Hemichannel Activity Research Articles

Increased Hemichannel Activity Displayed by a Connexin43 Mutation Causing a Familial Connexinopathy Exhibiting Hypotrichosis with Follicular Keratosis and Hyperostosis.

Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations.

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.

Inhibition of connexin hemichannels alleviates neuroinflammation and hyperexcitability in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is one of the most common types of epilepsy, yet approximately one-third of patients are refractory to current anticonvulsive drugs, which target neurons and synapses. Astrocytic and microglial dysfunction is commonly found in epileptic foci and has been shown to contribute to neuroinflammation and hyperexcitability in chronic epilepsy. Accumulating evidence points to a key role for glial hemichannels in epilepsy, but inhibiting both connexin (Cx) gap junctions and hemichannels can lead to undesirable side effects because the former coordinate physiological functions of cell assemblies. It would be a great benefit to use an orally available small molecule to block hemichannels to alleviate epileptic symptoms. Here, we explored the effect of D4, a newly developed compound that inhibits the Cx hemichannels but not Cx gap junctions using the pilocarpine mouse model of TLE. In vitro application of D4 caused a near-complete reduction in the pilocarpine-induced cell membrane permeability associated with increased Cx hemichannel activity. Moreover, preadministration of D4 invivo effectively reduced neuroinflammation and altered synaptic inhibition, which then enhanced the animal survival rate. Posttreatment with a single dose of D4 invivo has prolonged effects on suppressing the activation of astrocytes and microglia and rescued the changes in neuroinflammatory and synaptic gene expression induced by pilocarpine. Collectively, these results indicate that targeting Cx hemichannels by D4 is an effective and promising strategy for treating epilepsy in which neuroinflammation plays a critical role.

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

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

Blocking connexin 43 hemichannel-mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy.

Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions. Tonabersat partially negated glucose/TGFβ1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression. Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFβ1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.

The Bioactive Phenolic Agents Diaryl Ether CVB2-61 and Diarylheptanoid CVB4-57 as Connexin Hemichannel Blockers.

Inflammation mediators enhance the activity of connexin (Cx) hemichannels, especially in the epithelial and endothelial tissues. As potential release routes for injury signals, such as (oligo)nucleotides, Cx hemichannels may contribute to long-lasting inflammation. Specific inhibition of Cx hemichannels may therefore be a mode of prevention and treatment of long-lasting, chronic sterile inflammation. The activity of Cx hemichannels was analysed in N2A and HeLa cells transfected with human Cx26 and Cx46 as well as in Calu-3 cells, using dye uptake as functional assay. Moreover, the possible impacts of the bioactive phenolic agents CVB2-61 and CVB4-57 on the barrier function of epithelial cells was analysed using Calu-3 cells. Both agents inhibited the dye uptake in N2A cells expressing Cx26 (>5 µM) and Cx46 (>20 µM). In Calu-3 cells, CVB2-61 and CVB4-57 reversibly inhibited the dye uptake at concentrations as low as 5 µM, without affecting the gap junction communication and barrier function, even at concentrations of 20 µM. While CVB2-61 or CVB4-57 maintained a reduced dye uptake in Calu-3 cells, an enhancement of the dye uptake in response to the stimulation of adenosine signalling was still observed after removal of the agents. The report shows that CVB2-61 and CVB4-57 reversibly block Cx hemichannels. Deciphering the mechanisms of the interactions of these agents with Cx hemichannels could allow further development of phenolic compounds to target Cx hemichannels for better and safer treatment of pathologies that involve Cx hemichannels.

Reducing Atherosclerotic Plaque Development and Endothelial Hemichannel Activity with WIN-55,212-2: A Research Protocol

