-S Clusters in Rat Cardiac Cells.

Hydrogen sulfide (H2S) has been recently recognized as an important gasotransmitter with cardioprotections, and iron is vital for various cellular activities. This study explored the regulatory role of H2S on iron metabolism and mitochondrial functions in cultured rat cardiac cells. Rotenone, a mitochondrial complex I inhibitor, was used for establishing an in vitro model of ischemic cell damage. It was first found that rotenone induced oxidative stress and lipid peroxidation and decreased mitochondrial membrane potential and ATP generation, eventually causing cell death. The supplement of H2S at a physiologically relevant concentration protected from rotenone-induced ferroptotic cell death by reducing oxidative stress and mitochondrial damage, maintaining GPx4 expression and intracellular iron level. Deferiprone, an iron chelator, would also protect from rotenone-induced ferroptosis. Further studies demonstrated that H2S inhibited ABCB8-mediated iron efflux from mitochondria to cytosol and promoted NFS1-mediated Fe-S cluster biogenesis. It is also found that rotenone stimulated iron-dependent H2S generation. These results indicate that H2S would protect cardiac cells from ischemic damage through preserving mitochondrial functions and intracellular Fe-S cluster homeostasis.

Mitocentricity.

Worldwide, interest in mitochondria is constantly growing, as evidenced by scientific statistics, and studies of the functioning of these organelles are becoming more prevalent than studies of other cellular structures. In this analytical review, mitochondria are conditionally placed in a certain cellular center, which is responsible for both energy production and other non-energetic functions, without which the existence of not only the eukaryotic cell itself, but also the entire organism is impossible. Taking into account the high multifunctionality of mitochondria, such a fundamentally new scheme of cell functioning organization, including mitochondrial management of processes that determine cell survival and death, may be justified. Considering that this issue is dedicated to the memory of V. P. Skulachev, who can be called mitocentric, due to the history of his scientific activity almost entirely aimed at studying mitochondria, this work examines those aspects of mitochondrial functioning that were directly or indirectly the focus of attention of this outstanding scientist. We list all possible known mitochondrial functions, including membrane potential generation, synthesis of Fe-S clusters, steroid hormones, heme, fatty acids, and CO2. Special attention is paid to the participation of mitochondria in the formation and transport of water, as a powerful biochemical cellular and mitochondrial regulator. The history of research on reactive oxygen species that generate mitochondria is subject to significant analysis. In the section "Mitochondria in the center of death", special emphasis is placed on the analysis of what role and how mitochondria can play and determine the program of death of an organism (phenoptosis) and the contribution made to these studies by V. P. Skulachev.

Multipollutant reciprocal neurological hazard from smoke particulate matter and heavy metals cadmium and lead in brain nerve terminals

Heavy metals, Cd2+ and Pb2+, and carbonaceous air pollution particulate matter are hazardous neurotoxicants. Here, a capability of water-suspended smoke particulate matter preparations obtained from poplar wood (WPs) and polypropylene fibers (medical facemasks) (MPs) to influence Cd2+/Pb2+-induced neurotoxicity, and vice versa, was monitored using biological system, i.e. isolated presynaptic rat cortex nerve terminals. Combined application of Pb2+ and WPs/MPs to nerve terminals in an acute manner revealed that smoke preparations did not change a Pb2+-induced increase in the extracellular levels of excitatory neurotransmitter L-[14C]glutamate and inhibitory one [3H]GABA, thereby demonstrating additive result and no interference of neurotoxic effects of Pb2+ and particulate matter. Whereas, both smoke preparations decreased a Cd2+-induced increase in the extracellular level of L-[14C]glutamate and [3H]GABA in nerve terminals. In fluorimetric measurements, the metals and smoke preparations demonstrated additive effects on the membrane potential of nerve terminals causing membrane depolarisation. WPs/MPs-induced reduction of spontaneous ROS generation was mitigated by Cd2+ and Pb2+. Therefore, a potential variety of multipollutant heavy metal-/airborne particulate-induced effects on key presynaptic processes was revealed. Multipollutant reciprocal neurological hazard through disturbance of the excitation-inhibition balance, membrane potential and ROS generation was evidenced. This multipollutant approach and data contribute to up-to-date environmental quality/health risk estimation.

