Structures of human pannexin 1 reveal ion pathways and mechanism of gating.

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.

Synthesis of Novel 2-(Cyclopentylamino)thiazol-4(5H)-one Derivatives with Potential Anticancer, Antioxidant, and 11ß-HSD Inhibitory Activities.

In this study, a series of nine new 2-(cyclopentylamino)thiazol-4(5H)-one derivatives were synthesized, and their anticancer, antioxidant, and 11ß-hydroxysteroid dehydrogenase (11ß-HSD) inhibitory activities were tested. Anticancer activity has been assessed using the MTS (MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay against human colon carcinoma (Caco-2), human pancreatic carcinoma (PANC-1), glioma (U-118 MG), human breast carcinoma (MDA-MB-231), and skin melanoma (SK-MEL-30) cancer cell lines. Cell viability reductions, especially in the case of Caco-2, MDA-MB-231, and SK-MEL-30 lines, were observed for most compounds. In addition, the redox status was investigated and oxidative, but nitrosative stress was not noted at a concentration of 500 µM compounds tested. At the same time, a low level of reduced glutathione was observed in all cell lines when treated with compound 3g (5-(4-bromophenyl)-2-(cyclopentylamino)thiazol-4(5H)-one) that most inhibited tumor cell proliferation. However, the most interesting results were obtained in the study of inhibitory activity towards two 11ß-HSD isoforms. Many compounds at a concentration of 10 µM showed significant inhibitory activity against 11ß-HSD1 (11ß-hydroxysteroid dehydrogenase type 1). The compound 3h (2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one) showed the strongest 11ß-HSD1 inhibitory effect (IC50 = 0.07 µM) and was more selective than carbenoxolone. Therefore, it was selected as a candidate for further research.

Inhibition of Schwann cell pannexin 1 attenuates neuropathic pain through the suppression of inflammatory responses.

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.

Hyposmotic stress causes ATP release in a discrete zone within the outer cortex of rat lens.

Purpose: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release. Methods: Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca2+, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space. Results: The incubation of the rat lenses in the AAH that lacked Ca2+ induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid. Conclusions: Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.

Pannexin1 channels regulate mechanically stimulated but not spontaneous adenosine release.

Fast-scan cyclic voltammetry (FSCV) is a rapid technique to measure neuromodulators, and using FSCV, two modes of rapid adenosine have been discovered. Spontaneous transients occur randomly in the brain, while mechanical stimulation also causes a rapid adenosine event. Pannexin1 channels are membrane channels that transport ions, including ATP, out of the cell where it is rapidly broken down into adenosine. Pannexin 1 channels (Panx1) have a flickering mode of rapid opening and are also mechanically stimulated. Here, we test the extent to which pannexin channels, specifically pannexin1 (Panx1) channels, are responsible for rapid adenosine events. Spontaneous adenosine release or mechanosensitive adenosine release were measured using fast-scan cyclic voltammetry in hippocampal (CA1) brain slices. In global Panx1KO mice, there is no significant difference in the frequency or concentration of spontaneous adenosine release, indicating Panx1 is not a release mechanism for spontaneous adenosine. Spontaneous adenosine frequency decreased slightly after administration of a large (100 µM) dose of carbenoxolone, a nonspecific inhibitor of many pannexin and connexin channels, suggesting other hemichannels only play a small role at most. For mechanically stimulated adenosine release, the concentration of each adenosine event significantly decreased 30% in Panx1KO mice and the frequency of stimulations that evoked adenosine also decreased. The response was similar in WT mice with carbenoxolone. Thus, Panx1 is a release mechanism for mechanically stimulated adenosine release, but not the only mechanism. These results demonstrate that pannexin channels differentially regulate rapid adenosine release and could be targeted to differentially affect mechanically stimulated adenosine due to brain damage.

Carbenoxolone Ameliorates Allergic Airway Inflammation through NF-κB/NLRP3 Pathway in Mice.