Introduction: Endothelial cells (ECs) are critical regulators of vascular homeostasis, and their dysfunction leads to the development of atherosclerosis – the main underlying cause of cardiovascular diseases (CVDs). This dysfunction can be promoted by prolonged endothelial connexin43 hemichannel activity, which is caused by the combination of high glucose levels and cytokines IL-1β/TNF-α. WIN-55,212-2 (WIN) is a synthetic agonist of CB1/CB2 receptors and can counteract the proinflammatory effects of high glucose and IL-1β/TNF-α. We hypothesize that WIN treatment on ECs will reduce connexin43 hemichannel activity, thus preventing endothelial dysfunction and atherosclerotic progression. Methods: We will use the Apolipoprotein E Knockout (ApoE-/-) mouse model to assess the impact on atherosclerotic lesions. Hyperglycemia will be generated in these mice with Streptozotocin injections. The increased levels of glucose should induce IL-1β expression and stimulate prolonged hemichannel activity. ECs will be isolated from a subset of mice and cultured to test WIN-efficacy. ATP release will be assessed through an ATP viability assay. More in vitro assessments on subsets of ApoE-/- mice treated or not with WIN will be performed. Flow cytometry will evaluate monocyte-derived macrophage concentration and other pro and anti-inflammatory cytokines in tissue samples. Furthermore, atherosclerotic plaque volume in the aortic sinus will be quantified and characterized. Results: We expect that WIN-treated ECs will reduce ATP synthesis compared to those from the control group. Moreover, we expect to see a reduction in the inflammatory response with a consequent decrease in atherosclerotic progression. Discussion: This manuscript outlines the use of a novel compound that could prevent atherosclerosis progression. The results of this study could outline a potential mechanism that may be targeted to treat or forestall atherosclerosis progression. Conclusion: Overall, we aim to determine if WIN may not only hinder this pervasive condition but inhibit CVDs through curtailing atherosclerotic plaque development. The following steps include performing the experiment, confirming results through repetition, and using other animal models.

Synthesis and Biological Evaluation of Termini-Modified and Cyclic Variants of the Connexin43 Inhibitor Peptide5.

Peptide5 is a 12–amino acid mimetic peptide that corresponds to a region of the extracellular loop 2 (EL2) of connexin43. Peptide5 regulates both cellular communication with the cytoplasm (hemichannels) and cell-to-cell communication (gap junctions), and both processes are implicated in neurological pathologies. To address the poor in vivo stability of native peptide5 and to improve its activity, twenty-five novel peptide5 mimetics were designed and synthesized. All the analogues underwent biological evaluation as a hemichannel blocker and as a gap junction disruptor, and several were assessed for stability in human serum. From this study, it was established that several acylations on the N-terminus were tolerated in the hemichannel assay. However, the replacement of the L-Lys with an N-methylated L-Lys to give H-VDCFLSRPTE-N-MeKT-OH showed good hemichannel and gap junction activity and was more stable in human serum. The cyclic peptide variants generally were not tolerated in either the hemichannel and gap junction assay although several possessed outstanding stability in human serum.

The Ambivalence of Connexin43 Gap Peptides in Cardioprotection of the Isolated Heart against Ischemic Injury.

The present study investigates infarct-reducing effects of blocking ischemia-induced opening of connexin43 hemichannels using peptides Gap19, Gap26 or Gap27. Cardioprotection by ischemic preconditioning (IPC) and Gap peptides was compared, and combined treatment was tested in isolated, perfused male rat hearts using function and infarct size after global ischemia, high-resolution respirometry of isolated mitochondrial and peptide binding kinetics as endpoints. The Gap peptides reduced infarct size significantly when given prior to ischemia plus at reperfusion (Gap19 76.2 ± 2.7, Gap26 72.9 ± 5.8 and Gap27 71.9 ± 5.8% of untreated control infarcts, mean ± SEM). Cardioprotection was lost when Gap26, but not Gap27 or Gap19, was combined with triggering IPC (IPC 73.4 ± 5.5, Gap19-IPC 60.9 ± 5.1, Gap26-IPC 109.6 ± 7.8, Gap27-IPC 56.3 ± 8.0% of untreated control infarct). Binding stability of peptide Gap26 to its specific extracellular loop sequence (EL2) of connexin43 was stronger than Gap27 to its corresponding loop EL1 (dissociation rate constant Kd 0.061 ± 0.004 vs. 0.0043 ± 0.0001 s−1, mean ± SD). Mitochondria from IPC hearts showed slightly but significantly reduced respiratory control ratio (RCR). In vitro addition of Gap peptides did not significantly alter respiration. If transient hemichannel activity is part of the IPC triggering event, inhibition of IPC triggering stimuli might limit the use of cardioprotective Gap peptides.

TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1β increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1β treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1β-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell–cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Connexin-Based Channels in the Liver.

