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Aims: The development of novel and more effective medications for alcohol use disorder (AUD) is an important unmet medical need. Drug repositioning or repurposing is an appealing strategy to bring new therapies to the clinic because it greatly reduces the overall costs of drug development and expedites the availability of treatments to those who need them. Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, is a drug used clinically to treat hyperuricemia and gout due to its activity as an inhibitor of the kidneys' organic anion transporter that reclaims uric acid from urine. Probenecid also inhibits pannexin1 channels that are involved in purinergic neurotransmission and inflammation, which have been implicated in alcohol's effects and motivation for alcohol. Therefore, we tested the effects of probenecid on alcohol intake in rodents., Methods: We tested the effects of probenecid on operant oral alcohol self-administration in alcohol-dependent rats during acute withdrawal as well as in nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice., Results: Probenecid reduced alcohol intake in both dependent and nondependent rats and in the DID paradigm in mice without affecting water or saccharin intake, indicating that probenecid's effect was selective for alcohol and not the result of a general reduction in reward., Conclusions: These results raise the possibility that pannexin1 is a novel therapeutic target for the treatment of AUD. The clinical use of probenecid has been found to be generally safe, suggesting that it can be a candidate for drug repositioning for the treatment of AUD., (© The Author(s) 2019. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Prenatal fever could result in brain function impairments in the offspring. The present study investigated the effect of interleukin-6 (IL-6)-induced maternal fever on the offspring and the involvement of connexin 36 in this process. Pregnant C57BL/6J mice were injected with IL-6 on gestational day 15. The levels of iNOS and COX-2 were measured as an index of neuroinflammation in the brain of newborn pups. Offspring were treated with the connexin 36 (Cx36) inhibitor mefloquine at postnatal day (P)1-P3 or at P40-P42. Rotarod, grip traction, and foot fault tests were carried out to evaluate the motor behavior of adult offspring. Injection of IL-6 led to an elevation of the core temperature in the pregnant dams. Offspring of these dams showed significantly increased COX-2 and iNOS mRNA expression and protein levels in the whole-brain samples and significantly increased Cx36 in the cerebellum. Moreover, offspring of these dams showed motor deficits at an adult age. Neonatal administration of the Cx36 inhibitor mefloquine could prevent these motor deficits. Maternal fever during pregnancy induced by IL-6 injection could lead to neuroinflammation and motor deficits in the offspring. Neonatal inhibition of Cx36 could ameliorate the motor deficits in the offspring, indicating an involvement of Cx36 in the IL-6-induced maternal fever., (© 2019 S. Karger AG, Basel.)

In the domestic cat, nuclear maturation and embryo development after vitrification of immature oocytes have been obtained but developmental competence after warming remains low. It has been reported that during folliculogenesis, the association and communication between the oocyte and the surrounding cumulus cells through connexin-based gap junctions is essential for normal oocyte and follicular development. Gap junctions result from the head-to-head interaction of two hemichannels; however, there is always a population of hemichannels not incorporated into gap junctions. These unopposed hemichannels are normally closed but may open under certain stress conditions, potentially also during vitrification and warming, turning them into toxic pores inducing cell injury and cell death. The aim of our study was to test whether inhibiting connexin 37 (Cx37) and connexin 43 (Cx43) channels with the connexin-targeting peptide Gap26 during vitrification and warming of cat immature cumulus-oocyte-complexes (COCs) could improve oocyte maturation and competence of resultant blastocysts derived by parthenogenetic activation. In the first experiment, our immunostainings confirmed the presence of Cx43 protein in the cytoplasm of immature cat oocytes and in the plasma membranes of cumulus cells. In the second experiment, COCs were randomly divided in three different groups: a control group (control), a group vitrified without Gap26 (vitrified) and a group vitrified with Gap26 (vitrified-peptide). The maturation rate was checked and oocytes from all three different experimental groups were parthenogenetically activated and cultured in vitro until day 8. After vitrification and warming, 49% of the oocytes in the control group matured, while this was 8% and 19% in the vitrified and vitrified-peptide groups, respectively. Compared to the vitrified group, oocytes in the vitrified-peptide group had significantly larger maturation rates. No blastocysts were detected at day 8 in the vitrified group, while 2% and 13% of the oocytes further developed to blastocyst at day 8 in the vitrified-peptide and control non-vitrified group, respectively. We conclude that the use of Gap26 in vitrification and warming media to vitrify immature cat oocytes improves maturation success and allows such oocytes to reach the blastocyst stage (2%) at day 8 after parthenogenetic activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a K ATP channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca 2+ channel activity, cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and insulin secretion. Mefloquine was tested in the concentration range of 5-50 μM 25 μM mefloquine was as effective as 500 μM tolbutamide to depolarize the plasma membrane of beta cells, but did not induce action potentials. Rather, it abolished tolbutamide-induced action potentials and the associated increase of [Ca 2+ ] i . In the range of 5-50 μM mefloquine inhibited voltage-dependent Ca 2+ currents in primary beta cells as effectively as 1 μM nisoldipine, a specific blocker of L-type Ca 2+ channels. The Ca 2+ channel opening effect of Bay K8644 was completely antagonized by mefloquine. Likewise, the increase of [Ca 2+ ] i and of insulin secretion stimulated by 40 mM KCl, but not that by 30 mM glucose was antagonized by 50 μM mefloquine. Neither at 5 μM nor at 50 μM did mefloquin stimulate insulin secretion at basal glucose. In conclusion, mefloquine blocks K ATP channels and L-type Ca 2+ channels in pancreatic beta cells in the range from 5 to 50 μM. Thus it inhibits depolarization-induced insulin secretion, but in the presence of a stimulatory glucose concentration additional effects of mefloquine, possibly on intracellular Ca 2+ mobilization, and the metabolic amplification by glucose permit a sustained rate of secretion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny. SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH., (Copyright © 2018 the authors 0270-6474/18/387713-12$15.00/0.)