Asthma is a respiratory disease characterized by heterogeneous chronic airway inflammation. Activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome is involved in the development of many pulmonary inflammatory diseases. The role and regulatory mechanism of carbenoxolone (CBX) in ovalbumin (OVA)-induced asthma models are not fully clear. Therefore, the study investigated whether CBX ameliorates airway inflammation and remodeling, as well as its mechanism in OVA induced-inflammation in mice. Wright-Giemsa staining was used to count inflammatory cells in bronchoalveolar lavage fluid (BALF). The level of inflammatory cells infiltration, mucus cell proliferation, and collagen deposition in lung tissue were separately assessed by hematoxylin and eosin, periodic acid-Schiff, and Masson trichrome staining, respectively. Airway resistance (AR) was measured by non-invasive airway system. Immunohistochemical assay was used to observe NLRP3 expression area. The expression of nuclear factor-kappaB (NF-κB), p-NF-κB, inhibitor of kappaB (IκB)-α, p-IκB-α, NLRP3, pro-caspase-1, caspase-1, and interleukin (IL)-1ß in lung tissue were measured using quantitative real-time PCR or Western blotting. Our results showed that CBX can significantly attenuate the leukocyte count and the percentage of eosinophils and neutrophils in the BALF, peribronchial inflammation, airway mucus secretion, collagen deposition area, and AR in OVA-induced airway inflammation. In addition, the expression of p-NF-κB, p-IκB-α, NLRP3 and related factors were dramatically alleviated after CBX treatment. These data suggest that CBX has a significant protective effect on allergic airway inflammation by suppressing the activation of NLRP3 inflammasome through NF-κB pathway in asthmatic mice.

Comparative effect of vitamin D3 and carbenoxolone treatments in metabolic syndrome rats.

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including central obesity, hypertension, insulin resistance, dyslipidemia, and hyperglyemia. MetS is found to be a positive predictor of cardiovascular morbidity and mortality. The present study was planned to test the efficacy of vitamin D3 supplementation as compared with cortisol inhibition on MetS parameters. Wistar rats were allocated into four groups: control, untreated MetS, and MetS treated with either vitamin D3 (10 µg/kg) or carbenoxolone (50 mg/kg). MetS was induced by combination of high-fat diet and oral fructose. After the induction period (8 weeks), MetS was confirmed, and treatment modalities started for a further 4 weeks. Compared with untreated MetS, vitamin D3- and carbenoxolone-treated rats showed significant reduction in blood pressure, body mass index, Lee index, waist circumference, retroperitoneal fat, and improvement of dyslipidemia. Meanwhile, treatment with carbenoxolone significantly lowered the elevated liver enzymes, and vitamin D3 resulted in improved insulin sensitivity, enhanced glucose uptake by muscles, and replenished glycogen content in the liver and muscles near control levels. In conclusion, although treatment with vitamin D3 or carbenoxolone reduced the risk factors associated with MetS, vitamin D3 was effective in ameliorating insulin resistance which is the hallmark of MetS.

Exploration of Biomarkers of Psoriasis through Combined Multiomics Analysis.

Background: Aberrant DNA methylation patterns are of increasing interest in the study of psoriasis mechanisms. This study aims to screen potential diagnostic indicators affected by DNA methylation for psoriasis based on bioinformatics using multiple machine learning algorithms and to preliminarily explore its molecular mechanisms. Methods: GSE13355, GSE14905, and GSE73894 were collected from the gene expression omnibus (GEO) database. Differentially expressed genes (DEGs) and differentially methylated region- (DMR-) genes between psoriasis and control samples were combined to obtain differentially expressed methylated genes. Subsequently, a protein-protein interaction (PPI) network was established to analyze the interaction between differentially expressed methylated genes. Moreover, the hub genes of psoriasis were screened by the least absolute shrinkage and selection operator (LASSO), Random Forest (RF), and Support Vector Machine (SVM), which were further performed single-gene gene set enrichment analysis (GSEA) to clarify the pathogenesis of psoriasis. The druggable genes were predicted using DGIdb. Finally, the expressions of hub genes in psoriasis lesions and healthy controls were detected by immunohistochemistry (IHC) and quantitative real-time PCR (RT-qPCR). Results: In this study, a total of 767 DEGs and 896 DMR-genes were obtained. Functional enrichment showed that they were significantly associated with skin development, skin barrier function, immune/inflammatory response, and cell cycle. The combined transcriptomic and DNA methylation data resulted in 33 differentially expressed methylated genes, of which GJB2 was the final identified hub gene for psoriasis, with robust diagnostic power. IHC and RT-qPCR showed that GJB2 was significantly higher in psoriasis samples than those in healthy controls. Additionally, GJB2 may be involved in the development and progression of psoriasis by disrupting the body's immune system, mediating the cell cycle, and destroying the skin barrier, in addition to possibly inducing diseases related to the skeletal aspects of psoriasis. Moreover, OCTANOL and CARBENOXOLONE were identified as promising compounds through the DGIdb database. Conclusion: The abnormal expression of GJB2 might play a critical role in psoriasis development and progression. The genes identified in our study might serve as a diagnostic indicator and therapeutic target in psoriasis.

