Potassium Efflux Research Articles

Gallesia integrifolia (Spreng.) Harms: In vitro and in vivo antibacterial activities and mode of action

Ethnopharmacological relevanceGallesia integrifolia (Phytolaccaceae) is commonly known as “pau-d’alho” in Brazil or “garlic plant” due to the strong scent of garlic peculiar to all parts of the plant. The bark decoction is used for the treatment of microbial infections among other diseases by different ethnic groups in Brazil, Peruvian Amazonians, Bolivia and Mosetene Indians. This study aimed to advance in the antibacterial activity and characterize the mode of action of the hydroethanolic extract of the inner stem bark of G. integrifolia (HEGi) using in vivo and in vitro experimental models. Materials and methodsThe qualitative and quantitative phytochemical analyzes of HEGi were carried out using colorimetric and HPLC technique. The cytotoxic potential of HEGi was evaluated against CHO-K1 cells by Alamar blue assay and its acute toxicity was assessed by the Hippocratic screening test using Swiss-Webster mice. The antibacterial activity was evaluated by micro- dilution method against ten strains of Gram-positive and Gram-negative bacteria. The mode of action of HEGi was investigated by outer membrane permeability, nucleotide leakage and potassium efflux assays. In vivo infection model was established by using Staphylococcus aureus infection model Wistar rats. ResultsQualitative phytochemical analysis of HEGi revealed the presence of saponins, alkaloids, phenolic compounds and flavonoids. Phytochemical quantification of HEGi showed that higher total phenolic (80.10±0.62mg GAE/g) and flavonoid (16.10±0.03mg RE/g) contents. HPLC fingerprint analysis revealed the presence of gallic acid, rutin, and morin. In the Alamar blue assay no cytotoxic effect of HEGi in CHO-K1 cells was observed up to 200µg/mL, and no signs or symptoms of acute toxicity were observed in mice of both sexes at higher doses of up to 2000mg/kg, p.o. HEGi demonstrated bacteriostatic effect against selected Gram positive and Gram negative bacterial pathogens. Its mode of action is associated, at least partly, with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in Pseudomonas aeruginosa, intense K+ efflux and nucleotides leakage in Shigella flexneri, Streptococcus pyogenes and S. aureus. HEGi attenuated the experimental blood borne S. aureus infection in rats at all the tested doses levels (10, 50 and 250mg/kg). ConclusionHEGi is safe at the dose tested when used acutely, and it presented broad antibacterial effect, which support its traditional use in the treatment of bacterial infections. It contains well known important phytochemicals, recognized to be active against bacterial pathogens in vitro and might be collectively responsible for the antibacterial activity of HEGi. It is bacteriostatic in nature, with membrane perturbation being one of it mode of action. HEGi represent a potential phytotherapic antibacterial agent.

NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis

Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1β secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells.

A Near-Ir Fluorescent Sensor for Detecting Cellular Potassium Efflux

Our goal is to develop a fluorescence-based method to visualize potassium efflux and extracellular accumulation in cells and living organisms. Rapid, coordinated transport of potassium across the plasma membrane is vital for cardiac rhythmicity, muscle contraction and neuronal excitability. However, potassium selective electrodes and electrophysiological methods that are used conventionally to measure potassium transport provide minimal spatiotemporal information on potassium efflux from an entire cell. Our approach is to detect transient fluctuations in potassium concentration at the cell surface by attaching potassium-specific fluorescent sensors to the re-engineered glycocalyces of living cells. We have already synthesized and characterized a potassium-selective, near-IR fluorescent sensor suitable for measuring potassium under physiological conditions. Our fluorescent potassium sensor possesses a chemical handle that allows the sensor to be attached covalently to the re-engineered glycocalyces of cells using bio-orthogonal click chemistry. We are currently using this newly developed technology to visualize potassium transport through a variety of voltage-gated potassium channels. The synthesis of the potassium-selective, near-IR fluorescent sensor, its characterization, and application will be presented.

Quelling Inflammation with Ketosis and Steric Chemistry.

Quelling Inflammation with Ketosis and Steric Chemistry.

NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.

Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest.

We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment.

The effect of atp-dependent potassium uptake on mitochondrial functions under acute hypoxia.

The opening of mitochondrial K(+) АТР-channel (mtK(+) АТР-channel) is supposed to be important in the modulation of mitochondrial functions under hypoxia, but the underlying mechanisms have not been clarified yet. The aim of this work was to study the effect of acute hypoxia on mtK(+) АТР-channel activity and to estimate the contribution of the channel in the modulation of mitochondrial functions. MtK(+) АТР-channel activity was assessed polarographically from the rate of State 4 respiration and by potentiometric monitoring of potassium efflux from deenergized mitochondria. It was shown that hypoxia reliably increased mtK(+) АТР-channel activity, which resulted in the changes of respiration rates (increase of State 4 and suppression of State 3 respiration), uncoupling (the decrease of respiratory control ratio) and suppression of phosphorylation. These effects were well mimicked by mtK(+) АТР-channel opener diazoxide (DZ) in isolated rat liver mitochondria. MtK(+) АТР-channel opening in vitro suppressed phosphorylation too, but increased phosphorylation efficiency, while mtK(+) АТР-channel blockers reduced it dramatically. The correlation was established between mtK(+) АТР-channel activity and the endurance of the rats to physical training under hypoxia. Hypoxia improved physical endurance, but treatment by mtK(+) АТР-channel blockers glibenklamide and 5-hydroxydecanoate (5-HD) prior to hypoxia strongly reduced both the channel activity and the endurance limits. This was in accord with the observation that under glibenklamide and 5-HD administration hypoxia failed to restore mtK(+) АТР-channel activity. Based on the experiments, we came to the conclusion that mtK(+) АТР-channel opening played a decisive role in the regulation of energy metabolism under acute hypoxia via the modulation of phosphorylation system in mitochondria.

Platinum nanopetal-based potassium sensors for acute cell death monitoring

A novel potassium-selective electrode based on Pt nanopetals has been used for monitoring potassium efflux from cells as due to two death mechanisms: osmotic shock in DI water and necro-apoptosis by drug overdose.

The photochemical thiol-ene reaction as a versatile method for the synthesis of glutathione S-conjugates targeting the bacterial potassium efflux system Kef.

The thiol-ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione-S-conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K+ efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K+ homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs.

APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.

HIV Promotes NLRP3 Inflammasome Complex Activation in Murine HIV-Associated Nephropathy

Dysregulated growth and loss of podocytes are important features of HIV-associated nephropathy. Recently, HIV was reported to induce a new type of programed cell death, pyroptosis, in Tlymphocytes through induction of Nod-like receptor protein 3 (NLRP3) inflammasome complexes. We evaluated therole of HIV in podocyte NLRP3 inflammasome formation both invivo and invitro. Renal cortical sections of HIV-transgenic mice (Tg26) displayed increased expression of NLRP3, ASC (aCARD protein), caspase-1, and IL-1β proteins, confirming NLRP3 inflammasome complex formation in podocytes ofTg26 mice. Renal tissues of Tg26 mice also displayed enhanced mRNA levels and protein expressions of inflammasome markers (NLRP3, ASC, and caspase-1, and IL-1β). Serum of Tg26 mice also showed elevated concentrations of IL-1β cytokine compared with FVBN mice. HIV induced pyroptosis in a dose- and time-dependent manner within podocytes, a phenotype of inflammasome activation. Caspase-1 inhibitor not only attenuated podocyte expression of caspase-1 and IL-1β but also provided protection against pyroptosis, suggesting that HIV-induced podocyte injury was mediated by caspase-1 activation. Interestingly, HIV-induced podocyte pyroptosis could be partially inhibited by Tempol (asuperoxide dismutase-mimetic agent) and by glyburide (an inhibitor of potassium efflux). Thesefindings suggest that generation of reactive oxygen species and potassium efflux contribute to HIV-induced pyroptosis and NLRP3 inflammasome activation in podocytes.