Leonurine has been reported to play an important role in ameliorating cognitive dysfunction, inhibiting ischemic stroke, and attenuating perihematomal edema and neuroinflammation in intracerebral hemorrhage. However, the exact mechanism and potential molecular targets of this effect remain unclear. Thus, in this study we investigated the neuroprotective effects of leonurine on hypoxia ischemia injury and explored the underlying mechanisms. An in vitro model of oxygen-glucose deprivation (OGD)-induced PC12 cells was established to mimic ischemic-like conditions. Cell viability, apoptosis, Cx36 and pCaMKII/CaMKII expression levels were evaluated after treatment with leonurine. The Cx36-selective antagonist mefloquine and CaMKII Inhibitor KN-93 were used to investigate the neuroprotective effect of leonurine on and the involvement of Cx36/CaMKII in this process. The results revealed that cell viability decreased and cell apoptosis and the protein expression of Cx36 and pCaMKII/CaMKII increased in the OGD-induced PC12 cells. Leonurine significantly increased cell viability and decreased cell apoptosis and the protein expression of Cx36 and pCaMKII/CaMKII in the OGD-induced PC12 cells. The specific inhibitor of Cx36 and CaMKII displayed similar protective effects. Moreover, the inhibition of Cx36 reduced pCaMKII levels and the ratio of pCaMKII/CaMKII in the OGD-induced PC12 cells, and vice versa. Taken together, these results suggest that leonurine might have a protective effect on OGD-induced PC12 cells through targeting the Cx36/CaMKII pathway. Thus, leonurine appears to have potential as a preventive or therapeutic drug against ischemic-induced neuronal injury., Competing Interests: The authors have declared that no competing interests exist.

Background Airway mucociliary transport is an important function for the clearance of inhaled foreign particulates in the respiratory tract. The present study aimed at investigating the regulatory mechanism of acetylcholine (Ach)-induced ciliary beat of the human nasal mucosa in ex vivo. Methods The inferior turbinate mucosa was collected from patients with chronic hypertrophic rhinitis during endoscopic surgery. The mucosa was cut into thin strips, and ciliary movement was observed under a phase-contrast light microscope with a high-speed digital video camera. The sample was alternatively subjected to scanning electron microscopic observation. Results Cilia on the turbinate epithelium were well preserved at the ultrastructural level. The baseline ciliary beat frequency (CBF) was 6.45 ± 0.32 Hz. CBF was significantly increased by stimulation with 100 µM Ach and 100 µM adenosine triphosphate. The Ach-induced CBF increase was completely inhibited by removing extracellular Ca 2+ . Significant inhibition of the Ach-induced CBF was also observed by the addition of 1 µM atropine, 40 µM 2-aminoethoxydiphenyl borate (inositol trisphosphate [IP 3 ] receptor antagonist), 10 µM carbenoxolone (pannexin-1 blocker), 1 mM probenecid (pannexin-1 blocker), 100 µM pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid (P2X antagonist), and 300 µM flufenamic acid (connexin blocker). Meanwhile, 30 nM bafilomycin A1 (vesicular transport inhibitor) did not inhibit the Ach-induced CBF increase., Conclusions: These results indicate that the regulatory mechanism of the Ach-induced ciliary beat is dependent on extracellular Ca 2+ and involves the muscarinic Ach receptor, IP 3 receptor, pannexin-1 channel, purinergic P2X receptor, and connexin channel. We proposed a tentative intracellular signaling pathway of the Ach-induced ciliary beat, in which the pannexin-1-P2X unit may play a central role in ciliary beat regulation.

Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation. Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI. Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro , Panx1 -deficient proximal tubule cells released less and retained more ATP under hypoxic stress. Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI., (Copyright © 2018 by the American Society of Nephrology.)

A rational treatment strategy for glioma, the most common primary central nervous system tumor, should focus on early invasive growth and resistance to current therapeutics. Connexin 43 (Cx43), a gap junction protein, plays important roles not only in the development of the central nervous system and but also in the progression of glioma. The different structural domains of Cx43, including extracellular loops, transmembrane domains, and an intracellular carboxyl terminal, have distinct functions in the invasion and proliferation of gliomas. Targeting these domains of Cx43, which is expressed in distinct patterns in the heterogeneous glioma cell population, can inhibit tumor cell invasion and new tumor formation. Thus, this review summarizes the structural characteristics of Cx43, the effects of regulating different Cx43 domains on the biological characteristics of glioma cells, intervention strategies targeting different domains of Cx43, and future research directions.

Since their characterization more than five decades ago, gap junctions and their structural proteins-the connexins-have been associated with cancer cell growth. During that period, the accumulation of data and molecular knowledge about this association revealed an apparent contradictory relationship between them and cancer. It appeared that if gap junctions or connexins can down regulate cancer cell growth they can be also implied in the migration, invasion and metastatic dissemination of cancer cells. Interestingly, in all these situations, connexins seem to be involved through various mechanisms in which they can act either as gap-junctional intercellular communication mediators, modulators of signalling pathways through their interactome, or as hemichannels, which mediate autocrine/paracrine communication. This complex involvement of connexins in cancer progression is even more complicated by the fact that their hemichannel function may overlap with other gap junction-related proteins, the pannexins. Despite this complexity, the possible involvements of connexins and pannexins in cancer progression and the elucidation of the mechanisms they control may lead to use them as new targets to control cancer progression. In this review, the involvements of connexins and pannexins in these different topics (cancer cell growth, invasion/metastasis process, possible cancer therapeutic targets) are discussed.