Electrical synapses between mushroom body neurons are critical for consolidated memory retrieval in Drosophila.

Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Here, we show that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αß neurons disrupted ARM, suggesting a specific requirement of INX5 in αß neurons for ARM formation. Hyperpolarization of αß neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive αß neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, our results suggest INX5 gap junction channels in αß neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.

Astroglial Connexins Inactivation Increases Relapse of Depressive-like Phenotype after Antidepressant Withdrawal.

Studies suggest that astrocytic connexins (Cx) have an important role in the regulation of high brain functions through their ability to establish fine-tuned communication with neurons within the tripartite synapse. In light of these properties, growing evidence suggests a role of Cx in psychiatric disorders such as major depression but also in the therapeutic activity of antidepressant drugs. However, the real impact of Cx on treatment response and the underlying neurobiological mechanisms remain yet to be clarified. On this ground, the present study was designed to evaluate the functional activity of Cx in a mouse model of depression based on chronic corticosterone exposure and to determine to which extent their pharmacological inactivation influences the antidepressant-like activity of venlafaxine (VENLA). On the one hand, our results indicate that depressed mice have impaired Cx-based gap-junction and hemichannel activities. On the other hand, while VENLA exerts robust antidepressant-like activity in depressed mice; this effect is abolished by the pharmacological inhibition of Cx with carbenoxolone (CBX). Interestingly, the combination of VENLA and CBX is also associated with a higher rate of relapse after treatment withdrawal. To our knowledge, this study is one of the first to develop a model of relapse, and our results reveal that Cx-mediated dynamic neuroglial interactions play a critical role in the efficacy of monoaminergic antidepressant drugs, thus providing new targets for the treatment of depression.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

Blocking ATP-releasing channels prevents high extracellular ATP levels and airway hyperreactivity in an asthmatic mouse model.

Allergic asthma is a chronic airway inflammatory response to different triggers like inhaled allergens. Excessive ATP in fluids from patients with asthma is considered an inflammatory signal and an important autocrine/paracrine modulator of airway physiology. Here, we investigated the deleterious effect of increased extracellular ATP (eATP) concentration on the mucociliary clearance (MCC) effectiveness and determined the role of ATP releasing channels during airway inflammation in an ovalbumin (OVA)-sensitized mouse model. Our allergic mouse model exhibited high levels of eATP measured in the tracheal fluid with a luciferin-luciferase assay and reduced MCC velocity determined by microspheres tracking in the trachea ex vivo. Addition of ATP had a dual effect on MCC, where lower ATP concentration (µM) increased microspheres velocity, whereas higher concentration (mM) transiently stopped microspheres movement. Also, an augmented ethidium bromide uptake by the allergic tracheal airway epithelium suggests an increase in ATP release channel functionality during inflammatory conditions. The use of carbenoxolone, a nonspecific inhibitor of connexin and pannexin1 channels reduced the eATP concentration in the allergic mouse tracheal fluid and dye uptake by the airway epithelium, providing evidence that these ATP release channels are facilitating the net flux of ATP to the lumen during airway inflammation. However, only the specific inhibition of pannexin1 with 10Panx peptide significantly reduced eATP in bronchoalveolar lavage and decreased airway hyperresponsiveness in OVA-allergic mouse model. These data provide evidence that blocking eATP may be a pharmacological alternative to be explored in rescue therapy during episodes of airflow restriction in patients with asthma.

11ß hydroxysteroid dehydrogenases regulate circulating glucocorticoids but not central gene expression.

