The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity.

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.

Pannexin channel 1, P2×7 receptors, and Dimethyl Sulfoxide mediate pain responses in zebrafish.

The zebrafish has been considered an ideal model for studies of complex behaviors since its behavioral repertoire is well described. Therefore, this study evaluated the perceived pain through behavioral changes in zebrafish larvae. Here we investigated the Acetic Acid (AA) effects on zebrafish larvae exposed in a short-time period (60 s) and the preventive effect from routinely used compounds, Dimethyl Sulfoxide (DMSO), Ethanol (EtOH), Ibuprofen (IBP), and Paracetamol (PAR). In addition, the effect of P2×7 antagonist, A740003, and pannexin channel 1 (PANX-1) inhibitor Probenecid (PROB) on AA-induced behavioral changes were evaluated. AA impaired the distance covered, acceleration, movement, and latency to the first entry in the center from 5 dpf exposed larvae. At 0.050% AA, PAR prevented alterations from the distance covered, acceleration, and movement. Surprisingly, 0.3% DMSO prevented behavioral changes induced by AA. However, the effects from 0.2% DMSO were not prominent. We used 0.2% DMSO as a PROB diluent. PROB prevented the changes in distance and movement observed at both AA concentrations (0.0025% and 0.05%) tested. Since EtOH had no analgesic properties, we used it as an A740003 vehicle to observe the analgesic effects of this compound. As noted, A740003 did not prevent the behavioral changes in the AA-induced pain model. In contrast, 0.2% DMSO and PROB prevented AA-induced behavioral changes. These data enforce that zebrafish could be used in translational studies since this species has behavioral responses related to pain in the early stages of development and responses to analgesics similar to observed in mammals.

Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain.

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Over-activated hemichannels: A possible therapeutic target for human diseases.

In our body, all the cells are constantly sharing chemical and electrical information with other cells. This intercellular communication allows them to respond in a concerted way to changes in the extracellular milieu. Connexins are transmembrane proteins that have the particularity of forming two types of channels; hemichannels and gap junction channels. Under normal conditions, hemichannels allow the controlled release of signaling molecules to the extracellular milieu. However, under certain pathological conditions, over-activated hemichannels can induce and/or exacerbate symptoms. In the last decade, great efforts have been put into developing new tools that can modulate these over-activated hemichannels. Small molecules, antibodies and mimetic peptides have shown a potential for the treatment of human diseases. In this review, we summarize recent findings in the field of hemichannel modulation via specific tools, and how these tools could improve patient outcome in certain pathological conditions.

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.

AIM2-driven inflammasome activation in heart failure.

AIMS: Interleukin-1ß (IL-1ß) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 ß are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1ß. METHODS AND RESULTS: Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. CONCLUSIONS: This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.

Design and synthesis of the first indole-based blockers of Panx-1 channel.

Panx-1 is a membrane channel protein involved in some pathologies such as ischemic stroke, cancer and neuropathic pain, thus representing a promising therapeutic target. We present here a study aimed at obtaining the first class of selective Panx-1 blockers, a new topic for pharmaceutical chemistry, since all compounds used so far for the study of this channel have different primary targets. Among various scaffolds analyzed, the indole nucleous emerged, whose elaboration yielded interesting Panx-1 blockers, such as the potent 5-sulfamoyl derivatives 14c and 15b (I% = 100 at 50 µM). In vivo tests performed in the mouse model of oxaliplatin-induced neuropathy, demonstrated that the hypersensitivity was completely reverted by treatment with 15b (1 nmol, administered intrathecally), suggesting a relationship between this effect and the channel blocking ability. Finally, we decided to perform a virtual screening study on compounds 5b, 6l and 14c using a recently resolved cryo-EM structure of hPanx-1 channel, to try to relate the potency of our new inhibitors.

Human immunodeficiency virus-1/simian immunodeficiency virus infection induces opening of pannexin-1 channels resulting in neuronal synaptic compromise: A novel therapeutic opportunity to prevent NeuroHIV.

In healthy conditions, pannexin-1 (Panx-1) channels are in a close state, but in several pathological conditions, including human immunodeficiency virus-1 (HIV) and NeuroHIV, the channel becomes open. However, the mechanism or contribution of Panx-1 channels to the HIV pathogenesis and NeuroHIV is unknown. To determine the contribution of Panx-1 channels to the pathogenesis of NeuroHIV, we used a well-established model of simian immunodeficiency virus (SIV) infection in macaques (Macaca mulatta) in the presence of and absence of a Panx-1 blocker to later examine the synaptic/axonal compromise induced for the virus. Using Golgi's staining, we demonstrated that SIV infection compromised synaptic and axonal structures, especially in the white matter. Blocking Panx-1 channels after SIV infection prevented the synaptic and axonal compromise induced by the virus, especially by maintaining the more complex synapses. Our data demonstrated that targeting Panx-1 channels can prevent and maybe revert brain synaptic compromise induced by SIV infection.

Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation.

Deletion of pannexin-1 (Panx-1) leads not only to a reduction in endothelium-derived hyperpolarization but also to an increase in NO-mediated vasodilation. Therefore, we evaluated the participation of Panx-1-formed channels in the control of membrane potential and [Ca2+]i of endothelial cells. Changes in NO-mediated vasodilation, membrane potential, superoxide anion (O2 ·-) formation, and endothelial cell [Ca2+]i were analyzed in rat isolated mesenteric arterial beds and primary cultures of mesenteric endothelial cells. Inhibition of Panx-1 channels with probenecid (1 mM) or the Panx-1 blocking peptide 10Panx (60 µM) evoked an increase in the ACh (100 nM)-induced vasodilation of KCl-contracted mesenteries and in the phosphorylation level of endothelial NO synthase (eNOS) at serine 1177 (P-eNOSS1177) and Akt at serine 473 (P-AktS473). In addition, probenecid or 10Panx application activated a rapid, tetrodotoxin (TTX, 300 nM)-sensitive, membrane potential depolarization and [Ca2+]i increase in endothelial cells. Interestingly, the endothelial cell depolarization was converted into a transient spike after removing Ca2+ ions from the buffer solution and in the presence of 100 µM mibefradil or 10 µM Ni2+. As expected, Ni2+ also abolished the increment in [Ca2+]i. Expression of Nav1.2, Nav1.6, and Cav3.2 isoforms of voltage-dependent Na+ and Ca2+ channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O2 ·- production. Treatment with 10 µM TEMPOL or 100 µM apocynin prevented the increase in O2 ·- formation, ACh-induced vasodilation, P-eNOSS1177, and P-AktS473 observed in response to Panx-1 inhibition. These findings indicate that the Panx-1 channel blockade triggers a novel complex signaling pathway initiated by the sequential activation of TTX-sensitive Nav channels and Cav3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOSS1177, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.

Inhibition of astrocyte hemichannel improves recovery from spinal cord injury.

Spinal cord injury (SCI) causes severe disability, and the current inability to restore function to the damaged spinal cord leads to lasting detrimental consequences to patients. One strategy to reduce SCI morbidity involves limiting the spread of secondary damage after injury. Previous studies have shown that connexin 43 (Cx43), a gap junction protein richly expressed in spinal cord astrocytes, is a potential mediator of secondary damage. Here, we developed a specific inhibitory antibody, mouse-human chimeric MHC1 antibody (MHC1), that inhibited Cx43 hemichannels, but not gap junctions, and reduced secondary damage in 2 incomplete SCI mouse models. MHC1 inhibited the activation of Cx43 hemichannels in both primary spinal astrocytes and astrocytes in situ. In both SCI mouse models, administration of MHC1 after SCI significantly improved hind limb locomotion function. Remarkably, a single administration of MHC1 30 minutes after injury improved the recovery up to 8 weeks post-SCI. Moreover, MHC1 treatment decreased gliosis and lesion sizes, increased white and gray matter sparing, and improved neuronal survival. Together, these results suggest that inhibition of Cx43 hemichannel function after traumatic SCI reduces secondary damage, limits perilesional gliosis, and improves functional recovery. By targeting hemichannels specifically with an antibody, this study provides a potentially new, innovative therapeutic approach in treating SCI.

Connexin hemichannel inhibitors with a focus on aminoglycosides.

Connexins are membrane proteins involved directly in cell-to-cell communication through the formation of gap-junctional channels. These channels result from the head-to-head docking of two hemichannels, one from each of two adjacent cells. Undocked hemichannels are also present at the plasma membrane where they mediate the efflux of molecules that participate in autocrine and paracrine signaling, but abnormal increase in hemichannel activity can lead to cell damage in disorders such as cardiac infarct, stroke, deafness, cataracts, and skin diseases. For this reason, connexin hemichannels have emerged as a valid therapeutic target. Know small molecule hemichannel inhibitors are not ideal leads for the development of better drugs for clinical use because they are not specific and/or have toxic effects. Newer inhibitors are more selective and include connexin mimetic peptides, anti-connexin antibodies and drugs that reduce connexin expression such as antisense oligonucleotides. Re-purposed drugs and their derivatives are also promising because of the significant experience with their clinical use. Among these, aminoglycoside antibiotics have been identified as inhibitors of connexin hemichannels that do not inhibit gap-junctional channels. In this review, we discuss connexin hemichannels and their inhibitors, with a focus on aminoglycoside antibiotics and derivatives of kanamycin A that inhibit connexin hemichannels, but do not have antibiotic effect.

