Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci.

In onion thrips Thrips tabaci, reduced sensitivity of the sodium channel caused by several sodium channel mutations have been correlated with pyrethroid resistance. For this study, using mitochondrial cytochrome c oxidase subunit I gene sequences, we examined the phylogenetic relation among a total of 52 thelytokous and arrhenotokous strains with different genotypes of the sodium channel mutations. Then, we used flow cytometry to estimate their ploidy. Results showed that the strains are divisible into three groups: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky. Using 23 whole genome resequencing data obtained from 20 strains out of 52, we examined their genetic relation further using principal component analysis, admixture analysis, and a fixation index. Results showed that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by the respective group members (strains). The greatest genetic divergence was observed between thelytokous and arrhenotokous groups with a pair of T929I and K1774N. Nevertheless, they shared a genomic region with virtually no polymorphism around the sodium channel gene loci, suggesting a hard selective sweep. Based on these findings, we discuss the evolutionary origin and distribution of the sodium channel mutations in T. tabaci.

Identification, Characterization, and Modeling of a Bioinsecticide Protein Isolated from Scorpion Venom gland: A Three-Finger Protein.

Background: The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Methods: Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. Results: The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. Conclusion: This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.

Luteolin attenuates lipopolysaccharide-induced acute lung injury/acute respiratory distress syndrome by activating alveolar epithelial sodium channels via cGMP/PI3K pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Luteolin (Lut) was recently identified as the major active ingredient of Mosla scabra, which was a typical representative traditional Chinese medicine and had been used to treat pulmonary diseases for thousands of years. AIM OF THE STUDY: This study was to explore the effects and relative mechanisms of Lut in LPS-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS). The main characteristic of ALI/ARDS is pulmonary edema, and epithelial sodium channel (ENaC) is a key factor in effective removal of excessive alveolar edematous fluid, which is essential for repairing gas exchange and minimizing damage to the peripheral tissues. However, whether the therapeutic effects of Lut on respiratory diseases are relative with ENaC is still unknown. MATERIALS AND METHODS: Alveolar fluid clearance was calculated in BALB/c mice and ENaC function was measured in H441 cells. Moreover, ENaC membrane protein and mRNA were detected by Western blot and real-time PCR, respectively. We also studied the involvement of cGMP/PI3K pathway during the regulation of Lut on ENaC during LPS-induced ALI/ARDS by ELISA method and applying cGMP/PI3K inhibitors/siRNA. RESULTS: The beneficial effects of Lut in ALI/ARDS were evidenced by the alleviation of pulmonary edema, and enhancement of both amiloride-sensitive alveolar fluid clearance and short-circuit currents. Lut could alleviate the LPS decreased expression levels of ENaC mRNA and membrane protein in H441 cells and mouse lung. In addition, cGMP concentration was increased after the administration of Lut in ALI/ARDS mice, while the inhibition of cGMP/PI3K pathway could abrogate the enhanced AFC and ENaC protein expression of Lut. CONCLUSION: These results implied that Lut could attenuate pulmonary edema via enhancing the abundance of membrane ENaC at least partially through the cGMP/PI3K pathway, which could provide a promising therapeutic strategy for treating ALI/ARDS.

The neuronal potassium current IA is a potential target for pain during chronic inflammation.

Voltage-gated ion channels play a key role in the action potential (AP) initiation and its propagation in sensory neurons. Modulation of their activity during chronic inflammation creates a persistent pain state. In this study, we sought to determine how peripheral inflammation caused by complete Freund's adjuvant (CFA) alters the fast sodium (INa ), L-type calcium (ICaL ), and potassium (IK ) currents in primary afferent fibers to increase nociception. In our model, intraplantar administration of CFA induced mechanical allodynia and thermal hyperalgesia at day 14 post-injection. Using whole-cell patch-clamp recording in dissociated small (C), medium (Aδ), and large-sized (Aß) rat dorsal root ganglion (DRG) neurons, we found that CFA prolonged the AP duration and increased the amplitude of the tetrodotoxin-resistant (TTX-r) INa in Aß fibers. In addition, CFA accelerated the recovery of INa from inactivation in C and Aδ nociceptive fibers but enhanced the late sodium current (INaL ) only in Aδ and Aß neurons. Inflammation similarly reduced the amplitude of ICaL in each neuronal cell type. Fourteen days after injection, CFA reduced both components of IK (IKdr and IA ) in Aδ fibers. We also found that IA was significantly larger in C and Aδ neurons in normal conditions and during chronic inflammation. Our data, therefore, suggest that targeting the transient potassium current IA represents an efficient way to shift the balance toward antinociception during inflammation, since its activation will selectively decrease the AP duration in nociceptive fibers. Altogether, our data indicate that complex interactions between IK , INa , and ICaL reduce pain threshold by concomitantly enhancing the activity of nociceptive neurons and reducing the inhibitory action of Aß fibers during chronic inflammation.