Connexin proteins oligomerize in hexameric structures called connexin hemichannels, which then dock to form gap junctions. Gap junctions direct cell-cell communication by allowing the exchange of small molecules and ions between neighboring cells. In this way, hepatic gap junctions support liver homeostasis. Besides serving as building blocks for gap junctions, connexin hemichannels provide a pathway between the intracellular and the extracellular environment. The activation of connexin hemichannels is associated with acute and chronic liver pathologies. This article discusses the role of gap junctions and connexin hemichannels in the liver. © 2022 American Physiological Society. Compr Physiol 12:1-17, 2022.

Osteocytes regulate bone anabolic response to mechanical loading in male mice via activation of integrin α5

Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic β-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.

Connexin 32 overexpression increases proliferation, reduces gap junctional intercellular communication, motility and epithelial-to-mesenchymal transition in Hs578T breast cancer cells.

Connexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32's effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.

Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels.

Connexin (Cxs) hemichannels participate in several physiological and pathological processes, but the molecular mechanisms that control their gating remain elusive. We aimed at determining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in “permanently closed hemichannels”, which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.

A Short-Term Sucrose Diet Impacts Cell Proliferation of Neural Precursors in the Adult Hypothalamus.

Radial glia-like cells in the hypothalamus and dorsal vagal complex are neural precursors (NPs) located near subventricular organs: median eminence and area postrema, respectively. Their strategic position can detect blood-borne nutrients, hormones, and mitogenic signals. Hypothalamic NPs increase their proliferation with a mechanism that involves hemichannel (HC) activity. NPs can originate new neurons in response to a short-term high-fat diet as a compensatory mechanism. The effects of high carbohydrate Western diets on adult neurogenesis are unknown. Although sugars are usually consumed as sucrose, more free fructose is now incorporated into food items. Here, we studied the proliferation of both types of NPs in Sprague Dawley rats exposed to a short-term high sucrose diet (HSD) and a control diet. In tanycyte cultures, we evaluated the effects of glucose and fructose and a mix of both hexoses on HC activity. In rats fed an HSD, we observed an increase in the proliferative state of both precursors. Glucose, either in the presence or absence of fructose, but not fructose alone, induced in vitro HC activity. These results should broaden the understanding of the nutrient monitoring capacity of NPs in reacting to changes in feeding behavior, specifically to high sugar western diets.

Recent advances in connexin gap junction biology.

Connexins are assembled into dodecamer intercellular channels, a collection of which is termed a gap junction, and their canonical function allowing direct exchange of ions and metabolites has been unequivocally established. When initially assembled into undocked cell surface connexin hemichannels, healthy cells may also engage in cell signaling via a regulated small-molecule release. Recent advances in the field have led to an expanded view of the functional roles of intercellular channels and hemichannels in both physiology and pathology. As more of the 21-member human connexin family is intensely interrogated, mounting evidence points to the biological uniqueness of each member, and no longer can we confidently refer to all connexins engaging in the same cellular processes. Innovations in high-resolution cryo-electron microscopy have revealed important insights into the structure of functionally important domains of both hemichannels and channels. These and other studies have established a foundation of knowledge that should allow inhibitory smart drug design for situations where enhanced intercellular or hemichannel activity is at the root of a connexin-linked disease. Assessment of the connexin interactome, which varies widely for each connexin subtype, continues to provide regulatory insights into the assembly and function of connexins that exhibit a short half-life. As the most intensely studied, Cx43 is found in about 50% of all human cell types and is extensively regulated by multiple inhibitory and enhancing phosphorylation events that have direct implications on tissue function and outcomes of disease, including cancer. Here, we briefly discuss these advances and give our thoughts on where the field is headed.

Endothelial connexin hemichannels as a novel endothelial regulatory mechanism of vasodilation