A mitochondria-targeting heptamethine cyanine-chlorambucil formulated polymeric nanoparticle to potentiate native tumor chemotherapeutic efficacy.

Chlorambucil (Cbl) is a DNA alkylating drug in the nitrogen mustard family, but the clinical applications of nitrogen mustard antitumor drugs are frequently limited by their poor aqueous solubility, poor cellular uptake, lack of targeting, and severe side effects. Additionally, mitochondria are the energy factories for cells, and tumor cells are more susceptible to mitochondrial dysfunction than some healthy cells, thus making mitochondria an important target for tumor therapy. As a proof-of-concept, direct delivery of Cbl to tumor cells' mitochondria will probably bring about new opportunities for the nitrogen mustard family. Furthermore, IR775 chloride is a small-molecule lipophilic cationic heptamethine cyanine dye with potential advantages of mitochondria targeting, near-infrared (NIR) fluorescence imaging, and preferential internalization towards tumor cells. Here, an amphiphilic drug conjugate was facilely prepared by covalently coupling chlorambucil with IR775 chloride and further self-assembly to form a carrier-free self-delivery theranostic system, in which the two components are both functional units aimed at theranostic improvement. The theranostic IR775-Cbl potentiated typical "1 + 1 > 2" tumor inhibition through specific accumulation in mitochondria, which triggered a remarkable decrease in mitochondrial membrane potential and ATP generation. In vivo biodistribution and kinetic monitoring were achieved by real-time NIR fluorescence imaging to observe its transport inside a living body. Current facile mitochondria-targeting modification with clinically applied drugs was promising for endowing traditional drugs with targeting, imaging, and improved potency in disease theranostics.

Membrane potential generation by cytochrome <i>bd</i>

This treatise gives an overview of current thinking on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase. It is assumed that the main contribution to the Δψ formation is made by the movement of H+ across the membrane along the intraprotein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.

Investigation of the mechanism of membrane potential generation by heme-copper respiratory oxidases in real time mode

Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze the final stage of cellular respiration of eukaryotes and many prokaryotes: the transfer of electrons from cytochromes or quinols to molecular oxygen and the reduction of O2 to water. The free energy released in this redox reaction is converted by heme-copper respiratory oxidases into a transmembrane gradient of the electrochemical potential of hydrogen ions (ΔµH+). Heme-copper respiratory oxidases have a unique mechanism for generation ΔµH+ - a redox-coupled proton pump. The use of a combination of a direct electrometric method for measuring the kinetics of membrane potential generation with approaches and methods of prestationary kinetics and directed mutagenesis in the study of heme-copper oxidases makes it possible to obtain unique information about the movement of protons inside the protein in real time. The review summarizes the results of the use of the permitted time.

PCB126 impairs human sperm functions by affecting post-translational modifications and mitochondrial functions

Over the past few decades, there has been a consistent decline in semen quality across the globe, with environmental pollution being identified as the primary cause. Among the various contaminants present in the environment, persistent organic pollutants (POPs) have garnered significant attention due to their high toxicity, slow degradation, bio-accumulation, and long-range migration. PCBs, which include 210 congeners, are a crucial type of POPs that are known to have harmful effects on the environment and human health. Among the various PCB congeners, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) is a typical environmental endocrine-disrupting chemical that is widely distributed and has been associated with several health hazards. However, the impact and mechanism of PCB126 on human sperm function has not been fully elucidated. We aimed to investigate the effects of different concentrations of PCB126 (0.01, 0.1, 1, 10 μg/mL) on sperm motility, viability, hyperactivation, and acrosome reaction after incubation for different periods (1 and 2 h), delving deeper into the molecular mechanism of human sperm dysfunction caused by PCB126. First, we investigated the link between PCB126 treatment and the occurrence of protein modifications that are critical to sperm function regulation, such as tyrosine phosphorylation and lysine glutarylation. Second, we examined the potential impact of PCB126 on different parameters related to mitochondrial function, including reactive oxygen species, malondialdehyde levels, mitochondrial membrane potential, mitochondria respiration and adenosine triphosphate generation. Our findings indicate that exposure to environmental pollutants such as PCB126 in vitro may have a negative impact on human sperm functions by interfering with post-translational modifications and mitochondrial functions.

