Membrane Potential Of Cells Research Articles

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Studies on Membrane Potential and Ca2+ Oscillations in Rat Carotid Body Type I Cells

Carotid body (CB) glomus cells sense changes in arterial O2 pressure (PO2) and pH, and adjust the secretory and carotid sinus nerve activity and ventilation to help maintain normal levels of blood PO2 and pH. Our recent studies showed that isolated CB cell clusters perfused with physiological buffer solution generate spontaneous Ca2+ oscillations at low frequency in normoxia. Mild and moderate levels of hypoxia (2-5%O2) and acidosis (pHo7.2-7.3) increased the frequency and amplitude of Ca2+ oscillations. Inhibitors of voltage-dependent Ca2+ channels and removal of external Ca2+ abolished Ca2+ oscillations, indicating that Ca2+ influx was critical for generating Ca2+ oscillations. To better understand the phenomenon of Ca2+oscillations in glomus cells, we examined the potential role of oscillations in cell membrane potential (Em) in CB cells in triggering Ca2+ influx and Ca2+ oscillations. Using the cell-attached patch configuration, we assessed cell Em by recording TASK single channels, because a linear relationship exists between TASK amplitude and cell Em. Recording of TASK in CB cells in normoxia showed that many CB cells exhibit oscillations in TASK amplitude, indicating the presence of spontaneous oscillations in cell Em. The oscillation frequency of cell Em was similar to that of Ca2+ oscillations. Oscillations in TASK single channel amplitude were blocked by nifedipine (Ca2+ channel antagonist), by removal of extracellular Ca2+, and by 2-APB (an inhibitor of ER Ca2+ channel and store-operated Ca2+ entry). Mild hypoxia increased the frequency of oscillations of TASK amplitude, indicating that mild hypoxia increased the frequency of cell Em oscillations. These findings suggest that cell Em oscillations (that open voltage-dependent Ca2+ channels and increase Ca2+ influx) are an integral component of the cellular signaling mechanism by which Ca2+ oscillations are produced in CB cells. This work was funded by National Institutes of Health (NIH) grants to D.K. (HL111497) and C.W. (HL142906), and an award from Rosalind Franklin University of Medicine and Science. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pulmonary Fibrosis Augments CGRP-Dependent Hyperpolarization and Vasodilation of Mouse Pulmonary Arteries

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. PF was induced by intratracheal delivery of bleomycin (3 wk), and saline was used as a control (sham). Pulmonary arteries (PAs; 100-150 μm diameter) from male C57BL/6J mice (age, ~5 mo), were cannulated, pressurized to 16 cmH2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was similar for SMCs (bleomycin = -43±3 mV, sham = -44±3 mV; n = 5/group) and ECs (bleomycin = -45±4 mV, sham = -44±3 mV; n=5/group). Increasing concentrations of CGRP (10−10-10−6 M) evoked hyperpolarization of Vm with greater potency in SMCs (EC50: bleomycin = 2 nM, sham = 15 nM) and ECs (EC50: bleomycin = 3 nM, sham = 8 nM) from PF mice, without changing effcacy (SMCs: bleomycin ΔVm = -24±4 mV, sham ΔVm = -21±3 mV; ECs: bleomycin ΔVm = -26±2 mV, sham ΔVm=23±4 mV). Consistently, vasodilation to CGRP in PAs preconstricted with uridine triphosphate (UTP, 5 μM; EC50) was enhanced in PF (92±6% maximum dilation;) vs. sham (83±4% maximum dilation) mice (n=5/group). Greater vasodilation and hyperpolarization of SMCs to CGRP persisted in endothelium-disrupted PAs and during treatment with L-NAME (10−4 M) indicating that ECs are not required for greater responsiveness to CGRP in SMCs. With no effect on resting Vm, inhibition of KATP channels with 1 μM glibenclamide or PKA with protein kinase inhibitor [PKI-(14-22)-amide, myristoylated; 1 μM] significantly attenuated (p<0.05) hyperpolarization of SMCs and ECs (ΔVm ≤5 mV for all groups). Both glibenclamide and PKI attenuated vasodilation to CGRP in PAs and eliminated differences between groups. In a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity. AHA CDA#931652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integrating single-cell and multi-omic approaches reveals Euphorbiae Humifusae Herba-dependent mitochondrial dysfunction in non-small-cell lung cancer.