Regulation of glucocorticoids (GCs), important mediators of physiology and behavior at rest and during stress, is multi-faceted and dynamic. The 11ß hydroxysteroid dehydrogenases 11ß-HSD1 and 11ß-HSD2 catalyze the regeneration and inactivation of GCs, respectively, and provide peripheral and central control over GC actions in mammals. While these enzymes have only recently been investigated in just two songbird species, central expression patterns suggest that they may function differently in birds and mammals, and little is known about how peripheral expression regulates circulating GCs. In this study, we utilized the 11ß-HSD inhibitor carbenoxolone (CBX) to probe the functional effects of 11ß-HSD activity on circulating GCs and central GC-dependent gene expression in the adult zebra finch (Taeniopygia guttata). Peripheral CBX injection produced a marked increase in baseline GCs 60 min after injection, suggestive of a dominant role for 11ß-HSD2 in regulating circulating GCs. In the adult zebra finch brain, where 11ß-HSD2 but not 11ß-HSD1 is expressed, co-incubation of micro-dissected brain regions with CBX and stress-level GCs had no impact on expression of several GC-dependent genes. These results suggest that peripheral 11ß-HSD2 attenuates circulating GCs, whereas central 11ß-HSD2 has little impact on gene expression. Instead, rapid 11ß-HSD2-based regulation of local GC levels might fine-tune membrane GC actions in brain. These results provide new insights into the dynamics of GC secretion and action in this important model organism.

A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Carbenoxolon Is Capable to Regulate the Mitochondrial Permeability Transition Pore Opening in Chronic Alcohol Intoxication.

BACKGROUND: carbenoxolone, which is a derivative of glyceretic acid, is actively used in pharmacology for the treatment of diseases of various etiologies. In addition, we have shown carbenoxolone as an effective inducer of mitochondrial permeability transition pore in rat brain and liver mitochondria. METHODS: in the course of this work, comparative studies were carried out on the effect of carbenoxolone on the parameters of mPTP functioning in mitochondria isolated from the liver of control and alcoholic rats. RESULTS: within the framework of this work, it was found that carbenoxolone significantly increased its effect in the liver mitochondria of rats with chronic intoxication. In particular, this was expressed in a reduction in the lag phase, a decrease in the threshold calcium concentration required to open a pore, an acceleration of high-amplitude cyclosporin-sensitive swelling of mitochondria, as well as an increase in the effect of carbenoxolone on the level of mitochondrial membrane-bound proteins. Thus, as a result of the studies carried out, it was shown that carbenoxolone is involved in the development/modulation of alcohol tolerance and dependence in rats.

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt)max - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

A myogenic motor pattern in mice lacking myenteric interstitial cells of Cajal explained by a second coupled oscillator network.

The interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are a network of coupled oscillators in the small intestine that generate rhythmic electrical phase waves leading to corresponding waves of contraction, yet rhythmic action potentials and intercellular calcium waves have been recorded from c-kit-mutant mice that lack the ICC-MP, suggesting that there may be a second pacemaker network. The gap junction blocker carbenoxolone induced a "pinstripe" motor pattern consisting of rhythmic "stripes" of contraction that appeared simultaneously across the intestine with a period of ~4 s. The infinite velocity of these stripes suggested they were generated by a coupled oscillator network, which we call X. In c-kit mutants rhythmic contraction waves with the period of X traveled the length of the intestine, before the induction of the pinstripe pattern by carbenoxolone. Thus X is not the ICC-MP and appears to operate under physiological conditions, a fact that could explain the viability of these mice. Individual stripes consisted of a complex pattern of bands of contraction and distension, and between stripes there could be slide waves and v waves of contraction. We hypothesized that these phenomena result from an interaction between X and the circular muscle that acts as a damped oscillator. A mathematical model of two chains of coupled Fitzhugh-Nagumo systems, representing X and circular muscle, supported this hypothesis. The presence of a second coupled oscillator network in the small intestine underlines the complexity of motor pattern generation in the gut.NEW & NOTEWORTHY Physiological experiments and a mathematical model indicate a coupled oscillator network in the small intestine in addition to the c-kit-expressing myenteric interstitial cells of Cajal. This network interacts with the circular muscle, which itself acts as a system of damped oscillators, to generate physiological contraction waves in c-kit (W) mutant mice.

Cooling-induced dilatation of cutaneous arteries is mediated by increased myoendothelial communication.