Differential contribution of the subthreshold operating currents IT, Ih, and IKir to the resonance of thalamocortical neurons.

Membrane potential oscillations of thalamocortical (TC) neurons are believed to be involved in the generation and maintenance of brain rhythms that underlie global physiological and pathological brain states. These membrane potential oscillations depend on the synaptic interactions of TC neurons and their intrinsic electrical properties. These oscillations may be also shaped by increased output responses at a preferred frequency, known as intrinsic neuronal resonance. Here, we combine electrophysiological recordings in mouse brain slices, modern pharmacological tools, dynamic clamp, and computational modeling to study the ionic mechanisms that generate and modulate TC neuron resonance. We confirm findings of pioneering studies showing that most TC neurons display resonance that results from the interaction of the slow inactivation of the low-threshold calcium current IT with the passive properties of the membrane. We also show that the hyperpolarization-activated cationic current Ih is not involved in the generation of resonance; instead it plays a minor role in the stabilization of TC neuron impedance magnitude due to its large contribution to the steady conductance. More importantly, we also demonstrate that TC neuron resonance is amplified by the inward rectifier potassium current IKir by a mechanism that hinges on its strong voltage-dependent inward rectification (i.e., a negative slope conductance region). Accumulating evidence indicate that the ion channels that control the oscillatory behavior of TC neurons participate in pathophysiological processes. Results presented here points to IKir as a new potential target for therapeutic intervention.NEW & NOTEWORTHY Our study expands the repertoire of ionic mechanisms known to be involved in the generation and control of resonance and provides the first experimental proof of previous theoretical predictions on resonance amplification mediated by regenerative hyperpolarizing currents. In thalamocortical neurons, we confirmed that the calcium current IT generates resonance, determined that the large steady conductance of the cationic current Ih curtails resonance, and demonstrated that the inward rectifier potassium current IKir amplifies resonance.

ALDH2 deficiency induces atrial fibrillation through dysregulated cardiac sodium channel and mitochondrial bioenergetics: A multi-omics analysis.

Point mutation in alcohol dehydrogenase 2 (ALDH2), ALDH2*2 results in decreased catalytic enzyme activity and has been found to be associated with different human pathologies. Whether ALDH2*2 would induce cardiac remodeling and increase the attack of atrial fibrillation (AF) remains poorly understood. The present study evaluated the effect of ALDH2*2 mutation on AF susceptibility and unravelled the underlying mechanisms using a multi-omics approach including whole-genome gene expression and proteomics analysis. The in-vivo electrophysiological study showed an increase in the incidence and reduction in the threshold of AF for the mutant mice heterozygous for ALDH2*2 as compared to the wild type littermates. The microarray analysis revealed a reduction in the retinoic acid signals which was accompanied by a downstream reduction in the expression of voltage-gated Na+ channels (SCN5A). The treatment of an antagonist for retinoic acid receptor resulted in a decrease in SCN5A transcript levels. The integrated analysis of the transcriptome and proteome data showed a dysregulation of fatty acid ß-oxidation, adenosine triphosphate synthesis via electron transport chain, and activated oxidative responses in the mitochondria. Oral administration of Coenzyme Q10, an essential co-factor known to meliorate mitochondrial oxidative stress and preserve bioenergetics, conferred a protection against AF attack in the mutant ALDH2*2 mice. The multi-omics approach showed the unique pathophysiology mechanisms of concurrent dysregulated SCN5A channel and mitochondrial bioenergetics in AF. This inspired the development of a personalized therapeutic agent, Coenzyme Q10, to protect against AF attack in humans characterized by ALDH2*2 genotype.

