Maintaining Membrane Potential Research Articles

Dissecting structure and function of the monovalent cation/H+ antiporters Mdm38 and Ylh47 in Saccharomyces cerevisiae.

The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H+ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca2+/H+ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca2+ but instead are selective for K+ and Na+. Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.

Mitochondria as determinants of reproductive senescence and competence: implications for diagnosis of embryo competence in assisted reproduction.

Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future.

The anti-apoptotic role of Ginkgolide B via mitochondrial permeability transition pore inhibition in retinal ischemia-reperfusion

This research delves into the effectiveness of Ginkgolide B (GB), a compound from Ginkgo biloba, in combating cell death caused by glaucoma, with a focus on mitochondrial impairment and the mitochondrial permeability transition pore (mPTP). Utilizing models of high intraocular pressure and in vitro glaucoma simulations, the study investigates GB's impact on retinal progenitor cells (RPCs) under oxygen-glucose deprivation/reperfusion (OGD/R) and in a rat glaucoma model. The study methodologies included apoptosis assessment, apoptotic marker analysis via Western blot, and mitochondrial structure and function evaluation. The findings reveal that GB notably decreases apoptosis in RPCs exposed to OGD/R in vitro, and reduces ischemia-reperfusion damage in vivo. GB's protective role is attributed to its ability to preserve mitochondrial integrity, maintain membrane potential, regulate calcium levels, and inhibit mPTP opening. These results underscore GB's potential as a therapeutic agent for acute primary angle-closure glaucoma, highlighting its capability to alleviate mitochondrial damage and apoptosis in RPCs and retinal nerve fiber layer cells.

Mitochondria-derived vesicles: potential nano-batteries to recharge the cellular powerhouse.

Mitochondria dysfunction is increasingly recognized as a critical factor in various pathogenic processes. The mechanism governing mitochondrial quality control serves as an adaptive response, ensuring the preservation of mitochondrial morphology, quantity, and overall function, crucial for cell survival. The generation of mitochondria-derived vesicles (MDVs) is one of the processes of mitochondrial quality control. Recent literature has suggested MDV heterogeneity; however, the detailed characteristics of various MDV subtypes still need to be studied better. Recent studies have shown that MDVs also play a role in inter-organelle communication for mitochondria besides quality control. For instance, Hazan et al. demonstrated that functional mitochondria from Saccharomyces cerevisiae release vesicles independent of the fission machinery. These vesicles, falling within the typical size range of MDVs, were selectively loaded with mitochondrial proteins, especially with functional ATP synthase subunits. Intriguingly, these MDVs maintained membrane potential and could generate ATP. Moreover, MDVs could fuse with naïve mitochondria, transferring their ATP generation machinery. Lastly, this study revealed a potential delivery mechanism of ATP-producing vesicles, presenting a promising avenue to rejuvenate ATP-deficient mitochondria. Overall, this study unveils a novel mechanism for inter-organelle communication by vesicles, which is crucial for maintaining cellular homeostasis and could also be important in pathological conditions.

Discovery and in vivo evaluation of novel inhibitors of the hydrolytic activity of mitochondrial F1Fo ATP synthase to alleviate myocardial ischemia reperfusion injury