Euphorbiae Humifusae Herba (EHH) is a pivotal therapeutic agent with diverse pharmacological effects. However, a substantial gap exists in understanding its pharmacological properties and anti-tumour mechanisms. This study aimed to address this gap by exploring EHH's pharmacological properties, identifying NSCLC therapy-associated protein targets, and elucidating how EHH induces mitochondrial disruption in NSCLC cells, offering insights into novel NSCLC treatment strategies. String database was utilized to explore protein-protein interactions. Subsequently, single-cell analysis and multi-omics further unveiled the impact of EHH-targeted genes on the immune microenvironment of NSCLC, as well as their influence on immunotherapeutic responses. Finally, both invivo and invitro experiments elucidated the anti-tumour mechanisms of EHH, specifically through the assessment of mitochondrial ROS levels and alterations in mitochondrial membrane potential. EHH exerts its influence through engagement with a cluster of 10 genes, including the apoptotic gene CASP3. This regulatory impact on the immune milieu within NSCLC holds promise as an indicator for predicting responses to immunotherapy. Besides, EHH demonstrated the capability to induce mitochondrial ROS generation and perturbations in mitochondrial membrane potential in NSCLC cells, ultimately leading to mitochondrial dysfunction and consequent apoptosis of tumour cells. EHH induces mitochondrial disruption in NSCLC cells, leading to cell apoptosis to inhibit the progress of NSCLC.

Novel in vivo optogenetic tools reveal autonomous pacemaker control of renal hemodynamics by macula densa cells

Macula densa (MD) cells, the tubular component of the juxtaglomerular apparatus (JGA) are generally considered as sensor cells detecting tubular salt (NaCl) concentration and based on this information provide feedback control of glomerular hemodynamics (tubuloglomerular feedback, TGF). However, the neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the paradigm-shifting hypothesis that rather than being simply the sensory arm of TGF, MD cells function as the nephron’s autonomous pacemaker and the primary driver and oscillator of glomerular hemodynamics. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-GT mice that selectively express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in MD cells. Optogenetic MD stimulation was induced in vivo by blue light (470nm) exposure of MD-Ai27 mouse kidneys that express the depolarizing channelrhodopsin ChR2 specifically in MD cells. Conversely, MD inhibition was induced by yellow light (560nm) exposure of MD-Ai39 mouse kidneys that selectively express the hyperpolarizing halorhodopsin NpHR in MD cells. Cell membrane expression of ChR2 (in MD-Ai27 mice) and NpHR (in MD-Ai39 mice) only in the MD among all renal cell types was confirmed using immunohistochemistry. Pacemaker-like regular Ca2+ oscillations (4-fold elevations in baseline Ca2+ and average frequency of 0.03 Hz) and their cell-to-cell propagation within the MD plaque via long cell processes were observed in MD-GT mice. These Ca2+ oscillations were MD cell autonomous, as they were preserved in freshly dissected single MD cells and in the in vitro dissected and microperfused JGA, and were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high MD expression of genes related to pacemaker function, cell membrane depolarization, voltage-dependent ion channels, Ca2+ signaling and synaptic transmission that was validated by immunohistochemistry. Blue light exposure of single glomeruli/JGAs using ROI scan in MD-Ai27 mouse kidneys that previously underwent superficial corticotomy (open loop nephrons) acutely and reversibly increased capillary and afferent (AA)/efferent arteriole (EA) blood flow by approx. 50% in the light-exposed glomerulus but not in adjacent glomeruli. In contrast, yellow light exposure of single glomeruli/JGAs in MD-Ai39 mouse kidneys acutely and reversibly decreased blood flow but increased glomerular capillary diameter (suggesting preferential EA vs AA vasoconstriction) in the exposed but not in adjacent single nephrons. In addition, yellow light stimulation of single glomeruli in MD-Ai39 mouse kidneys augmented the spontaneous glomerular blood flow oscillations in these open-loop nephrons (frequency of 0.03 Hz), but not in adjacent control nephrons. Altogether, these studies identified MD cells as the nephron’s central pacemaker and oscillator of single nephron blood flow, and emphasized the importance of MD cell membrane potential and Ca2+-dependent vasodilator release as a major driver of nephron function. DK064234 DK123564 DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial Membrane Potential Changes are Required for Propagated Vasodilation