Rhein suppresses African swine fever virus replication in vitro via activating the caspase-dependent mitochondrial apoptosis pathway

African swine fever (ASF) is a virulent infectious diseases of pigs caused by the African swine fever virus (ASFV) that can spread widely and cause high fatality rates. Currently, there is no effective way to treat the disease, and there is no effective vaccine to prevent it. Rhein, an anthraquinone compound extracted from many traditional Chinese medicines, exhibits anti-inflammatory, anti-tumor, and anti-viral activities. However, the anti-viral effects of rhein on ASFV remain unclear. Therefore, this study aimed to investigate the anti-ASFV activity of rhein in porcine alveolar macrophages (PAMs) and the underlying mechanisms. In this study, we confirmed that rhein inhibits ASFV replication significantly in a dose-dependent manner in vitro. Moreover, rhein could alter the susceptibility of PAMs to ASFV and promoted the production of superoxide in the mitochondria, which induced the loss of mitochondrial membrane potential, leading to the activation of caspase-9, caspase-3, and apoptosis. Mito-TEMPO, a mitochondria-targeted antioxidant, blocked rhein-induced mitochondrial superoxide generation and loss of mitochondrial membrane potential, prevented caspase-9 and caspase-3 activation, alleviated apoptosis, and suppressed the anti-ASFV activity of rhein. Altogether, our results suggested that rhein could play an anti-ASFV role by inducing apoptosis through the activation of the caspase-dependent mitochondrial apoptotic pathway and may provide a novel compound for developing anti-ASFV drugs.

Життєздатність ацинарних клітин підшлункової залози та рівень мембранного потенціалу мітохондрій за дії жовчевої кислоти TLC-S

To investigate the mechanism of action of bile acids, TLC-S, the viability of pancreatic acinar cells, and the level of mitochondrial membrane potential were studied using various oxidation substrates. The experiments were conducted on male Wistar rats weighing 250–300 g. A suspension of isolated pancreatic acinar cells was obtained using collagenase (type IV, 0.2 mg/ml). The trypan blue exclusion test assessed cell viability after isolation and the amount was >90 %. The survival of acinar cells after prolonged incubation in different environments was determined by fluorescent dyes propidium iodide (0.5 mg/ml) and Hoechst 33258 (5 mg/ml) dyes. Mitochondrial membrane potential was recorded by adding the dye TMRM (50 nM). Cells were photographed using an inverted microscope Olympus IX73 with a digital camera DP-74. Cell counting and fluorescence intensity of TMRM were analyzed using ImageJ software. It was found that the presence of TLC-S in the medium at a concentration of 0.5 mmol/L, when used as an oxidation substrate for glucose (10 mmol/L), pyruvate (2 mmol/L), or alanine (2 mmol/L), did not significantly affect the proportion of live cells after 2 and 4 hours of incubation. However, when TLC-S was added to the medium at a concentration of 2 mmol/L, the proportion of live cells significantly decreased. This decrease was least pronounced during pyruvate oxidation. Under the action of TLC-S (after 25 min) in the presence of glucose alone, the mitochondrial membrane potential decreased by 9.8 % (P<0.05, n=3), in the presence of alanine (in the presence of glucose) – by 23.5 % (P<0.05, n=4). When pyruvate was added to the medium (in the presence of glucose), the mitochondrial membrane potential likely did not change significantly. These results suggest the presence of several different mechanisms of TLC-S’s negative effects on pancreatic acinar cells: at low concentrations (or at early stages), it disrupts alanine transamination reactions, leading to the disruption of mitochondrial membrane potential generation, while at high concentrations, it affects the integrity of plasma and/or intracellular membranes.