Abstract Background F1Fo ATP-synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyze ATP leading to energetic deficit and cardiac injury. We have previously discovered via in-silico virtual screening selective hydrolase inhibitors with pyrazole-pyridine scaffold and verified their inhibitory effect on hydrolysis on isolated murine mitochondria. Purpose In the present work we aimed to 1) synthesize novel hydrolase inhibitors, 2) select the best candidates to evaluate their effect against myocardial ischemia (I) reperfusion (R) injury in vivo and 3) investigate the underlying molecular mechanisms of cardioprotection. Methods The inhibitory effect on ATP hydrolysis for all compounds was evaluated on isolated murine heart mitochondria and at a cellular level on H9C2 cells in the presence of rotenone where their ability to maintain membrane potential was monitored. The previously described ATP hydrolase inhibitor BTB06584 (BTB) was used in vitro and in vivo as a reference and three novel compounds were selected for in vivo evaluation namely 1117, 1119 and STK-71. In the first cohort, C57Bl6 mice were randomized into 5 groups that received intravenously: 1) vehicle, 2) 1117, 3)1119, 4) STK-71 and 5) BTB. The administration was performed 10 min prior to 30 min of I and 2 hours of R and infarct size (IS) was determined. The dose of each compound was based on the effective concentration needed at a cellular level to inhibit the ATP hydrolysis. In a second cohort of mice, the best candidate and vehicle were given 10 min after the induction of I and IS was measured to test the intervention in a more translational setting. Finally, in the third cohort, the cardioprotective mechanism was investigated at the 10th min of R. Results We discovered three candidates that inhibit the ATP hydrolytic activity with comparable IC50 values to BTB. These three compounds inhibited ATP hydrolysis on the H9C2 cells at 50μΜ while BTB was inactive at the same concentration. In vivo, the three novel inhibitors reduced IS compared to the control group with STK-71 having the most remarkable cardioprotective effect while BTB did not result in attenuation of IS. STK-71 when given after the induction of I, also reduced IS indicating that its cardioprotective effect is pertained in a translational protocol. Regarding the mechanism of cardioprotection, STK-71 increased T173 phosphorylation and activation of PKA which led to the downstream activation of phospholamban. STK-71 also led to the significant activation of the RISK pathway, increased the expression of the antiapoptotic protein Bcl-Xl and reduced apoptosis. Conclusion We discovered a novel pyrazolopyridine derivative that acts as an inhibitor of ATP hydrolysis and reduces myocardial IS in vivo. Its cardioprotective mechanism involves the activation of PKA signaling and alleviation of apoptosis.

Ion channels and transporters regulate nutrient absorption in health and disease.

Ion channels and transporters are ubiquitously expressed on cell membrane, which involve in a plethora of physiological process such as contraction, neurotransmission, secretion and so on. Ion channels and transporters is of great importance to maintaining membrane potential homeostasis, which is essential to absorption of nutrients in gastrointestinal tract. Most of nutrients are electrogenic and require ion channels and transporters to absorb. This review summarizes the latest research on the role of ion channels and transporters in regulating nutrient uptake such as K+ channels, Ca2+ channels and ion exchangers. Revealing the mechanism of ion channels and transporters associated with nutrient uptake will be helpful to provide new methods to diagnosis and find potential targets for diseases like diabetes, inflammatory bowel diseases, etc. Even though some of study still remain ambiguous and in early stage, we believe that ion channels and transporters will be novel therapeutic targets in the future.

Ena Proteins Respond to PacC-Mediated pH Signaling Pathway and Play a Crucial Role in Patulin Biosynthesis.

Penicillium expansum is a main producer of patulin that causes severe postharvest decay and food safety issues in the fruit industry. Development, pathogenicity, and patulin production of P. expansum are strongly influenced by the PacC-pH signaling pathway. Global transcription factor PacC regulates various fungal biological processes through a complicated molecular network. In the present study, three Ena family genes (PeEnas), PeEnaA, PeEnaB, and PeEnaC, as important downstream targets of PePacC, were identified in P. expansum. Deletion of PeEnaA, PeEnaB, and PeEnaC showed little effect on mycelial growth under alkaline or high salinity conditions, but double and triple deletion of these genes impaired the virulence of P. expansum on apple fruit. Notably, patulin biosynthesis of P. expansum was distinctly inhibited in the deletion mutants of PeEnas. PeEnas regulated expressions of the patulin gene cluster, AP1, CreA, Sge1, and Hog1 at the transcriptional level and played roles in maintaining membrane potential. Overexpression of PeEnaC in ΔPePacC restored the patulin production defect of ΔPePacC. Our results indicated that, as downstream targets of PePacC, the PeEna family proteins play a crucial role in patulin biosynthesis in P. expansum.

Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M.tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M.tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M.tuberculosis survival in hypoxic lung granulomas.

Study of the effect of protein synthesis inhibitors on growing <i>Escherichia coli</i> bacteria using electrochemical sensors