Introduction: The coordination of blood flow in response to localized increases in metabolic activity is vital for tissue function. This adaptive response typically involves the release of vasoactive factors leading to local vasodilation. This local vasodilation must propagate upstream to proximal feed arterioles and arteries to ensure a coordinated increase in blood flow and adequate tissue perfusion. But the mechanisms underlying such propagated vasodilation remain to be fully understood. This study aims to identify the conduction pathway(s) that facilitate this response. The hypothesis is that endothelial membrane potential changes are required for propagated vasodilation to occur. Methods: To dissect the pathways involved in propagated vasodilation, we employed micropuffng techniques to locally apply vasoactive agents to precontracted rat 2nd order mesenteric arteries. Using a 50um tip micropipette activators were applied against a flowing perfusate, to limit the action of the drug, and the precise site of activators determined by co-application of the fluorophore sulforhodamine. Vasodilator responses were recorded at the site of application and at intervals upstream from the application site. Results: Local application of the muscarinic receptor agonist, acetylcholine (ACh), evoked robust vasodilator responses that propagated upstream from the site of application. The propagation of vasodilation terminated at the site of removal of a 100uM wide strip of endothelial cells. A high-potassium solution, which induces depolarization and ‘clamps’ the membrane potential, also inhibited propagated vasodilation. In contrast, the nitric oxide donor, sodium nitroprusside did not initiate propagated vasodilation in arteries with an intact endothelium. Conclusion: Collectively, these results show that the endothelium serves as the primary signalling pathways for propagated vasodilation. Furthermore, our results highlight the necessity of endothelial cell membrane potential change in the initiation of this vasodilator signal. These insights reinforce the endothelium’s pivotal role in coordinating vascular function. British Heart Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondria control IP3-evoked Ca2+ release triggered by voltage-dependent Ca2+ entry in intact resistance arteries