A high-fat diet (HFD) causes obesity by promoting excessive energy intake, and simultaneously, by disturbing the timing of energy intake. Restoring the feeding pattern is sufficient to prevent HFD-induced obesity in mice. However, the molecular mechanism(s) underlying HFD-induced feeding pattern disturbances remain elusive. Saturated fatty acids activate microglia and cause hypothalamic inflammation. Activated microglia cause neuroinflammation, which spreads via inflammatory cytokines and gap-junction hemichannels. However, the role of gap-junction hemichannels in HFD-induced obesity remains unaddressed. We used a novel, central-acting connexin inhibitor, INI-0602, which has high affinity for gap junction hemichannels and does not affect the induction of inflammatory cytokines. We analyzed ad libitum feeding behavior and locomotor activity in mice that were fed normal chow (NC), a HFD with elevated saturated fatty acids (SFAs), or a HFD with very high SFAs. We found that HFD feeding induced acute hyperphagia, mainly during the light cycle. Feeding pattern disturbances were more pronounced in mice that consumed the HFD with very high SFAs than in mice that consumed the HFD with elevated SFAs. When INI-0602 was administered before the HFD was introduced, it blocked the feeding pattern disturbance, but not locomotor activity disturbances; moreover, it prevented subsequent diet-induced obesity. However, when INI-0602 was administered after the HFD had disturbed the feeding pattern, it failed to restore the normal feeding pattern. Therefore, we propose that SFAs in HFDs played a major role in disrupting feeding patterns in mice. Moreover, the feeding pattern disturbance required the function of central, gap junction hemichannels at the initiation of a HFD. However, altering hemichannel function after the feeding pattern disturbance was established had no effect. Thus, preventing the occurrence of a feeding pattern disturbance by blocking the hemichannel pathway was associated with the prevention of the HFD-induced obesity in mice.

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone., Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone., Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure., Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition., (© 2017 American Heart Association, Inc.)

Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Coordination of vascular smooth muscle cell tone in resistance arteries plays an essential role in the regulation of peripheral resistance and overall blood pressure. Recent observations in animals have provided evidence for a coupling between adrenoceptors and Panx1 (pannexin-1) channels in the regulation of sympathetic nervous control of peripheral vascular resistance and blood pressure; however, evidence for a functional coupling in humans is lacking. We determined Panx1 expression and effects of treatment with the pharmacological Panx1 channel inhibitor probenecid on the vasoconstrictor response to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle. Probenecid treatment increased ( P <0.05) leg vascular conductance at baseline by ≈15% and attenuated ( P <0.05) the leg vasoconstrictor response to arterial infusion of tyramine (α1- and α2-adrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition of α1-adrenoceptors prevented the probenecid-induced increase in baseline leg vascular conductance, but did not alter the effect of probenecid on the vascular response to tyramine. No differences with probenecid treatment were detected in the forearm. These observations provide the first line of evidence in humans for a functional role of Panx1 channels in setting resting tone via α1-adrenoceptors and in the constrictive effect of noradrenaline via α2-adrenoceptors, thereby contributing to the regulation of peripheral vascular resistance and blood pressure in humans., (© 2017 American Heart Association, Inc.)

While gap junctions support the exchange of a number of molecules between neighboring cells, connexin hemichannels provide communication between the cytosol and the extracellular environment of an individual cell. The latter equally holds true for channels composed of pannexin proteins, which display an architecture reminiscent of connexin hemichannels. In physiological conditions, gap junctions are usually open, while connexin hemichannels and, to a lesser extent, pannexin channels are typically closed, yet they can be activated by a number of pathological triggers. Several agents are available to inhibit channels built up by connexin and pannexin proteins, including alcoholic substances, glycyrrhetinic acid, anesthetics and fatty acids. These compounds not always strictly distinguish between gap junctions, connexin hemichannels and pannexin channels, and may have effects on other targets as well. An exception lies with mimetic peptides, which reproduce specific amino acid sequences in connexin or pannexin primary protein structure. In this paper, a state-of-the-art overview is provided on inhibitors of cellular channels consisting of connexins and pannexins with specific focus on their mode-of-action and therapeutic potential., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis., Methods: Thioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5-10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5-10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA., Results: Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1., Conclusions: These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.

Connexins are ubiquitous channel forming proteins that assemble as plasma membrane hemichannels and as intercellular gap junction channels that directly connect cells. In the heart, gap junction channels electrically connect myocytes and specialized conductive tissues to coordinate the atrial and ventricular contraction/relaxation cycles and pump function. In blood vessels, these channels facilitate long-distance endothelial cell communication, synchronize smooth muscle cell contraction, and support endothelial-smooth muscle cell communication. In the central nervous system they form cellular syncytia and coordinate neural function. Gap junction channels are normally open and hemichannels are normally closed, but pathologic conditions may restrict gap junction communication and promote hemichannel opening, thereby disturbing a delicate cellular communication balance. Until recently, most connexin-targeting agents exhibited little specificity and several off-target effects. Recent work with peptide-based approaches has demonstrated improved specificity and opened avenues for a more rational approach toward independently modulating the function of gap junctions and hemichannels. We here review the role of connexins and their channels in cardiovascular and neurovascular health and disease, focusing on crucial regulatory aspects and identification of potential targets to modify their function. We conclude that peptide-based investigations have raised several new opportunities for interfering with connexins and their channels that may soon allow preservation of gap junction communication, inhibition of hemichannel opening, and mitigation of inflammatory signaling., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA A receptors. To test for effects on GABA A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a 'docked' counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.

Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: The present study aimed at investigating ATP release in response to acetylcholine (Ach) and pharmacologically elucidating the intracellular signal transduction pathway of this reaction in an ex vivo experiment., Methods: The inferior turbinate mucosa was collected from 21 patients with chronic hypertrophic rhinitis who underwent endoscopic turbinectomy. The mucosa was shaped into a filmy round piece, and incubated with chemical(s) in Hank's balanced salt solution for 10min. After incubation, the ATP concentration was measured by a luciferin-luciferase assay., Results: The baseline release of ATP without stimulus was 57.2±10.3fM. The ATP release was significantly increased by stimulation with 100μM Ach. The Ach-induced ATP release was completely inhibited by removing extracellular Ca 2+ . Significant inhibition of the Ach-induced ATP release was also observed by the addition of 1μM atropine, 40μM 2-APB, 10μM CBX, and 100μM PPADS, whereas 30nM bafilomycin A1 did not affect the ATP release., Conclusion: These results indicate that the Ach-induced ATP release from the human nasal mucosa is dependent on the pannexin-1 channel and purinergic P2X7 receptor, suggesting that these two molecules constitute a local autocrine/paracrine signaling system in the human nasal epithelium., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Current evidence indicates that transient receptor potential (TRP) channel activity involves a relationship between opening of pannexin-1 and release of ATP into the extracellular space. We examined the effects of agonists of thermosensitive TRP channels (TRPM8, TRPA1, TRPV1, and TRPV2) on ATP release from rat nasal mucosa, and measured ciliary beat frequency (CBF) using digital high-speed video imaging. Single-cell patch clamping from dissociated rat nasal columnar epithelial cells was performed to confirm the relationship between pannexin-1 and TRP. We demonstrated that ATP release and CBF were significantly potentiated by the heat-sensitive TRPV1 agonist capsaicin (10 μM), but not by other TRP agonists. Capsaicin-induced ATP release and CBF increase were significantly inhibited by the pannexin-1 blockers carbenoxolone (10 μM) and probenecid (300 μM). In addition, the voltage step-evoked currents in the presence of capsaicin were inhibited by the pannexin-1 blockers in single-cell patch clamping. Our results suggest the participation of TRPV1 and pannexin-1 in the physiologic functions of rat nasal mucosa.

Key Points: 5-HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH). In the present study, we show that, in addition to type I cells, adjacent glial-like type II cells express functional, ketanserin-sensitive 5-HT 2 receptors, and their stimulation increases cytoplasmic Ca 2+ derived from intracellular stores. In type II cells, 5-HT activated a ketanserin-sensitive inward current (I 5-HT ) that was similar to that (I UTP ) activated by the P2Y2R agonist, UTP. As previously shown for I UTP , I 5-HT was inhibited by BAPTA-AM and carbenoxolone (5 μm), a putative blocker of ATP-permeable pannexin (Panx)-1 channels; I UTP was reversibly inhibited by the specific Panx-1 mimetic peptide channel blocker, 10 Panx peptide. Paracrine stimulation of type II cells by 5-HT, leading to ATP release via Panx-1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea)., Abstract: Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5-HT and increased autocrine-paracrine 5-HT 2 receptor signalling contributes to sensory long-term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial-like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca 2+ ] i ) and activating carbenoxolone-sensitive, ATP-permeable, pannexin (Panx)-1-like channels. In the present study, using dissociated rat CB cultures, we found that 5-HT induced Δ[Ca 2+ ] i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5-HT-induced Ca 2+ response in type II cells was dose-dependent (EC 50 ∼183 nm) and largely inhibited by the 5-HT 2A receptor blocker, ketanserin (1 μm), and also arose mainly from intracellular stores. 5-HT also activated an inward current (I 5-HT ) in type II cells (EC 50 ∼200 nm) that was reversibly inhibited by ketanserin (1-10 nm), the Ca 2+ chelator BAPTA-AM (5 μm), and low concentrations of carbenoxolone (5 μm), a putative Panx-1 channel blocker. I 5-HT reversed direction at approximately -11 mV and was indistinguishable from the UTP-activated current (I UTP ). Consistent with a role for Panx-1 channels, I UTP was reversibly inhibited by the specific Panx-1 mimetic peptide blocker 10 Panx (100 μm), although not by its scrambled control peptide ( sc Panx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5-HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx-1 channels., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The membrane channel Pannexin 1 (Panx1) mediates apoptotic and inflammatory signaling cascades in injured neurons, responses previously shown to be sexually dimorphic under ischemic conditions. We tested the hypothesis that Panx1 plays an underlying role in mediating sex differences in stroke outcome responses. Middle-aged, 8-9 month old male and female wild type and Panx1 KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocyte and microglia activation were assessed 4 days later. The sexually dimorphic nature of Panx1 deletion was also explored by testing the effect of probenecid a known Panx1 blocker to alter stroke volume. Panx1 KO females displayed significantly smaller infarct volumes (~ 50 % reduction) compared to their wild-type counterparts, whereas no such KO effect occurred in males. This sex-specific effect of Panx1 KO was recapitulated by significant reductions in peri-infarct inflammation and astrocyte reactivity, as well as smaller infarct volumes in probenecid treated females, but not males. Finally, females showed overall, higher Panx1 protein levels than males under ischemic conditions. These findings unmask a deleterious role for Panx1 in response to permanent MCA occlusion, that is unique to females, and provide several new frameworks for understanding sex differences in stroke outcome.