Differentiation of the 50B11 dorsal ganglion cells into NGF and GDNF responsive nociceptor subtypes.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids.

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Cardiac protection of Bauhinia championii against reperfusion injury.

This study aims to investigate the protective effects of the Bauhinia championii (BC) against ischemia/reperfusion (I/R)-induced injury in an isolated heart model. Langendorff-perfused C57BL/6JNarl mice hearts were performed with 30 minutes ischemia and 60 minutes reperfusion by left anterior descending artery ligation. Before reperfusion, boiling water extracts of BC (10 mg/L) was pretreated for 15 minutes. During reperfusion, BC significantly decreased the occurrence of ventricular arrhythmias by lead II electrocardiogram (ECG). Electrophysiological effect of BC was further determined in isolated ventricular myocytes by whole-cell patch clamp technique. The underlying mechanism may result from its Na+ channel blocking activity characterized with reduced rise slope of action potential and Na+ current density. Moreover, BC dramatically reduced I/R-caused infarct size, which was accessed by 2,3,5-triphenyltetrazolium chloride (TTC) assay. Since BC decreased I/R-induced myoglobin release and oxidation of Ca2+ -calmodulin-dependent protein kinase, inhibition of myocardial necroptosis may account for the protective effects of BC on myocytes lose. This study indicated that BC may prevent I/R induced ventricular arrhythmias and myocyte death by blocking Na+ channels and decreasing necroptosis, respectively. Since most of the available antiarrhythmic remedies have unwanted adverse actions, BC could be a novel candidate for the treatment of myocardial infarction and ventricular arrhythmia.

CC chemokine ligand 2 (CCL2) enhances TTX-sensitive sodium channel activity of primary afferent neurons in the complete Freud adjuvant-induced inflammatory pain model.

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2-CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

Brugada syndrome trafficking-defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels.

Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.

Essential Oils and Their Constituents Targeting the GABAergic System and Sodium Channels as Treatment of Neurological Diseases.

Essential oils and the constituents in them exhibit different pharmacological activities, such as antinociceptive, anxiolytic-like, and anticonvulsant effects. They are widely applied as a complementary therapy for people with anxiety, insomnia, convulsion, pain, and cognitive deficit symptoms through inhalation, oral administration, and aromatherapy. Recent studies show that essential oils are emerging as a promising source for modulation of the GABAergic system and sodium ion channels. This review summarizes the recent findings regarding the pharmacological properties of essential oils and compounds from the oils and the mechanisms underlying their effects. Specifically, the review focuses on the essential oils and their constituents targeting the GABAergic system and sodium channels, and their antinociceptive, anxiolytic, and anticonvulsant properties. Some constituents target transient receptor potential (TRP) channels to exert analgesic effects. Some components could interact with multiple therapeutic target proteins, for example, inhibit the function of sodium channels and, at the same time, activate GABAA receptors. The review concentrates on perspective compounds that could be better candidates for new drug development in the control of pain and anxiety syndromes.

Role of sodium ion transporters and osmotic adjustments in stress alleviation of Cynodon dactylon under NaCl treatment: a parallel investigation with rice.