Membrane damage by lipid peroxidation retains the cadmium constraint and is not the primary cause of K+ extrusion in yeast

Yeast cells exposed to cadmium (Cd) generate free radicals that cause disruption of membranes, possibly induced by elevated levels of lipid peroxidation with associated K+ efflux. The influence of lipid peroxidation on cadmium incorporation and the relationship of lipid oxidation with sodium/potassium movement was investigated. Mutant ahp1∆ [alkyl hydroperoxidase reductase (AHP1) gene deletion] was used. ahp1∆ mutant showed a lipid peroxidation index higher than wild type (WT) independently of the presence of cadmium. Despite lipid peroxidation being generally correlated to membrane permeabilization, cadmium incorporation was similar in both strains. The Na+ level was higher in ahp1∆ mutant compared to WT cells. Cd treatment tended to restore the Na+ level in mutant cells to similar levels determined in WT cells. Cd induced a lower K+ efflux than that for sodium exit. We point out the possible influence of glutathione on potassium efflux as the first cause of ionic movement and the influence of the AHP1 gene on ionic movement.

Ion efflux and influenza infection trigger NLRP3 inflammasome signaling in human dendritic cells

The nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, a multiprotein complex, is an essential intracellular mediator of antiviral immunity. In murine dendritic cells, this complex responds to a wide array of signals, including ion efflux and influenza A virus infection, to activate caspase-1-mediated proteolysis of IL-1β and IL-18 into biologically active cytokines. However, the presence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells, in response to various triggers, including viral infection, has not been defined clearly. Here, we delineate the contribution of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome to the secretion of IL-1β, IL-18, and IL-1α by human dendritic cells (monocyte-derived and primary conventional dendritic cells). Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells by various synthetic activators resulted in the secretion of bioactive IL-1β, IL-18, and IL-1α and induction of pyroptotic cell death. Cellular IL-1β release depended on potassium efflux and the activity of proteins nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1. Likewise, influenza A virus infection of dendritic cells resulted in priming and activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and secretion of IL-1β and IL-18 in an M2- and nucleotide-binding oligomerization domain-like receptor protein 3-dependent manner. The magnitude of priming by influenza A virus varied among different strains and inversely corresponded to type I IFN production. To our knowledge, this is the first report describing the existence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells and the ability of influenza A virus to prime and activate this pathway in human dendritic cells, with important implications for antiviral immunity and pathogenesis.

Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3-ASC-caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1-deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans-induced activation of either the canonical NLRP3-ASC-caspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3-ASC-caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.

Activation of the NLRP3 inflammasome by cellular labile iron

Cellular labile iron, which contains chelatable redox-active Fe(2+), has been implicated in iron-mediated cellular toxicity leading to multiple organ dysfunction. Iron homeostasis is controlled by monocytes/macrophages through their iron recycling and storage capacities. Furthermore, iron sequestration by monocytes/macrophages is regulated by pro-inflammatory cytokines including interleukin-1, highlighting the importance of these cells in the crosstalk between inflammation and iron homeostasis. However, a role for cellular labile iron in monocyte/macrophage-mediated inflammatory responses has not been defined. Here we describe how cellular labile iron activates the NLRP3 inflammasome in human monocytes. Stimulation of lipopolysaccharide-primed peripheral blood mononuclear cells with ferric ammonium citrate increases the level of cellular Fe(2+) levels in monocytes and induces production of interleukin-1β in a dose-dependent manner. This ferric ammonium citrate-induced interleukin-1β production is dependent on caspase-1 and is significantly inhibited by an Fe(2+)-specific chelator. Ferric ammonium citrate consistently induced interleukin-1β secretion in THP1 cells, but not in NLRP3-deficient THP1 cells, indicating a requirement for the NLRP3 inflammasome. Additionally, activation of the inflammasome is mediated by potassium efflux, reactive oxygen species-mediated mitochondrial dysfunction, and lysosomal membrane permeabilization. Thus, these results suggest that monocytes/macrophages not only sequestrate iron during inflammation, but also mediate inflammation in response to cellular labile iron, which provides novel insights into the role of iron in chronic inflammation.

K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.

P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.