Spreading depolarization is a wave of neuronal and glial depolarization. Within minutes after spreading depolarization, the neuronal hemichannel pannexin 1 (PANX1) opens and forms a pore complex with the ligand-gated cation channel P2X7, allowing the release of excitatory neurotransmitters to sustain spreading depolarization and activate neuroinflammation. Here, we explore the hypothesis that the P2X7-PANX1 pore complex is a critical determinant of spreading depolarization susceptibility with important consequences for neuroinflammation and trigeminovascular activation. We found that genetic loss of function or ablation of the P2x7 gene inhibits spreading depolarization. Moreover, pharmacological suppression of the P2X7-PANX1 pore complex inhibits spreading depolarization in mice carrying the human familial hemiplegic migraine type 1 R192Q missense mutation as well as in wild-type mice and rats. Pore inhibitors elevate the electrical threshold for spreading depolarization, and reduce spreading depolarization frequency and amplitude. Pore inhibitors also suppress downstream consequences of spreading depolarization such as upregulation of interleukin-1 beta, inducible nitric oxide synthase and cyclooxygenase-2 in the cortex after spreading depolarization. In addition, they inhibit surrogates for trigeminovascular activation, including expression of calcitonin gene-related peptide in the trigeminal ganglion and c-Fos in the trigeminal nucleus caudalis. Our results are consistent with the hypothesis that the P2X7-PANX1 pore complex is a critical determinant of spreading depolarization susceptibility and its downstream consequences, of potential relevance to its signature disorders such as migraine., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Spreading depolarization-induced focal dendritic swelling (beading) is an early hallmark of neuronal cytotoxic edema. Pyramidal neurons lack membrane-bound aquaporins posing a question of how water enters neurons during spreading depolarization. Recently, we have identified chloride-coupled transport mechanisms that can, at least in part, participate in dendritic beading. Yet transporter-mediated ion and water fluxes could be paralleled by water entry through additional pathways such as large-pore pannexin-1 channels opened by spreading depolarization. Using real-time in vivo two-photon imaging in mice with pharmacological inhibition or conditional genetic deletion of pannexin-1, we showed that pannexin-1 channels are not required for spreading depolarization-induced focal dendritic swelling.

Pannexins constitute a relatively new family of transmembrane proteins that form channels linking the cytoplasmic compartment with the extracellular environment. The presence of pannexin1 in the liver has been documented previously, where it underlies inflammatory responses, such as those occurring upon ischemia-reperfusion injury. In the present study, we investigated whether pannexin1 plays a role in acute drug-induced liver toxicity. Hepatic expression of pannexin1 was characterized in a mouse model of acetaminophen-induced hepatotoxicity. Subsequently, mice were overdosed with acetaminophen followed by treatment with the pannexin1 channel inhibitor 10 Panx1. Sampling was performed 1, 3, 6, 24 and 48 h after acetaminophen administration. Evaluation of the effects of pannexin1 channel inhibition was based on a number of clinically relevant readouts, including protein adduct formation, measurement of aminotransferase activity and histopathological examination of liver tissue as well as on a series of markers of inflammation, oxidative stress and regeneration. Although no significant differences were found in histopathological analysis, pannexin1 channel inhibition reduced serum levels of alanine and aspartate aminotransferase. This was paralleled by a reduced amount of neutrophils recruited to the liver. Furthermore, alterations in the oxidized status were noticed with upregulation of glutathione levels upon suppression of pannexin1 channel opening. Concomitant promotion of regenerative activity was detected as judged on increased proliferating cell nuclear antigen protein quantities in 10 Panx1-treated mice. Pannexin1 channels are important actors in liver injury triggered by acetaminophen. Inhibition of pannexin1 channel opening could represent a novel approach for the treatment of drug-induced hepatotoxicity.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide ( 10 panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

Neuropathic pain symptoms respond poorly to available therapeutics, with most treated patients reporting unrelieved pain and significant impairment in daily life. Here, we show that Pannexin 1 (Panx1) in hematopoietic cells is required for pain-like responses following nerve injury in mice, and a potential therapeutic target. Panx1 knockout mice (Panx1 -/- ) were protected from hypersensitivity in two sciatic nerve injury models. Bone marrow transplantation studies show that expression of functional Panx1 in hematopoietic cells is necessary for mechanical hypersensitivity following nerve injury. Reconstitution of irradiated Panx1 knockout mice with hematopoietic Panx1 -/- cells engineered to re-express Panx1 was sufficient to recover hypersensitivity after nerve injury; this rescue required expression of a Panx1 variant that can be activated by G protein-coupled receptors (GPCRs). Finally, chemically distinct Panx1 inhibitors blocked development of nerve injury-induced hypersensitivity and partially relieved this hypersensitivity after it was established. These studies indicate that Panx1 expressed in immune cells is critical for pain-like effects following nerve injury in mice, perhaps via a GPCR-mediated activation mechanism, and suggest that inhibition of Panx1 may be useful in treating neuropathic pain., Competing Interests: The authors declare no competing financial interests.

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.

Aims: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca 2+ /purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca 2+  entry and ATP release contributes to smooth muscle cell (SMC) Ca 2+  dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca 2+  and ATP signalling to investigate their influence on SMC Ca 2+  dynamics and vessel contractility., Methods and Results: Confocal Ca 2+  imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca 2+  oscillations were inhibited by blocking IP 3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca 2+  concentration, indicating it facilitated Ca 2+  entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC., Conclusions: These results indicate that Cx43 hemichannels contribute to SMC Ca 2+  dynamics and contractility, by facilitating Ca 2+  entry, ATP release, and purinergic signalling., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.)