The vascular endothelium regulates vasomotor tone in arterioles through intercellular conduction of electrical signals by gap junctions. Endothelial vasodilation is mediated by nitric oxide (NO) and by a hyperpolarizing current initiated in the endothelium. Both signals are transmitted to smooth muscle cells evoking relaxation. Endothelial cells express connexin (Cx) 37,40,43. Cx hemichannels allow the exchange of ions and molecules between intracellular and extracellular compartments that may be associated with endothelial electrical activity. Whether or not Cx hemichannels regulate vasomotor tone is unknown. We evaluated the role of Cx‐hemichannels in the vasodilation process by measuring dye uptake in primary culture of endothelial cells and in isolated mouse mesenteric arterioles. We detected that both acetylcholine (ACh) and bradykinin (BK) increase dye permeability, which was inhibited by connexin mimetic peptides that block Cx43 hemichannels (Gap19). We determined that NO mediates Cx43 hemichannels activity inasmuch as blocking NOS inhibits hemichannel activity. Using biotin switch assay, we observed that ACh induced S‐nitrosylation only in Cx43 at cysteine 271. ACh and BK open endothelial Cx43 hemichannels activity and promote endothelial Ca2+entry from the extracellular space and lead to endothelium hyperpolarization signaling in isolated mesenteric arteries. Blocking Cx43‐hemichannels reduces calcium entry and hyperpolarization. To further assess the physiological role of Cx43 endothelial hemichannels activity, we cannulated isolated mesenteric arterioles and studied ACh‐induced arteriolar relaxation in the presence and absence of connexin mimetic peptides that block Cx43 hemichannels (Gap19) and Cx40 (40Gap 27, a connexin mimetic peptide). Gap19 attenuates ACh‐induced relaxation, but 40Gap27 fails to reduce relaxation. These results indicate that endothelial Cx43 hemichannels participate in arteriolar dilatation. In addition, we replaced cysteine 271 by serine to create a Cx43C271S mouse that failed to cause S‐nitrosylation of Cx43. We detected that this knockin mouse displayed hypertension compared to wild type mice, indicating that Cx43 hemichannels and S‐nitrosylated Cx43 play a role in vascular resistance. Our results suggest that Cx43 hemichannels are a potential therapeutic target in hypertension and vascular disease.

Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.

Gap Junction Protein, Connexin 43 is a Key Regulator of Metastasis in Syngeneic Ovarian Cancer Mouse Model

Ovarian cancer is the 7th most common cancer of women and the most lethal gynecological cancer that demands rigorous treatments involving increased radiation cycles and higher drug concentration that triggers toxicity and eventually mortality of patients. Understanding of the disease's progression warrants effective therapeutic inventions. Epithelial Ovarian Cancer cell lines ID8 derived from C57Bl/6 mice and ID8 transfected with Vascular Endothelial Growth Factor‐A (ID8‐VEGF) were used in vitro to generate a Syngeneic Ovarian Cancer Mouse Model. Vascular Endothelial Growth Factor‐A (VEGF‐A/VEGF) is the critical regulator of tumor angiogenesis that supports the transport of nutrients, hormones and O2 to the tumor cells and removal of waste products and CO2. It regulates vascular permeability, actin reorganization, cell migration, enhances tumor growth and progression, which may be associated with aberrant cellular metabolism due to altered gap junction intercellular communication (GJIC) contributing to the tumorigenesis. The transmembrane protein Connexin forms hexameric hemichannels known as connexons and docks with another connexon of adjacent cells to form a functional GJIC. Connexin 43 (Cx43) is one of the most ubiquitously expressed connexins and its C‐terminus interacts with various molecules to regulate many biological functions, including modulation of hemichannel activity, apoptotic signaling, cell cycle regulation and, cytoskeletal rearrangements. Our study aims to understand the effect of VEGF expression on the spatial expression of Cx43 and its nexus with other cellular factors contributing to consequent GJIC formation in the ovarian cancer cells as well as in the mouse model. Our findings suggest that upregulation of VEGF in ID8‐VEGF cell lines is associated with accelerated tumor formation in syngeneic mouse models. A series of biochemical and cellular studies revealed that Sodium Phenylbutyrate(4PBA) treatment induced differential upregulation of Cx43 trafficking to the cell surface, rearrangement of cytoskeletal proteins, cell migration, and the formation of GJIC in ID8 and ID8‐VEGF cells. 4PBA treatment on ID8‐VEGF cells significantly reduced the rate of tumor formation. These differential cellular activities denote that VEGF may directly or indirectly interact with cytoskeletal proteins that regulate Cx43 trafficking and its GJIC formation. Overall, The increased capacity of GJIC formation post‐4PBA treatment is associated with reduction in cell migration in vitro (ID8 and ID8‐VEGF) and tumor formation in vivo (ID8‐VEGF). Interactome studies combining RNASeq and IPA are designed to understand the nexus between several cellular pathways, including oxidative stress in the regulation of Cx43 in the context of VEGF, to dissect the cellular and molecular mechanism of metastasis.

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.