Background. The study of the mechanisms of action of antibiotics requires the integrated use of traditional microbiological and physicochemical methods. The aim. To study the response of Escherichia coli bacteria to the action of four antibiotics, inhibitors of protein synthesis, using combined approach. Methods. Bacteria were grown under aerobic conditions on minimal M9 medium with glucose. Tetracycline, kanamycin, streptomycin and chloramphenicol have been tested. The effect of antibiotics on survival (CFU) and growth rate was determined. Respiratory activity, sulfide production, extracellular potassium, as well as pH and Eh of the medium were measured using electrochemical sensors directly in a growing culture in the “real time”, membrane potential was measured using a DiBAC dye and a Leica DM2000 fluorescent microscope. Results. The tested antibiotics were divided into two groups according to their properties. Tetracycline and chloramphenicol showed a pronounced bacteriostatic effect, growth inhibition began immediately after the addition of antibiotics and occurred at a high rate. Both antibiotics inhibited respiration, stimulated sulfide production and an Eh jump. Bacteria treated with tetracycline and chloramphenicol retained the ability to maintain membrane potential and intracellular potassium better. Inhibition of respiration led to a decrease in glucose catabolism, as evidenced by a lower rate of acidification of the medium compared to the control. Growth inhibition with streptomycin and kanamycin was initiated with a 30-minute delay. Both antibiotics showed a bactericidal effect, did not stimulate sulfide production and Eh jump, did not inhibit respiration, but caused a drop in membrane potential and intracellular potassium. High respiratory activity promoted glucose catabolism, as evidenced by the rapid acidification of the medium. Of interest is the detection of kanamycin-induced sulfide production during E. coli growth on MOPS medium. Conclusion. An analysis of the data obtained indicates that the use of electrochemical sensors in combination with traditional methods is a promising approach to studying the mechanisms of action of antibiotics.

Bioinformatics approaches for classification and investigation of the evolution of the Na/K-ATPase alpha-subunit

BackgroundNa,K-ATPase is a key protein in maintaining membrane potential that has numerous additional cellular functions. Its catalytic subunit (α), found in a wide range of organisms from prokaryotes to complex eukaryote. Several studies have been done to identify the functions as well as determining the evolutionary relationships of the α-subunit. However, a survey of a larger collection of protein sequences according to sequences similarity and their attributes is very important in revealing deeper evolutionary relationships and identifying specific amino acid differences among evolutionary groups that may have a functional role.ResultsIn this study, 753 protein sequences using phylogenetic tree classification resulted in four groups: prokaryotes (I), fungi and various kinds of Protista and some invertebrates (II), the main group of invertebrates (III), and vertebrates (IV) that was consisted with species tree. The percent of sequences that acquired a specific motif for the α/β subunit assembly increased from group I to group IV. The vertebrate sequences were divided into four groups according to isoforms with each group conforming to the evolutionary path of vertebrates from fish to tetrapods. Data mining was used to identify the most effective attributes in classification of sequences. Using 1252 attributes extracted from the sequences, the decision tree classified them in five groups: Protista, prokaryotes, fungi, invertebrates and vertebrates. Also, vertebrates were divided into four subgroups (isoforms). Generally, the count of different dipeptides and amino acid ratios were the most significant attributes for grouping. Using alignment of sequences identified the effective position of the respective dipeptides in the separation of the groups. So that 208GC is apparently involved in the separation of vertebrates from the four other organism groups, and 41DH, 431FK, and 451KC were involved in separation vertebrate isoform types.ConclusionThe application of phylogenetic and decision tree analysis for Na,K-ATPase, provides a better understanding of the evolutionary changes according to the amino acid sequence and its related properties that could lead to the identification of effective attributes in the separation of sequences in different groups of phylogenetic tree. In this study, key evolution-related dipeptides are identified which can guide future experimental studies.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

Inward rectifier potassium (Kir) channels in the retina: living our vision.

Channel proteins are vital for conducting ions throughout the body and are especially relevant to retina physiology. Inward rectifier potassium (Kir) channels are a class of K+ channels responsible for maintaining membrane potential and extracellular K+ concentrations. Studies of the KCNJ gene (that encodes Kir protein) expression identified the presence of all of the subclasses (Kir 1-7) of Kir channels in the retina or retinal-pigmented epithelium (RPE). However, functional studies have established the involvement of the Kir4.1 homotetramer and Kir4.1/5.1 heterotetramer in Müller glial cells, Kir2.1 in bipolar cells, and Kir7.1 in the RPE cell physiology. Here, we propose the potential roles of Kir channels in the retina based on the physiological contributions to the brain, pancreatic, and cardiac tissue functions. There are several open questions regarding the expressed KCNJ genes in the retina and RPE. For example, why does not the Kir channel subtype gene expression correspond with protein expression? Catching up with multiomics or functional "omics" approaches might shed light on posttranscriptional changes that might influence Kir subunit mRNA translation within the retina that guides our vision.

Contribution of Neuronal and Glial Two-Pore-Domain Potassium Channels in Health and Neurological Disorders.