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels., Competing Interests: The authors declare no competing financial interests.

Background: In Alzheimer's disease (AD), modification of astrocytic properties is a well-known and documented fact, but their involvement in pathophysiology has only been examined in recent years. One distinct hallmark of AD is reactive gliosis which are represented in microglial and astrocytic phenotype changes. This reactive gliosis has been associated with changes in the expression and function of connexins. Connexins are proteins that can form gap junction channels and hemichannels, and in a disease context, have shown increased expression in astrocytes that contact amyloid plaques in vivo. Amyloid plaques are aggregates of the amyloid-beta protein, which present in the AD brain in patients and in animal models., Methods: Murine AD models demonstrate changes in connexin channel activity which mirror in cell culture systems treated with amyloid-beta peptide. This has been closely studied in the familial AD mouse model APPSwe/ PS1dE9 where the implications of connexin channel functions have been examined., Results: These studies demonstrate that while gap junctional communication was unaffected, hemichannel activation could be detected in the astrocytes of hippocampal slices containing amyloid-beta plaques. Most critically, the activation of hemichannels is associated with the release of gliotransmitters (such as ATP and glutamate) which results in the maintenance of a high intracellular free Ca2+ concentration within astrocytes, which initiates the start of a vicious cycle. Strategies that target astroglial connexin hemichannels include the knocking out of the connexin 43 gene in astrocytes of the APPSwe/PS1dE9 mice, as well as using various pharmacological tools. This results in the decrease of gliotransmitter release and the alleviation of neuronal damage. This includes the reduction of oxidative stress and neuritic dystrophies in neurons that are typically associated with plaque formation in the hippocampus. Concusion: In this review, we summarize recent findings on astroglial connexin channels in the neurodegenerative process of Alzheimer's disease, and discuss how this can be a strategy in AD treatment to block the activity of hemichannels in astrocytes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Alpha-type retinal ganglion cells (alpha cells) of the same class in mammalian retina are connected by gap junctions. Electrical synapses between alpha cells were examined using combined techniques of dual patch-clamp recordings, intracellular labeling and electron microscopy in the albino rat retina. In simultaneous dual whole-cell recordings from pairs of neighboring alpha cells, bidirectional electrical synapses with symmetrical junction conductance were observed in pairs with cells of the same morphological type. Regulatory domains of gap junction protein subunit connexins in electrical synapses between alpha cells by extracellular and intracellular ligands investigated by dual whole-patch clamp recordings. I examined how passage currents through electrical synapses between alpha cells are modulated by specific antibodies against connexin36 proteins, and extracellular or intracellular application of ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance (Gj) (1.35[Formula: see text][Formula: see text][Formula: see text]0.51[Formula: see text]nS). Experimental results show that high level of intracellular cyclic AMP within examined cells suppress electrical synapses between the neighboring cells. Gj between examined cells reduced to 0.15[Formula: see text][Formula: see text][Formula: see text]0.04[Formula: see text]nS. Under application of dopamine (1.25[Formula: see text][Formula: see text][Formula: see text]0.06[Formula: see text]nS) or intracellular cyclic GMP (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS), however, Gj also remains as in the control level. Intracellular application of an antibody against the cytoplasmic loop of connexin36 reduced G j (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS). Cocktail of the antibody against cytoplasmic connexin36 and intracellular cyclic AMP leaves Gj as in the level by single involvement of the cytoplasmic antibody. The elimination of Gj by the cytoplasmic antibody was in a dose-dependent manner. These results suggest that binding domains against cyclic AMP may be present in the cytoplasmic sites of connexin proteins to regulate channel opening of gap junctions between mammalian retinal alpha ganglion cells.