The contractility of vascular smooth muscle cells (VSMCs) is a major contributor of vascular tone and blood pressure. In VSMCs, intracellular Ca2+ level determines the contractile activity that generates the force to contract arteries. A major source of Ca2+ is via influx through voltage dependent Ca2+ channels (VDCCs). While mitochondria are now recognised as key regulators of intracellular Ca2+ homeostasis in several cell types, their role in modulating Ca2+ signalling mediated by VDCC in VSMCs is unresolved. Given the potential physiological significance, and unclarified interaction between mitochondria and VDCCs in VSMCs, we hypothesize that mitochondria directly regulate Ca2+ signalling mediated by VDCCs. The interplay between mitochondria and VDCCs was investigated by imaging and analysing intracellular Ca2+ signals in smooth muscle cells in intact arteries from rat mesentery. Depolarization of the plasma membrane potential, by high potassium (20 mM) physiological saline solution, triggered Ca2+ influx through VDCCs and a sustained increase in intracellular Ca2+ on which repetitive Ca2+ oscillations occurred. All Ca2+ signals were abolished by removal of external Ca2+ and by dihydropyridine inhibitors of VDCCs. Significantly, the repetitive Ca2+ oscillations, but not the sustained Ca2+ signals, were blocked by the IP3 receptor inhibitor 2-APB and SERCA inhibitor cyclopiazonic acid. Neither the repetitive Ca2+ oscillations or the sustained Ca2+ signals were altered by the ryanodine receptor inhibitors ryanodine and dantrolene. These results suggest that Ca2+ influx via VDCC triggers Ca2+-induced Ca2+ release at IP3 receptors in intact mesenteric arteries. Depolarization of the mitochondrial membrane potential (ψm), with the uncoupler CCCP or complex I inhibitor rotenone, but not ATP deprivation with the ATP synthesis blocker oligomycin, inhibited VDCC evoked IP3 mediated Ca2+ oscillations but not the sustained Ca2+ signals. Furthermore, in intact arteries, depolarization of ψm directly suppressed inositol triphosphate receptors (IP3Rs) mediated Ca2+ release from the internal Ca2+ store evoked by photolysis of caged IP3. These results suggest that mitochondria regulate Ca2+ release via IP3R triggered by Ca2+ entry via VDCC. In return, Ca2+ entry via VDCCs did not alter ψm, but upregulated ROS production. Together, these results suggest that mitochondria regulate Ca2+-induced Ca2+ release at IP3 receptors triggered by Ca2+ influx via VDCCs but do not directly regulate VDCC activity. British Heart Foundation (RG/F/20/110007; PG/20/9/34859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Discovery of Urea Derivatives of Celastrol as Selective Peroxiredoxin 1 Inhibitors against Colorectal Cancer Cells.

Peroxiredoxin (PRDX1) is a tumor-overexpressed antioxidant enzyme for eliminating excessive reactive oxygen species (ROS) to protect tumor cells from oxidative damage. Herein, a series of celastrol urea derivatives were developed based on its cocrystal structure with PRDX1, with the aim of pursuing a PRDX1-specific inhibitor. Among them, derivative 15 displayed potent anti-PRDX1 activity (IC50 = 0.35 μM) and antiproliferative potency against colon cancer cells. It covalently bound to Cys-173 of PRDX1 (KD = 0.37 μM), which was secured by the cocrystal structure of PRDX1 with an analogue of 15 while exhibiting weak inhibitory effects on PRDX2-PRDX6 (IC50 > 50 μM), indicating excellent PRDX1 selectivity. Treatment with 15 dose-dependently decreased the mitochondria membrane potential of SW620 cells, probably due to ROS induced by PRDX1 inhibition, leading to cell apoptosis. In colorectal cancer cell xenograft model, it displayed potent antitumor efficacy with superior safety to celastrol. Collectively, 15 represents a promising PRDX1 selective inhibitor for the development of anticolorectal cancer agents.

Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1.

Cardiac disorders in cancer patients pose significant challenges to disease prognosis. While it has been established that these disorders are linked to cancer cells, the precise underlying mechanisms remain elusive. In this study, we investigated the impact of cancerous ascites from the rat colonic carcinoma cell line RCN9 on H9c2 cardiomyoblast cells. We found that the ascites reduced mitochondrial volume, increased oxidative stress, and decreased membrane potential in the cardiomyoblast cells, leading to apoptosis and autophagy. Although the ascites fluid contained a substantial amount of high-mobility group box-1 (HMGB1), we observed that neutralizing HMGB1 with a specific antibody mitigated the damage inflicted on myocardial cells. Our mechanistic investigations revealed that HMGB1 activated both nuclear factor κB and phosphoinositide 3-kinases-AKT signals through HMGB1 receptors, namely the receptor for advanced glycation end products and toll-like receptor-4, thereby promoting apoptosis and autophagy. In contrast, treatment with berberine (BBR) induced the expression of miR-181c-5p and miR-340-5p while suppressing HMGB1 expression in RCN9 cells. Furthermore, BBR reduced HMGB1 receptor expression in cardiomyocytes, consequently mitigating HMGB1-induced damage. We validated the myocardial protective effects of BBR in a cachectic rat model. These findings underscore the strong association between HMGB1 and cancer cachexia, highlighting BBR as a promising therapeutic agent for myocardial protection through HMGB1 suppression and modulation of the signaling system.