Comparative analyses of the responses to NaCl in Cynodon dactylon and a sensitive crop species like rice could effectively unravel the salt tolerance mechanism in the former. C. dactylon, a wild perennial chloridoid grass having a wide range of ecological distribution is generally adaptable to varying degrees of salinity stress. The role of salt exclusion mechanism present exclusively in the wild grass was one of the major factors contributing to its tolerance. Salt exclusion was found to be induced at 4 days when the plants were treated with a minimum conc. of 200 mM NaCl. The structural peculiarities of the salt exuding glands were elucidated by the SEM and TEM studies, which clearly revealed the presence of a bicellular salt gland actively functioning under NaCl stress to remove the excess amount of Na+ ion from the mesophyll tissues. Moreover, the intracellular effect of NaCl on the photosynthetic apparatus was found to be lower in C. dactylon in comparison to rice; at the same time, the vacuolization process increased in the former. Accumulation of osmolytes like proline and glycine betaine also increased significantly in C. dactylon with a concurrent check on the H2O2 levels, electrolyte leakage and membrane lipid peroxidation. This accounted for the proper functioning of the Na+ ion transporters in the salt glands and also in the vacuoles for the exudation and loading of excess salts, respectively, to maintain the osmotic balance of the protoplasm. In real-time PCR analyses, CdSOS1 expression was found to increase by 2.5- and 5-fold, respectively, and CdNHX expression increased by 1.5- and 2-fold, respectively, in plants subjected to 100 and 200 mM NaCl treatment for 72 h. Thus, the comparative analyses of the expression pattern of the plasma membrane and tonoplast Na+ ion transporters, SOS1 and NHX in both the plants revealed the significant role of these two ion transporters in conferring salinity tolerance in Cynodon.

Capsaicin Used on Skin Influences Ion Transport Pathways: An in vitro Study.

Acute, adverse skin effects to capsaicin can be activated by inhibition of sodium transport not only in nociceptive neurons, but also in keratinocytes. The aim of the current study was to describe and compare immediate (15 s) and prolonged (30 min) effects of capsaicin on epidermal (not neural) sodium transport using a rabbit skin model. Skin fragments (n = 169) were incubated in 4 conditions: undisturbed ion transport (U; n = 48); inhibited sodium transport (INa; n = 34) with amiloride used as sodium transport blocker; long-term irritation by capsaicin with undisturbed ion transport (CAPSA-U; n = 43) and with inhibited sodium transport (CAPSA-INa; n = 35). After 30 min of incubation, a solution of capsaicin was applied directly to the skin fragments. The study demonstrated that sodium transport inhibition eliminated the effects of both immediate and prolonged capsaicin application. The results could be the basis for future research considering selective sodium transport inhibitors for human skin to reduce the side effects of capsaicin, related to activation of sodium channels in keratinocytes.

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Effects of Tetrodotoxin in Mouse Models of Visceral Pain.

Visceral pain is very common and represents a major unmet clinical need for which current pharmacological treatments are often insufficient. Tetrodotoxin (TTX) is a potent neurotoxin that exerts analgesic actions in both humans and rodents under different somatic pain conditions, but its effect has been unexplored in visceral pain. Therefore, we tested the effects of systemic TTX in viscero-specific mouse models of chemical stimulation of the colon (intracolonic instillation of capsaicin and mustard oil) and intraperitoneal cyclophosphamide-induced cystitis. The subcutaneous administration of TTX dose-dependently inhibited the number of pain-related behaviors in all evaluated pain models and reversed the referred mechanical hyperalgesia (examined by stimulation of the abdomen with von Frey filaments) induced by capsaicin and cyclophosphamide, but not that induced by mustard oil. Morphine inhibited both pain responses and the referred mechanical hyperalgesia in all tests. Conditional nociceptor­specific Nav1.7 knockout mice treated with TTX showed the same responses as littermate controls after the administration of the algogens. No motor incoordination after the administration of TTX was observed. These results suggest that blockade of TTX-sensitive sodium channels, but not Nav1.7 subtype alone, by systemic administration of TTX might be a potential therapeutic strategy for the treatment of visceral pain.

Effects of Late Sodium Current Blockade on Ventricular Refibrillation in a Rabbit Model.