Capillaries communicate with the arteriolar microvascular network by a pannexin/purinergic-dependent pathway in hamster skeletal muscle.

We sought to determine if a pannexin/purinergic-dependent intravascular communication pathway exists in skeletal muscle microvasculature that facilitates capillary communication with upstream arterioles that control their perfusion. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries and observed the vasodilatory response in the associated upstream 4A arteriole. We stimulated capillaries with vasodilators relevant to muscle contraction: 10-6 M S-nitroso-N-acetyl-dl-penicillamine (SNAP; nitric oxide donor), 10-6 M adenosine, 10 mM potassium chloride, 10-5 M pinacidil, as well as a known initiator of gap-junction-dependent intravascular communication, acetylcholine (10-5 M), in the absence and the presence of the purinergic membrane receptor blocker suramin (10-5 M), pannexin blocker mefloquine (2 × 10-5 M), or probenecid (5 × 10-6 M) and gap-junction inhibitor halothane (0.07%) applied in the transmission pathway, between the capillary stimulation site and the upstream 4A observation site. Potassium chloride, SNAP, and adenosine-induced upstream vasodilations were significantly inhibited by suramin, mefloquine, and probenecid but not halothane, indicating the involvement of a pannexin/purinergic-dependent signaling pathway. Conversely, SNAP-induced upstream vasodilation was only inhibited by halothane indicating that communication was facilitated by gap junctions. Both pinacidil and acetylcholine were inhibited by suramin but only acetylcholine was inhibited by halothane. These data demonstrate the presence of a pannexin/purinergic-dependent communication pathway between capillaries and upstream arterioles controlling their perfusion. This pathway adds to the gap-junction-dependent pathway that exists at this vascular level as well. Given that vasodilators relevant to muscle contraction can use both of these pathways, our data implicate the involvement of both pathways in the coordination of skeletal muscle blood flow.NEW & NOTEWORTHY Blood flow control during increased metabolic demand in skeletal muscle is not fully understood. Capillaries have been implicated in controlling blood flow to active skeletal muscle, but how capillaries communicate to the arteriolar vascular network is not clear. Our study uncovers a novel pathway through which capillaries can communicate to upstream arterioles to cause vasodilation and therefore control perfusion. This work implicates a new vascular communication pathway in blood flow control in skeletal muscle.

Connexins as therapeutic targets in neurological and neuropsychiatric disorders.

Astrocytes represent the reticular part of the central nervous system; gap junctions formed by connexins Cx43, Cx30- and Cx26 provide for homocellular astrocyte-astrocyte coupling, whereas connexins Cx30, Cx32, Cx43, and Cx47 connect astrocytes and oligodendrocytes. Astroglial networks are anatomically and functionally segregated being homologous to neuronal ensembles. Connexons, gap junctions and hemichannels (unpaired connexons) are affected in various neuropathologies from neuropsychiatric to neurodegenerative diseases. Manipulation of astrocytic connexins modulates the size and outreach of astroglial syncytia thus affecting astroglial homeostatic support. Modulation of astrocytic connexin significantly modifies pharmacological profile of many CNS drugs, which represents an innovative therapeutic approach for CNS disorders; this approach is now actively tested in pre-clinical and clinical studies. Wide combination of connexin modulators with CNS drugs open new promising perspectives for fundamental studies and therapeutic strategies.

Differential Action of Connexin Hemichannel and Pannexin Channel Therapeutics for Potential Treatment of Retinal Diseases.

Dysregulation of retinal function in the early stages of light-induced retinal degeneration involves pannexins and connexins. These two types of proteins may contribute to channels that release ATP, leading to activation of the inflammasome pathway, spread of inflammation and retinal dysfunction. However, the effect of pannexin channel block alone or block of both pannexin channels and connexin hemichannels in parallel on retinal activity in vivo is unknown. In this study, the pannexin channel blocker probenecid and the connexin hemichannel blocker tonabersat were used in the light-damaged rat retina. Retinal function was evaluated using electroretinography (ERG), retinal structure was analyzed using optical coherence tomography (OCT) imaging and the tissue response to light-induced injury was assessed immunohistochemically with antibodies against glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1 (Iba-1) and Connexin43 (Cx43). Probenecid did not further enhance the therapeutic effect of connexin hemichannel block in this model, but on its own improved activity of certain inner retina neurons. The therapeutic benefit of blocking connexin hemichannels was further evaluated by comparing these data against results from our previously published studies that also used the light-damaged rat retina model. The analysis showed that treatment with tonabersat alone was better than probenecid alone at restoring retinal function in the light-damaged retina model. The results assist in the interpretation of the differential action of connexin hemichannel and pannexin channel therapeutics for potential treatment of retinal diseases.