The Role of Ion Channels and Chemokines in Cancer Growth and Metastasis: A Proposed Mode of Action Using Peptides in Cancer Therapy.

Metastasis (Met) largely contributes to the major cause of cancer deaths throughout the world, rather than the growth of the tumor mass itself. The present report brings together several of the pertinent contributors to cancer growth and metastatic processes from an activity standpoint. Such biological activities include the following: (1) cell adherence and detachment; (2) cell-to-cell contact; (3) contact inhibition; (4) the cell interfacing with the extracellular matrix (ECM); (5) tumor cell-to-stroma communication networks; (6) chemotaxis; and (7) cell membrane potential. Moreover, additional biochemical factors that contribute to cancer growth and metastasis have been shown to comprise the following: (a) calcium levels in the extracellular matrix and in intracellular compartments; (b) cation voltage and ATP-regulated potassium channels; (c) selective and non-selective cation channels; and (d) chemokines (cytokines) and their receptors, such as CXCL12 (SDF-1) and its receptor/binding partner, CXCR4. These latter molecular components represent a promising group of an interacting and synchronized set of candidates ideal for peptide therapeutic targeting for cancer growth and metastasis. Such peptides can be obtained from naturally occurring proteins such as alpha-fetoprotein (AFP), an onco-fetal protein and clinical biomarker.

The Impact of Potassium Dynamics on Cardiomyocyte Beating in Hemodialysis Treatment.

Background: Observational studies of intermittent hemodialysis therapy have reported that the excess decrease in K+ concentration in plasma (KP) during treatment is associated with the destabilization of cardiac function. Elucidating the mechanism by which the decrease in KP impairs myocardial excitation is indispensable for a deeper understanding of prescription design. Methods: In this study, by using an electrophysiological mathematical model, we investigated the relationship between KP dynamics and cardiomyocyte excitability for the first time. Results: The excess decrease in KP during treatment destabilized cardiomyocyte excitability through the following events: (1) a decrease in KP led to the prolongation of the depolarization phase of ventricular cells due to the reduced potassium efflux rate of the Kr channel, temporarily enhancing contraction force; (2) an excess decrease in KP activated the transport of K+ and Na+ through the funny channel in sinoatrial nodal cells, disrupting automaticity; (3) the excess decrease in KP also resulted in a significant decrease in the resting membrane potential of ventricular cells, causing contractile dysfunction. Avoiding an excess decrease in KP during treatment contributed to the maintenance of cardiomyocyte excitability. Conclusions: The results of these mathematical analyses showed that it is necessary to implement personal prescription or optimal control of K+ concentration in dialysis fluid based on predialysis KP from the perspective of regulatory science in dialysis treatment.