Two-pore-domain potassium (K2P) channels are widespread in the nervous system and play a critical role in maintaining membrane potential in neurons and glia. They have been implicated in many stress-relevant neurological disorders, including pain, sleep disorder, epilepsy, ischemia, and depression. K2P channels give rise to leaky K+ currents, which stabilize cellular membrane potential and regulate cellular excitability. A range of natural and chemical effectors, including temperature, pressure, pH, phospholipids, and intracellular signaling molecules, substantially modulate the activity of K2P channels. In this review, we summarize the contribution of K2P channels to neuronal excitability and to potassium homeostasis in glia. We describe recently discovered functions of K2P channels in glia, such as astrocytic passive conductance and glutamate release, microglial surveillance, and myelin generation by oligodendrocytes. We also discuss the potential role of glial K2P channels in neurological disorders. In the end, we discuss current limitations in K2P channel researches and suggest directions for future studies.

Side‐Chain Polarity of Amino Acids within the Kir7.1 Channel Pore Lining Determine Permeability and Function

Background Inwardly rectifying potassium (K+) channels (Kir) maintain membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene encoding for Kir7.1 protein, in the retinal pigmented epithelium (RPE), cause pediatric blindness. One point mutation in the KCNJ13 gene c.458C>T Threonine (T) to Isoleucine (I) at amino acid position 153 lines the inner pore of the tetrameric protein. We sought to elucidate the impact of pore region amino-acid hydrophobicity and side-chain length on the channel function. Hypothesis Amino acid side-chain polarity and length at position T153 are important determinants of Kir7.1 channel structure/function. Methods HEK293 cells in culture were transfected with GFP tagged wildtype (WT) Kir7.1, T153I, T153G, T153A, T153L, T153C, or T153S plasmid DNA. Live-cell imaging, using the Nikon-C2 confocal system, mapped the expression of GFP fused protein. Hoescht and WGA-Alexa 594 labeled the nucleus and plasma membrane, respectively. Off-line image analysis was performed with Nikon NIS Elements. Whole-cell patch-clamp electrophysiology with either extracellular Ringer's or Rb+ determined channel function. Extracellular K+-gradient was used to determine chord conductance. Data analyzed using the Clampfit program. Results Live-cell imaging indicated that Kir7.1 WT and all mutants studied are trafficked to the membrane. The IV plot for the Kir7.1 WT showed inward current measured at -150 mV with a mean amplitude of -863.67 ± 142.44 pA (n = 8) compared to Ile -68.54 ± 10.49 pA (n = 9, P = 2.79x10-5). Extracellular K+ dependent chord conductance further confirmed that T153I is nonfunctional. The current amplitude rank order for the mutant channel K+ permeability was Cys > WT > Ser > Gly > Ile > Ala> Leu. The membrane potential, measured as zero-current potential, was -57.75 ± 3.23 mV (n = 8) for the WT compared to-5.46 ± 6.01 mV (n = 9, P = 2.27x10-6) for the disease mutant with a rank order of Cys > WT > Ser > Leu > Gly > Ile > Ala. Rb+ ion, selectively permeates through Kir7.1 channel with a Rb+-current fold-change of7.28 ± 1.63 (n = 8) for WT versus 6.48 ± 2.78 (n = 9, P=0.81) for the disease mutant. The rank order of Rb+-current fold-change was Leu > Ser > WT > Ile > Cys > Gly > Ala. Conclusion Even though all the mutant channels traffick normally to the membrane, T153I was non-functional as determined by K+ ion permeability. Upon testing a more permeable ion, Rb+, it was deemed that T153I is actually dysfunctional. Based on the rank order, a substitution of polar amino acid at position 153 is required for Kir7.1 channel function. Permeability of all the mutant channels except T153A to Rb+ indicates that channel function is dependent on size of the cation. Since aa position T153 is within the inner pore-lining of the channel, a hydrophobic aa might alter protein-lipid interaction to affect channel function. Additionally, short-chain amino acids may further alter protein-protein interactions within the channel.

The voltage-gated potassium channel KV1.3 regulates neutrophil recruitment during inflammation.

AimsNeutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling.Methods and resultsUsing in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3.ConclusionWe show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.

Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase

Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.