BACKGROUND: After defibrillation of initial ventricular fibrillation (VF), it is crucial to prevent refibrillation to ensure successful resuscitation outcomes. Inability of the late Na+ current to inactivate leads to intracellular Ca2+ dysregulation and arrhythmias. Our aim was to determine the effects of ranolazine and GS-967, inhibitors of the late Na+ current, on ventricular refibrillation. METHODS AND RESULTS: Long-duration VF was induced electrically in Langendorff-perfused rabbit hearts (n=22) and terminated with a defibrillator after 6 minutes. Fibrillating hearts were randomized into 3 groups: treatment with ranolazine, GS-967, or nontreated controls. In the treated groups, hearts were perfused with ranolazine or GS-967 at 2 minutes of VF. In control experiments, perfusion solution was supplemented with isotonic saline in lieu of a drug. Inducibility of refibrillation was assessed after initial long-duration VF by attempting to reinduce VF. Sustained refibrillation was successful in fewer ranolazine-treated (29.17%; P=0.005) or GS-967-treated (45.83%, P=0.035) hearts compared with that in nontreated control hearts (84.85%). In GS-967-treated hearts, significantly more spontaneous termination of initial long-duration VF was observed (66.67%; P=0.01). Ca2+ transient duration was reduced in ranolazine-treated hearts compared with that in controls (P=0.05) and also Ca2+ alternans (P=0.03). CONCLUSIONS: Late Na+ current inhibition during long-duration VF reduces the susceptibility to subsequent refibrillation, partially by mitigating dysregulation of intracellular Ca2+. These results suggest the potential therapeutic use of ranolazine and GS-967 and call for further testing in cardiac arrest models.

Zn2+ reduction induces neuronal death with changes in voltage-gated potassium and sodium channel currents.

In the present study, cultured rat primary neurons were exposed to a medium containing N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a specific cell membrane-permeant Zn2+ chelator, to establish a model of free Zn2+ deficiency in neurons. The effects of TPEN-mediated free Zn2+ ion reduction on neuronal viability and on the performance of voltage-gated sodium channels (VGSCs) and potassium channels (Kvs) were assessed. Free Zn2+ deficiency 1) markedly reduced the neuronal survival rate, 2) reduced the peak amplitude of INa, 3) shifted the INa activation curve towards depolarization, 4) modulated the sensitivity of sodium channel voltage-dependent inactivation to a depolarization voltage, and 5) increased the time course of recovery from sodium channel inactivation. In addition, free Zn2+ deficiency by TPEN notably enhanced the peak amplitude of transient outward K+ currents (IA) and delayed rectifier K+ currents (IK), as well as caused hyperpolarization and depolarization directional shifts in their steady-state activation curves, respectively. Zn2+ supplementation reversed the effects induced by TPEN. Our results indicate that free Zn2+ deficiency causes neuronal damage and alters the dynamic characteristics of VGSC and Kv currents. Thus, neuronal injury caused by free Zn2+ deficiency may correlate with its modulation of the electrophysiological properties of VGSCs and Kvs.

Antiepileptic drugs prevent seizures in hyperbaric oxygen: A novel model of epileptiform activity.

Breathing oxygen at sufficiently elevated pressures can trigger epileptiform seizures. Therefore, we tested the hypothesis that pre-treatment with FDA-approved antiepileptic drugs could prevent seizure onset in hyperoxia at 5 atmospheres absolute. We selected drugs from two putative functional categories, Na+-channel antagonists and GABA enhancers, each administered intraperitoneally at four doses in separate groups of C57BL/6 mice. The drugs varied in efficacy at the doses used. Of the five tested Na+-channel antagonists, carbamazepine and lamotrigine more than tripled seizure latency compared to values seen in vehicle controls. Primidone, zonisamide and oxcarbazepine were less effective. Of the four GABA reuptake inhibitors, tiagabine and vigabatrin also increased seizure latency by more than three times control values; valproic acid was less effective, and the GABA synthesis promoter gabapentin was intermediate in effectiveness. We infer that Na+-channel function and GABA neurotransmission may be critical targets in the pathophysiology of CNS O2 toxicity. Because these essential components of neuronal excitation and inhibition are also implicated in the pathogenesis of other seizure disorders, including generalized epilepsy, we propose that, at some level, common pathways are involved in these pathologies, although the initiating insults differ. Furthermore, hyperoxic exposures are not known to cause the spontaneously-recurring seizures that characterize true clinical epilepsy. Nonetheless, experimental studies of hyperbaric oxygen toxicity could provide new insights into molecular mechanisms of seizure disorders of various etiologies. In addition, the neuropathology of hyperbaric oxygen is particularly relevant to the hypothesis held by some investigators that oxidative stress is an etiological factor in clinical epilepsies.

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport.

Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10-8 cm/s vs (738 ± 190) ×10-8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI-H441 cells as a model for investigating ion and water transport in the human alveolar epithelium and also identify the pathways of sodium and chloride transport.