Fusobacterium nucleatum-induced imbalance in microbiome-derived butyric acid levels promotes the occurrence and development of colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent malignant tumors globally. Recent reports suggest that Fusobacterium nucleatum (F. nucleatum) contributes to the initiation, progression, and prognosis of CRC. Butyrate, a short-chain fatty acid derived from the bacterial fermentation of soluble dietary fiber, is known to inhibit various cancers. This study is designed to explore whether F. nucleatum influences the onset and progression of CRC by impacting the intestinal metabolite butyric acid. To investigate the mechanism by which F. nucleatum affects CRC occurrence and development. Alterations in the gut microbiota of BALB/c mice were observed following the oral administration of F. nucleatum. Additionally, DLD-1 and HCT116 cell lines were exposed to sodium butyrate (NaB) and F. nucleatum in vitro to examine the effects on proliferative proteins and mitochondrial function. Our research indicates that the prevalence of F. nucleatum in fecal samples from CRC patients is significantly greater than in healthy counterparts, while the prevalence of butyrate-producing bacteria is notably lower. In mice colonized with F. nucleatum, the population of butyrate-producing bacteria decreased, resulting in altered levels of butyric acid, a key intestinal metabolite of butyrate. Exposure to NaB can impair mitochondrial morphology and diminish mitochondrial membrane potential in DLD-1 and HCT116 CRC cells. Consequently, this leads to modulated production of adenosine triphosphate and reactive oxygen species, thereby inhibiting cancer cell proliferation. Additionally, NaB triggers the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, blocks the cell cycle in HCT116 and DLD-1 cells, and curtails the proliferation of CRC cells. The combined presence of F. nucleatum and NaB attenuated the effects of the latter. By employing small interfering RNA to suppress AMPK, it was demonstrated that AMPK is essential for NaB's inhibition of CRC cell proliferation. F. nucleatum can promote cancer progression through its inhibitory effect on butyric acid, via the AMPK signaling pathway.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Development of Cu(II) 4-hydroxybenzoylhydrazone complexes that induce mitochondrial DNA damage and mitochondria-mediated apoptosis in liver cancer

Cisplatin remains the most widely used chemotherapeutic agent in cancer treatment; however, its inherent drawbacks have fueled the development of novel metalloanticancer drugs. In this study, two novel Cu(II) complexes (Cu1 and Cu2) were designed and synthesized. Notably, these Cu(II) complexes showed higher cytotoxicity against HL-7402 cells than cisplatin. Moreover, Cu(II) complexes significantly inhibited liver cancer growth in a xenograft model. A mechanism study revealed that the Cu(II) complexes reduced the mitochondrial membrane potential of cancer cells, produced excessive reactive oxygen species (ROS), induced mitochondrial DNA (mtDNA) damage, and ultimately facilitated cancer cell apoptosis.

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10−5 and 10−6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041.

Depletion of SLC7A11 Sensitizes Nasopharyngeal Carcinoma Cells to Ionizing Radiation.

Radiotherapy is the primary treatment choice for Nasopharyngeal Carcinoma (NPC). However, its efficacy is compromised due to radioresistance. Ferroptosis, a novel iron-dependent regulated cell death induced by Ionizing Radiation (IR), plays a role in promoting cancer cell death. Yet, the relationship between enhanced ferroptosis and increased sensitivity of NPC cells to IR remains poorly understood. This study aimed to explore the association between IR and ferroptosis in NPC, as well as the role of the ferroptosis repressor SLC7A11 in IR-treated NPC cells. CNE1 and HNE-2 NPC cells were subjected to IR treatment. We performed qPCR and western blotting to evaluate the expression of ferroptosis-related genes in both control and IR-treated NPC cells. Additionally, we used the MTT assay to measure the viability of these NPC cells. JC-1 and DCFH-DA staining were employed to assess mitochondrial membrane potential and Reactive Oxygen Species (ROS) levels in both control and IR-treated NPC cells. Furthermore, we examined the levels of Fe2+, Malondialdehyde (MDA), reduced Glutathione (GSH), and oxidized glutathione (GSSG) in these cells. Moreover, we depleted SLC7A11 in IR-treated NPC cells to investigate its impact on the ferroptosis of these cells. IR upregulated the expression of ferroptosis-related genes, including SLC7A11, ACSL4, COX2, FTH1, and GPX4, in CNE1 and HNE-2 cells. IR treatment also resulted in decreased cell viability, disrupted mitochondrial membrane potential, increased ROS levels, altered glutathione levels, and elevated Fe2+ levels. Knockdown of SLC7A11 enhanced the sensitivity of NPC cells to IR. IR may induce ferroptosis in NPC cells, and stimulating ferroptosis could potentially serve as a therapeutic strategy to enhance the efficacy of IR in treating NPC patients.

CTBP2 contributes to cisplatin resistance in lung adenocarcinoma by inhibiting generation of reactive oxygen species.