Biological substrates of migraine

Migraine episodes are probably originated by central mechanisms in brain areas able to cause the classical neurological symptoms of prodromes and aura. Conversely, the headache phase begins with the activation of meningeal nociceptors at the origin of the trigeminovascular system. Recent information has postulated that the occurrence of aura can activate nociceptors in the meninges. Conversely, the mechanisms by which common prodromes initiate the headache phase or what sequence of events triggers activation of the meningeal nociceptors is not yet understood. However, a mechanistic analysis for a common factor in migraine clinical features strongly suggests a genetic predisposition to generalized neuronal hyperexcitability. Hypothalamic neurons are sensitive to changes in physiological and emotional homeostasis and they might activate meningeal nociceptors by altering the balance between parasympathetic and sympathetic influence in the meninges. Accordingly, hypothalamic neurons that contain dopamine, histamine, and orexin, and brainstem neurons that contain noradrenaline and serotonin send inputs to trigemino-thalamic neurons in sensory thalamic nuclei. These neurotransmitters can shift the activity of thalamic neurons. Clinical and preclinical studies suggest that migraine aura is caused by cortical spreading depression (CSD), a slowly propagating wave of depolarization/excitation followed by inhibition in cortical neurons and glia. In the cortex, the initial membrane depolarization is associated with a large efflux of potassium, influx of sodium and calcium and release of glutamate. Interestingly, endogenous CGRP is released in the cortical tissue during CSD, and CGRP receptor antagonists have an inhibitory effect on CSD, suggesting a critical role of CGRP in this phenomenon. The demonstration that CGRP antagonism reduces CSD supports the possible use of drugs targeting central CGRP receptors as antimigraine agents. Finally, among the various pathophysiological conditions controlling the expression and the features of headache in migraine, medication-overuse headache (MOH) plays a central role. MOH is a clinically important entity and it is now well documented that the regular use of acute symptomatic medication by people with migraine increases the risk of aggravation of the primary headache. MOH is one of the most common causes of chronic migraine-like syndrome. We have analyzed the possible mechanisms underlying sensitization in MOH by comparing these mechanisms with those reported for other forms of drug addiction. Recent data support the evidence for cognitive impulsivity in drug overuse in headache and in other forms of addiction associated with dysfunction of the frontostriatal system and an integrative hypothesis for compulsive reward-seeking in MOH.

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways.

Murine caspase-11 and its human orthologues, caspase-4 and caspase-5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram-negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL-1α, as well as the production of IL-1β and IL-18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase-11, -4, and -5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS-dependent IL-1β production requires the NLRP3 inflammasome and that its activation is dependent on K(+) efflux, whereas IL-1α release and pyroptotic cell death pathways are NLRP3-independent. These findings expand on previous research implicating K(+) efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.

Mediated electrochemical method for the analysis of membrane damage effects of phenolic compounds to Staphylococcus aureus

Abstract In this study, the mediated electrochemical method and conventional methods, including the atomic force microscopy, respiration inhibition, and potassium efflux methods were integrated for the analysis of the membrane damage effects of phenolic compounds to the food contamination bacteria Staphylococcus aureus (S. aureus). Hydrophilic ferricyanide, acting as both the permeability indicator and intracellular redox activity indicator, was employed as the mediator. Microelectrode voltammetric method was used to assay the ferrocyanide accumulations arising from the reduction of ferricyanide by the NADH: Fe3 + reductase embedded in the cell membrane. When the samples with S. aureus suspensions were incubated for 2 h, the mediated electrochemical response increased by 71.9% due to the presence of 3 mM carvacrol, which was consistent with the significant potassium efflux attributed to the presence of the carvacrol, indicating the outer membrane damage of the bacteria. However, the relative respiration activities of the S. aureus suspensions remained 32.5% of the controlled sample, which suggested the damage of the respiration chain due to the presence of 3 mM carvacrol. The mediated electrochemical method was also applicable to the other three phenolic compounds including eugenol, thymol, and 3-isopropylphenol, exhibiting the applicability of the mediated electrochemical method for the membrane damage effect of the phenolic compounds to bacteria.