Harnessing the therapeutic potential of antibodies targeting connexin hemichannels.

BACKGROUND: Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations. METHODS: We analysed the literature focusing on antibodies capable of modulating hemichannel function, highlighting generation methods, applications to basic biomedical research and translational potential. RESULTS: Anti-hemichannel antibodies generated over the past 3 decades targeted mostly connexin 43, with a focus on cancer treatment. A slow transition from relatively unselective polyclonal antibodies to more selective monoclonal antibodies resulted in few products with interesting characteristics that are under evaluation for clinical trials. Selection of antibodies from combinatorial phage-display libraries, has permitted to engineer a monoclonal antibody that binds to and blocks pathological hemichannels formed by connexin 26, 30 and 32. CONCLUSIONS: All known antibodies that modulate connexin hemichannels target the two small extracellular loops of the connexin proteins. The extracellular region of different connexins is highly conserved, and few residues of each connexins are exposed. The search for new antibodies may develop an unprecedented potential for therapeutic applications, as it may benefit tremendously from novel whole-cell screening platforms that permit in situ selection of antibodies against membrane proteins in native state. The demonstrated efficacy of mAbs in reaching and modulating hemichannels in vivo, together with their relative specificity for connexins overlapping epitopes, should hopefully stimulate an interest for widening the scope of anti-hemichannel antibodies. There is no shortage of currently incurable diseases for which therapeutic intervention may benefit from anti-hemichannel antibodies capable of modulating hemichannel function selectively and specifically.

Rapamycin relieves the cataract caused by ablation of Gja8b through stimulating autophagy in zebrafish.

Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens.Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; AV: autophagic vacuoles; Dpf: days post fertilization; GJA1: gap junction protein alpha 1; GJA3: gap junction protein alpha 3; GJA8: gap junction protein alpha 8; Hpf: hours post fertilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; WT: wild type.

Targeting connexins with Gap27 during cold storage of the human donor uterus protects against cell death.

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority.

Connexins-Therapeutic Targets in Cancers.

Connexins (Cx) are members of a protein family that forms intercellular channels localised in gap junction (GJ) plaques and single transmembrane channels called hemichannels. They participate in intercellular communication or communication between the intracellular and extracellular environments. Connexins affect cell homeostasis, growth and differentiation by enabling the exchange of metabolites or by interfering with various signalling pathways. Alterations in the functionality and the expression of connexins have been linked to the occurrence of many diseases. Connexins have been already linked to cancers, cardiac and brain disorders, chronic lung and kidney conditions and wound healing processes. Connexins have been shown either to suppress cancer tumour growth or to increase tumorigenicity by promoting cancer cell growth, migration and invasiveness. A better understanding of the complexity of cancer biology related to connexins and intercellular communication could result in the design of novel therapeutic strategies. The modulation of connexin expression may be an effective therapeutic approach in some types of cancers. Therefore, one important challenge is the search for mechanisms and new drugs, selectively modulating the expression of various connexin isoforms. We performed a systematic literature search up to February 2020 in the electronic databases PubMed and EMBASE. Our search terms were as follows: connexins, hemichannels, cancer and cancer treatment. This review aims to provide information about the role of connexins and gap junctions in cancer, as well as to discuss possible therapeutic options that are currently being studied.

Connexin 36 Mediates Orofacial Pain Hypersensitivity Through GluK2 and TRPA1.

Trigeminal neuralgia is a debilitating condition, and the pain easily spreads to other parts of the face. Here, we established a mouse model of partial transection of the infraorbital nerve (pT-ION) and found that the Connexin 36 (Cx36) inhibitor mefloquine caused greater alleviation of pT-ION-induced cold allodynia compared to the reduction of mechanical allodynia. Mefloquine reversed the pT-ION-induced upregulation of Cx36, glutamate receptor ionotropic kainate 2 (GluK2), transient receptor potential ankyrin 1 (TRPA1), and phosphorylated extracellular signal regulated kinase (p-ERK) in the trigeminal ganglion. Cold allodynia but not mechanical allodynia induced by pT-ION or by virus-mediated overexpression of Cx36 in the trigeminal ganglion was reversed by the GluK2 antagonist NS102, and knocking down Cx36 expression in Nav1.8-expressing nociceptors by injecting virus into the orofacial skin area of Nav1.8-Cre mice attenuated cold allodynia but not mechanical allodynia. In conclusion, we show that Cx36 contributes greatly to the development of orofacial pain hypersensitivity through GluK2, TRPA1, and p-ERK signaling.