Cold exposure causes cutaneous vasoconstriction via a reflex increase in sympathetic activity and a local effect to augment adrenergic constriction. Local cooling also initiates cutaneous dilatation, which may function to restrain cold-induced constriction. However, the underlying mechanisms and physiological role of cold-induced dilatation have not been defined. Experiments were performed to assess the role of endothelial-derived mediators in this response. In isolated pressurized cutaneous mouse tail arteries, cooling (28°C) did not affect the magnitude of dilatation to acetylcholine in preconstricted arteries. However, inhibition of nitric oxide (NO) [NG-nitro-l-arginine methyl ester (l-NAME)] and prostacyclin (PGI2) (indomethacin) reduced acetylcholine-induced dilatation at 37°C but not at 28°C, suggesting that cooling increased NO/PGI2-independent dilatation. This NO/PGI2-independent dilatation was reduced by inhibition of endothelial SK (UCL1684) and IK (TRAM34) Ca2+-activated K+-channels (KCa), consistent with endothelium-derived hyperpolarization (EDH). Cooling also increased dilatation to direct activation of KCa channels (SKA31, CyPPA) but did not affect dilatation to exogenous NO (DEA-NONOate). This cooling-induced increase in EDH-type dilatations was associated with divergent effects on potential downstream EDH mechanisms: cooling reduced dilatation to K+, which mimics an intercellular K+ cloud, but increased direct communication between endothelial and smooth muscle cells (myoendothelial coupling), assessed by cellular transfer of biocytin. Indeed, inhibition of gap junctions (carbenoxolone) abolished the EDH-type component of dilatation to acetylcholine during cooling but did affect NO-dominated dilatation at 37°C. Cooling also inhibited U46619 constriction that was prevented by inhibition of IK and SK KCa channels or inhibition of gap junctions. The results suggest that cooling dilates cutaneous arteries by increasing myoendothelial communication and amplifying EDH-type dilatation.NEW & NOTEWORTHY Cold causes cutaneous vasoconstriction to restrict heat loss. Although cold also initiates cutaneous dilatation, the mechanisms and role of this dilatation have not been clearly defined. This study demonstrates that cooling increases myoendothelial coupling between smooth muscle and endothelial cells in cutaneous arteries, which is associated with increased endothelium-derived hyperpolarization (EDH)-type dilatation. Dysfunction in this process may contribute to excessive cold-induced constriction and tissue injury.

LRRC8 channel activation and reduction in cytosolic chloride concentration during early differentiation of C2C12 myoblasts.

Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl- concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl- concentration by extracellular Cl- depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl- efflux early on during myogenic differentiation, and a moderate intracellular Cl- concentration is necessary for myoblast fusion.

A heterozygous mutation in GJB2 (Cx26F142L) associated with deafness and recurrent skin rashes results in connexin assembly deficiencies.

Mutations in GJB2 encoding Connexin 26 (CX26) are associated with hearing loss and hyperproliferative skin disorders of differing severity including keratitis-ichthyosis-deafness (KID) and Vohwinkel syndrome. A 6-year-old Caucasian girl who presented with recurrent skin rashes and sensorineural hearing loss harboured a heterozygous point mutation in GJB2 (c.424T > C; p.F142L). To characterize the impact of CX26F142L on cellular events. Plasmids CX26WT, CX26F142L, CX26G12R (KID) or CX26D66H (Vohwinkel) were transfected into HeLa cells expressing Cx26 or Cx43 or into HaCaT cells, a model keratinocyte cell line. Confocal microscopy determined protein localization. MTT assays assessed cell viability in the presence or absence of carbenoxolone, a connexin-channel blocker. Co-immunoprecipitation/Western blot analysis determined Cx43:Cx26 interactions. Quantitative real-time polymerase chain reaction assessed changes in gene expression of ER stress markers. Dye uptake assays determined Connexin-channel functionality. F142L and G12R were restricted to perinuclear areas. Collapse of the microtubule network, rescued by co-treatment with paclitaxel, occurred. ER stress was not involved. Cell viability was reduced in cells expressing F142L and G12R but not D66H. Unlike G12R that forms "leaky" hemichannels, F142L had restricted permeability. Cell viability of F142L and G12R transfected cells was greater in HeLa cells expressing Cx43 than in native Cx-free HeLa cells. Co-immunoprecipitation suggested a possible interaction between Cx43 and the three mutations. Expression of CX26F142L and G12R results in microtubule collapse, rescued by interaction with Cx43. The GJB2 mutations interacted with Cx43 suggesting that unique Cx43:Cx26 channels are central to the diverse phenotype of CX26 skin-related channelopathies.