Inflammation is extremely important in the development of cerebral ischemia/reperfusion injury. Pannexin 1 (Panx1) channel has been reported to activate inflammasome in astrocytes and be involved in ischemic injury, but this damage effect is reversed by a Panx1 inhibitor-probenecid. However, the mechanism of probenecid protects against cerebral ischemia/reperfusion injury remains unclear. In present study, we hypothesized that probenecid protected astrocytes from ischemia/reperfusion injury in vitro by modulating the inflammasome. Primary cultured neocortical astrocytes were exposed to oxygen-glucose deprivation/reoxygenation (OGD/RX) and probenecid was added in this model. Viability and nuclear morphology of astrocytes, production of reactive oxygen species (ROS), protein expressions of NLRP3 (NOD-like receptor protein 3), caspase-1, and AQP4 (Aquaporins 4), as well as release of cellular HMGB1 and IL-1β were observed to evaluate the effect and mechanisms of probenecid on OGD/reoxygenated astrocytes. Probenecid did not affect cell viability at concentrations of 1, 5, 10, and 100μM but induced significant astrocytes death at 500μM. Probenecid inhibited cell death and ROS generation in astrocytes subjected to 6h of OGD and 24h of reoxygenation. The expression levels of NLRP3, caspase-1, and AQP4 increased after 6h of OGD, but probenecid treatment attenuated this increase. Moreover, the extracellular release of IL-1β and HMGB1 from OGD/reoxygenated astrocytes increased significantly. However, treatment by probenecid resulted in substantial reduction of these proteins levels in extracellular space. In conclusion, The Panx1 inhibitor, probenecid, which was administered before OGD, provided protective effects on the OGD/reoxygenation model of cultured astrocytes by modulating inflammasome activity and downregulating AQP4 expression., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Study Objectives: Modafinil is a non-amphetaminic wake-promoting compound used as therapy against sleepiness and narcolepsy. Its mode of action is complex, but modafinil has been recently proposed to act as a cellular-coupling enhancer in glial cells, through modulation of gap junctions constituted by connexins. The present study investigated in mice the impact of connexins on the effects of modafinil using connexin inhibitors., Methods: Modafinil was administered alone or combined with inhibitors of astrocyte connexin, meclofenamic acid, or flecainide, respectively, acting on Cx30 and Cx43. Sleep-wake states were monitored in wild-type and narcoleptic orexin knockout mice. A spontaneous alternation task was used to evaluate working memory in wild-type mice. The effects of the compounds on astroglial intercellular coupling were determined using dye transfer in acute cortical slices., Results: Meclofenamic acid had little modulation on the effects of modafinil, but flecainide enhanced the wake-promoting and pro-cognitive effects of modafinil. Co-administration of modafinil/flecainide resulted in a marked decrease in the number and duration of direct transitions to rapid eye movement sleep, which are characteristic of narcoleptic episodes in orexin knockout mice. Furthermore, modafinil enhanced the connexin-mediated astroglial cell coupling, whereas flecainide reduced it. Finally, this modafinil-induced effect was reversed by co-administration with flecainide., Conclusions: Our study indicates that flecainide impacts the pharmacological effects of modafinil, likely through the normalization of Cx30-dependent gap junctional coupling in astroglial networks. The enhancement of the wake-promoting, behavioral, and cognitive outcomes of modafinil demonstrated here with flecainide would open new perspectives in the management of sleep disorders such as narcolepsy., Commentary: A commentary on this article appears in this issue on page 1175., (© 2016 Associated Professional Sleep Societies, LLC.)

Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40-/-), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40-/- but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40-/- mice. As a result, Cx40-/- mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment.

The cardiovascular system is extensively innervated by the autonomic nervous system, and the autonomic modulation including sympathetic innervation is crucial to the function of heart during normal and ischemic conditions. Severe myocardial ischemia could cause acute myocardial infarction, which is one of the leading diseases in the world. Thus studying the sympathetic modulation during ischemia could reduce the probability of myocardial infarction and further heart failure. The neurotransmitter ATP is released by myocardial cells during ischemia; however, the effect of ATP release remains elusive. We examined whether ATP released during ischemia functions as a neurotransmitter that activates sympathetic nerve in the heart. A novel technique of recording the sympathetic fiber calcium imaging in mouse cardiac tissue slices was used. We have applied the Cre/loxP system to specifically express GCaMP3, a genetically encoded calcium indicator, in the sympathetic nerve. Using this technique, we found that ATP released by myocardial cells through Pannexin-1 channel during ischemia could evoke calcium responses in cardiac sympathetic nerve fibers. Our study provides a new approach to study the cell and nerve interaction in the cardiac system, as well as a new understanding of ATP function during ischemia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating., (© 2016 Michalski and Kawate.)

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking., (© 2016 Society for Reproduction and Fertility.)

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Neurological impairments are caused by axonal damage due to demyelination and neuroinflammation within the central nervous system. T cells mediate the neuroinflammation. The activation of T cells is induced by the release of adenosine triphosphate and involves purinergic receptors as well as pannexin (Panx) proteins. As Panx1 is expressed on T cells, we here propose that application of probenecid, a known Panx inhibitor, will prevent the onset of clinical symptoms in a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE) model. EAE-induced mice received daily injections of probenecid. Disease scores, T cell numbers, and microglia activation were compared between experimental groups. Probenecid treatment resulted in lower disease scores as compared to EAE animals. Probenecid-treated animals also displayed fewer inflammatory lesions. Microglia activation was not altered by treatment. In conclusion, probenecid prevented the onset of EAE.

Hypothermia is partially neuroprotective after neonatal hypoxic-ischemic encephalopathy. Blockade of connexin hemichannels can improve recovery of brain activity and cell survival after ischemia in near-term fetal sheep. In this study, we investigated whether combining delayed hypothermia with connexin hemichannel blockade with intracerebroventricular infusion of a mimetic peptide can further improve outcomes after cerebral ischemia. Fetal sheep (0.85 gestation) received 30 minutes of cerebral ischemia followed by a 3-hour recovery period before treatment was started. Fetuses were randomized to one of the following treatment groups: normothermia (n=8), hypothermia for 3 days (n=8), connexin hemichannel blockade (50 μmol/L intracerebroventricular over 1 hour followed by 50 μmol/L over 24 hours, n=8) or hypothermia plus hemichannel blockade (n=7). After 7 days recovery, hypothermia was associated with reduced seizure burden, improved electroencephalographic (EEG) power, and a significant increase in neuronal and oligodendrocyte survival and reduced induction of Iba1-positive microglia. In contrast, although hemichannel blockade reduced seizure burden, there was no effect on EEG power or histology (P<0.05). There was no further improvement in outcomes with combined hypothermia plus hemichannel blockade. In conclusion, these data show that there is no additive neuroprotection with combined hypothermia and hemichannel blockade after cerebral ischemia in near-term fetal sheep.