Generation of Membrane Potential by Cytochrome bd.

An overview of current notions on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase is presented in this work. It is suggested that the main contribution to Δψ formation is made by the movement of H+ across the membrane along the intra-protein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.

Investigation of the Mechanism of Membrane Potential Generation by Heme-Copper Respiratory Oxidases in a Real Time Mode.

Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze the final step of cellular respiration in eukaryotes and many prokaryotes: the transfer of electrons from cytochromes or quinols to molecular oxygen and oxygen reduction to water. The free energy released in this redox reaction is converted by heme-copper respiratory oxidases into the transmembrane gradient of the electrochemical potential of hydrogen ions H+). Heme-copper respiratory oxidases have a unique mechanism for generating H+, namely, a redox-coupled proton pump. A combination of direct electrometric method for measuring the kinetics of membrane potential generation with the methods of prestationary kinetics and site-directed mutagenesis in the studies of heme-copper oxidases allows to obtain a unique information on the translocation of protons inside the proteins in real time. The review summarizes the data of studies employing time-resolved electrometry to decipher the mechanisms of functioning of these important bioenergetic enzymes.

The Integral Role of Chloride & With-No-Lysine Kinases in Cell Volume Regulation & Hypertension.

Chloride anions are the most abundant in humans. For many years, it has been believed that chloride is simply a counterion of all other cations, ensuring the electroneutrality of the extracellular space. Recent data suggests that chloride anions possess a broad spectrum of important activities that regulate vital cellular functions. It is now evident that, apart from its contribution to the electroneutrality of the extracellular space, it acts as an osmole and contributes to extracellular and intracellular volume regulation. Its anionic charge also contributes to the generation of cell membrane potential. The most interesting action of chloride anions is their ability to regulate the activity of with-no-lysine kinases, which in turn regulate the activity of sodium chloride and potassium chloride cotransporters and govern the reabsorption of salt and excretion of potassium by nephron epithelia. Chloride anions seem to play a crucial role in cell functions, such as cell volume regulation, sodium reabsorption in the distal nephron, potassium balance, and sodium sensitivity, which lead to hypertension. All of these functions are accomplished on a molecular level via complicated metabolic pathways, many of which remain poorly defined. We attempted to elucidate some of these pathways in light of recent advances in our knowledge, obtained mainly from experimental studies.

STF-083010 an inhibitor of IRE1α endonuclease activity affects mitochondrial respiration and generation of mitochondrial membrane potential

STF-083010 is an inhibitor of endonuclease activity of inositol requiring-enzyme 1α (IRE1α) that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. STF-083010 was tested as a possible antitumor agent in some previous studies exhibiting the ability either to induce death of tumour cells or to increase sensitivity of tumours cells to other neoplastic agents. STF-083010 exhibits also hepatoprotective effects in different models of liver injury and hepatic steatohepatitis. We have shown that STF-083010 has significant impact on mitochondrial functions that is not dependent on the way of STF-083010 application. We have observed that STF-083010 decrease of both maximal respiration (representing maximal electron transfer capacity of mitochondrial respiratory chain) and spare respiratory capacity after either incubation of the SH-SY5Y cells with STF-083010 or direct addition of STF-083010 to the respiration medium. In addition, we have documented impact of STF-083010 on generation of mitochondrial membrane potential (ΔΨm) that could be a result of decreased mitochondrial substrate level phosphorylation. Finally, increased sensitivity of ΔΨm to uncoupler in the presence of STF-083010 was documented. Our results indicate that STF-083010 has important impact on mitochondrial functions independently of its ability to inhibit endonuclease activity of IRE1α that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. The impact of STF-083010 on mitochondrial functions could be associated with its possible off-target effect.