Reappraisal of anoxic spreading depolarization as a terminal event during oxygen\u2013glucose deprivation in brain slices in vitro

Anoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen–glucose deprivation (OGD) in submerged cortical slices in vitro. However, mechanical artifacts caused by aSD-triggered edema may introduce error in the assessment of neuronal viability. Here, using continuous patch-clamp recordings from submerged rat cortical slices, we first confirmed that vast majority of L4 neurons permanently lost their membrane potential during OGD-induced aSD. In some recordings, spontaneous transition from whole-cell to out-side out configuration occurred during or after aSD, and only a small fraction of neurons survived aSD with reperfusion started shortly after aSD. Secondly, to minimize artifacts caused by OGD-induced edema, cells were short-term patched following OGD episodes of various duration. Nearly half of L4 cells maintained membrane potential and showed the ability to spike-fire if reperfusion started less than 10 min after aSD. The probability of finding live neurons progressively decreased at longer reperfusion delays at a rate of about 2% per minute. We also found that neurons in L2/3 show nearly threefold higher resistance to OGD than neurons in L4. Our results suggest that in the OGD ischemia model, aSD is not a terminal event, and that the “commitment point” of irreversible damage occurs at variable delays, in the range of tens of minutes, after OGD-induced aSD in submerged cortical slices.

Hydrolytic activity of mitochondrial F1Fo ATP synthase as a target for myocardial ischemia: discovery and in vitro evaluation of novel inhibitors

Abstract Background/Introduction F1Fo ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyse ATP leading to cellular energetic deficit, contracture and lethal cardiomyocyte injury. Therefore, inhibition of ATP hydrolase is of major significance and the discovery of selective hydrolase inhibitors could be of particular interest in terms of cardiac response to ischemia. Purpose We performed an in vitro screening in order to identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase. Methods Initially, we identified inhibitors of the hydrolytic activity of ATP synthase using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase of mus musculus that was modelled for the first time. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on our in-house library of 2000 compounds, Pharmalab, plus 266,151 compounds of the National Cancer Institute (NCI) database. The best candidates were evaluated in vitro on isolated murine heart mitochondria to verify their inhibitory effect on ATP synthase hydrolytic activity. Moreover, we confirmed their action at a cellular level. H9C2 cells were treated with the three best candidates in the presence of rotenone and their ability to maintain membrane potential was monitored. The experiment was repeated in absence of rotenone in order to detect toxic or non-specific effects (n=5 independent experiments). Finally, the compounds were tested using an assay in which primary adult rat ventricular cardiomyocytes (ARVCs) were challenged with the mitochondrial uncoupler CCCP to induce reverse ATPase activity, and time until contracture was determined. For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls. Results Different steps of filtering through the virtual screening provided 38 molecules from Pharmalab and 15 candidates form NCI database. From the 53 candidates in total, five compounds displayed in vitro inhibitory activity at 200μM on isolated mitochondria and their IC50 values were determined. Among them, three synthetic derivatives possessing a central pyrazolopyridine core displayed the best IC50 values (81.7±1.3 μM, 99.8±1.2 μM, 144.8±1.3μM) and were evaluated on the H9C2 cells. They exhibited significant inhibitory activity at 50μM (p<0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the final novel inhibitors significantly extended (p<0.001) the time until shortening of the ARVCs. Conclusion We have discovered two novel agents that act as selective inhibitors of ATP hydrolase and can be tested against myocardial ischemia. Funding Acknowledgement Type of funding source: None

Potassium channels on smooth muscle as a molecular target for plant-derived Resveratrol

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a phytoalexin present in a variety of plant species. Resveratrol has a wide spectrum of pharmacologic properties, and it exhibits versatile biological effects on different human and animal models. The studies have shown that potassium (K) channels can be potential targets in the mechanism of resveratrol action. K channels play a crucial role in maintaining membrane potential. Inhibition of K channels causes membrane depolarization and then contraction of smooth muscles, while the activation leads to membrane hyperpolarization and subsequently, relaxation. Five diverse types of K channels have been identified in smooth muscle cells in different tissue: ATP-sensitive K channels (KATP), voltage-dependent K channels (Kv), Ca2+ - and voltage-dependent K channels (BKCa), inward rectifier K channels (Kir), and tandem two-pore K channels (K2P). The expression and activity of K channels altered in many types of diseases. Aberrant function or expression of K channels can be underlying in pathologies such as cardiac arrhythmia, diabetes mellitus, hypertension, preterm birth, preeclampsia, and various types of cancer. Modulation of K channel activity by molecular approaches and selective drug development may be a novel treatment modality for these dysfunctions in the future. The plant-derived non-toxic polyphenols, such as resveratrol, can alter K channel activity and lead to the desired outcome. This review presents the basic properties, physiological, pathophysiological functions of K channels, and pharmacological roles of resveratrol on the major types of K channels that have been determined in smooth muscle cells.