Mutations in connexin-31 (Cx31) are associated with multiple human diseases, including familial erythrokeratodermia variabilis (EKV). The pathogenic mechanism of EKV-associated Cx31 mutants remains largely elusive. Here, we show that EKV-pathogenic Cx31 mutants are un/misfolded and temperature sensitive. In Drosophila, expression of pathogenic Cx31, but not wild-type Cx31, causes depigmentation and degeneration of ommatidia that are rescued by expression of either dBip or dHsp70. Ectopic expression of Cx31 in mouse skin results in skin abnormalities resembling human EKV. The affected tissues show remarkable disrupted gap junction formation and significant upregulation of chaperones Bip and Hsp70 as well as AP-1 proteins c-Fos and JunB, in addition to molecular signatures of skin diseases. Consistently, c-Fos, JunB, Bip and Hsp70 are strikingly higher in keratinocytes of EKV patients than their matched control individuals. Furthermore, a druggable AP-1 inhibitory small molecule suppresses skin phenotype and pathological abnormalities of transgenic Cx31 mice. The study suggests that Cx31 mutant proteins are un/misfolded to cause EKV likely via an AP-1-mediated mechanism and identifies a small molecule with therapeutic potential of the disease., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Leukemia encompasses several hematological malignancies with shared phenotypes that include rapid proliferation, abnormal leukocyte self-renewal, and subsequent disruption of normal hematopoiesis. While communication between leukemia cells and the surrounding stroma supports tumor survival and expansion, the mechanisms underlying direct leukemia cell-cell communication and its contribution to tumor growth are undefined. Gap junctions are specialized intercellular connections composed of connexin proteins that allow free diffusion of small molecules and ions directly between the cytoplasm of adjacent cells. To characterize homotypic leukemia cell communication, we employed in vitro models for both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and measured gap junction function through dye transfer assays. Additionally, clinically relevant gap junction inhibitors, carbenoxolone (CBX) and 1-octanol, were utilized to uncouple the communicative capability of leukemia cells. Furthermore, a qRT-PCR screen revealed several connexins with higher expression in leukemia cells compared with normal hematopoietic stem cells. Cx25 was identified as a promising adjuvant therapeutic target, and Cx25 but not Cx43 reduction via RNA interference reduced intercellular communication and sensitized cells to chemotherapy. Taken together, our data demonstrate the presence of homotypic communication in leukemia through a Cx25-dependent gap junction mechanism that can be exploited for the development of anti-leukemia therapies.

Oxaliplatin-induced neurotoxicity (OIN) is a common complication of chemotherapy without effective treatment. In order to clarify the mechanisms of both acute and chronic OIN, we used an ex-vivo mouse sciatic nerve model. Exposure to 25 μM oxaliplatin caused a marked prolongation in the duration of the nerve evoked compound action potential (CAP) by nearly 1200% within 300 min while amplitude remained constant for over 20 h. This oxaliplatin effect was almost completely reversed by the gap junction (GJ) inhibitor octanol in a concentration-dependent manner. Further GJ blockers showed similar effects although with a narrower therapeutic window. To clarify the target molecule we studied sciatic nerves from connexin32 (Cx32) and Cx29 knockout (KO) mice. The oxaliplatin effect and neuroprotection by octanol partially persisted in Cx29 better than in Cx32 KO nerves, suggesting that oxaliplatin affects both, but Cx32 GJ channels more than Cx29 hemichannels. Oxaliplatin also accelerated neurobiotin uptake in HeLa cells expressing the human ortholog of Cx29, Cx31.3, as well as dye transfer between cells expressing the human Cx32, and this effect was blocked by octanol. Oxaliplatin caused no morphological changes initially (up to 3 h of exposure), but prolonged nerve exposure caused juxtaparonodal axonal edema, which was prevented by octanol. Our study indicates that oxaliplatin causes forced opening of Cx32 channels and Cx29 hemichannels in peripheral myelinated fibers leading to disruption of axonal K(+) homeostasis. The GJ blocker octanol prevents OIN at very low concentrations and should be further studied as a neuroprotectant., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Anticancer therapy based on the repeated administration of oxaliplatin is limited by the development of a neuropathic syndrome difficult to treat. Oxaliplatin neurotoxicity is based on complex nervous mechanisms, the comprehension of the role of single neurotransmitters and the knowledge of the signal flow among cells is matter of importance to improve therapeutic chances. In a rat model of oxaliplatin-induced neuropathy, we report increased P2X7-evoked glutamate release from cerebrocortical synaptosomes. The release was abolished by the P2X7 receptor (P2X7R) antagonists Brilliant-Blue-G (BBG) and A-438079, and significantly reduced by Carbenoxolone and the Pannexin 1 (Panx1) selective inhibitors Erioglaucine and (10)Panx suggesting the recruitment of Panx1. Aimed to evaluate the significance of P2X7R-Panx1 system activation in pain generated by oxaliplatin, pharmacological modulators were spinally infused by intrathecal catheter in oxaliplatin-treated animals. BBG, Erioglaucine and (10)Panx reverted oxaliplatin-dependent pain. Finally, the influence of the P2X7R-Panx1 system blockade on oxaliplatin anticancer activity was evaluated on the human colon cancer cell line HT-29. Prevention of HT-29 apoptosis and mortality was dependent by kind and concentration of P2X7R antagonists. On the contrary, the inhibition of Panx1 did not alter oxaliplatin lethality in tumor cells. It is concluded that glutamate release dependent on P2X7R is increased in cerebrocortical nerve terminals from oxaliplatin-treated rats; the increase is mediated by functional recruitment of Panx1; P2X7R antagonists and Panx1 inhibitors revert oxaliplatin-induced neuropathic pain; Panx1 inhibitors do not alter the oxaliplatin-induced mortality of cancer cells HT-29. The inhibition of Panx1 channel is suggested as a new and safe pharmacological target., (Copyright © 2015 Elsevier Ltd. All rights reserved.)