Cisplatin (CP) is commonly used for the initial treatment of lung adenocarcinoma (LUAD). Resistance to CP has long been recognized as a significant obstacle to achieving improved therapeutic outcomes. Nevertheless, the intricate molecular mechanisms underlying the phenomenon remain incompletely understood. The present study utilized the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) and Gene Expression Profiling Interactive Analysis (GEPIA) databases to conduct an analysis of the expression of C-terminal binding protein 2 (CTBP2) in LUAD. The correlation between CTBP2 expression and survival data was investigated by the Kaplan-Meier (K-M) plotter. Subsequently, the roles of CTBP2 in CP resistance were explored by analyzing cell viability, cell apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) in CP-resistant cells (A549/DDP). Our data indicated that the CTBP2 expression in LUAD exhibited a significant increase compared to the non-malignant tissues. CTBP2 overexpression showed a correlation to poor survival. CTBP2 knockdown significantly enhanced cell sensitivity to CP in A549/DDP cells. The underlying mechanism is related to promoting ROS production and decreasing MMP after CP treatment. CTBP2 expression has been identified as a novel biomarker for resistance to CP, and its downregulation has been found to enhance sensitivity to CP. Therefore, CTBP2 can serve as a predictor related to CP resistance and a viable therapeutic target for CP resistance in LUAD.

Synergistic antifungal mechanism of eugenol and citral against Aspergillus niger: Molecular Level

Previous studies have demonstrated that the combination of eugenol and citral (CEC) synergistically damages the Aspergillus niger cell membrane, yet the mechanism remains elusive. This study investigated the molecular perspective of the synergistic damage mechanism of CEC on A. niger cell membrane. Both eugenol and citral are highly lipophilic, with eugenol exhibiting higher lipophilicity. Analysis of glycerophospholipids, ergosterol, fatty acid content, as well as intracellular pH and potassium ions leakage, suggested that eugenol plays a leading role in CEC. In contrast, citral had a greater effect on cell membrane potential and energy metabolism than eugenol. Furthermore, CEC regulated the expression of proteins and genes related to cell membrane synthesis, with eugenol exerting a more pronounced regulatory effect on ergosterol synthesis-related proteins than citral. These results provided new insights for future investigations into the synergistic mechanism of essential oil small molecules or antifungal agents.

Modulating oxidative stress, apoptosis, and mitochondrial dysfunctions on cardiotoxicity induced by aluminum phosphide pesticide using resveratrol

The agricultural fumigant pesticide aluminum phosphide (AlP) is cardiotoxic. Water causes AlP to emit phosphine gas, a cardiac toxin that affects heart function and causes cardiogenic shock. AlP poisoning’s high fatality rate is due to cardiotoxicity. This study examines how resveratrol reduces oxidative stress, mitochondrial activity, and apoptosis in human cardiac myocyte (HCM) cells. After determining the optimal doses of resveratrol using the MTT test, HCM cells were subjected to a 24-h treatment of resveratrol following exposure to AlP (2.36 μM). The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) activity, mitochondrial swelling, mitochondrial cytochrome c release, and mitochondrial membrane potential (MMP) in HCM cells were investigated. Also, the expression of Bax and Bcl-2, caspace-3 activity, and apoptosis were assessed. The present investigation revealed that AlP substantially increased the level of ROS and decreased SOD activation, which were significantly modulated by resveratrol in a dose-dependent manner. Moreover, AlP induced an elevation of mitochondrial swelling, cytochrome c release, and MMP collapse. Co-administration of resveratrol significantly reduced above mitochondrial markers. AlP also significantly upregulated BAX and downregulated Bcl-2 expression, elevated caspace-3 activity, and apoptosis. Resveratrol co-administration was able to meaningfully modulate the mentioned parameters and finally reduce apoptosis. In conclusion, resveratrol, via its pleotropic properties, significantly demonstrated cytoprotective effects on HCM cytotoxicity induced by AlP.