Hemichannel-mediated ATP Release Research Articles

Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury

BackgroundWithout a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined.MethodsUsing the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD.ResultsIncreased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity.ConclusionAberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD.-uWW98vNU7-T3Uscg54sCsVideo

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.

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.

Basic and clinical science posters: Basic science of diabetes complications

Aims: Glucose evoked changes in profibrotic transforming growth factor-beta1 (TGFs1), increase connexin hemichannel mediated ATP release in kidney proximal tubule cells. With ATP linked to inflammation, we evaluated the efficacy of hemichannel blocker Peptide 5 in negating ATP induced inflammatory cytokine secretion in proximal tubule cells. Methods: Primary human proximal tubular epithelial cells (hPTECs), and immortalised human kidney (HK2) cells were treated with TGFs1 (10ng/ml) ± Peptide5 (25µM) for 48hrs. Carboxyfluorescein (200µM) dye uptake and ATP biosensing determined hemichannel activity and ATP release respectively. Proteome arrays determined cytokine secretion. Results: In TGFs1 treated hPTECs, carboxyfluorescein dye uptake decreased from 347.4±11.9%, (P<0.001, N=3) to 158.6±7.9% when co-incubated with Peptide 5. Parallel to this, ATP release increased from 0.16±0.1µM to 2.5±0.14µM (P<0.001, N=3) in TGF1 treated cells as compared to control. Peptide 5 significantly blunted this response, to 0.62±0.17µM (P<0.001, N=3). A proteome array determined the impact of Peptide 5 on secretion of 105 candidate chemokines and cytokines in TGFs1 treated primary cells. As an example, Peptide 5 significantly restored secretion of adiponectin from 163±8.0% (P<0.001, N=4) to 136±6.1% (P<0.05, N=4), granulocyte-macrophage colony-stimulating factor from 183±8.0% (P<0.001, N=4) to 111.3±5.9% (P<0.001, N=4) and interleukin 1-beta from 189±14.9 (P<0.001, N=4) to 109±9.6% (P<0.001, N=4) in TGF-s1 treated hPTECs, as compared to control. Conclusion: Glucose evoked changes in TGFs1 increase hemichannel mediated ATP release. Hemichannel blocker; Peptide 5 blocks ATP release and downstream cytokine and chemokine secretion, suggesting that Peptide 5 may be of future therapeutic potential in targeting inflammation of the diabetic kidney.

Paracellular permeability changes induced by multi-walled carbon nanotubes in brain endothelial cells and associated roles of hemichannels

Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 μg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine.

Purinergic receptor (P2X7) activation reduces cell–cell adhesion between tubular epithelial cells of the proximal kidney

Loss of epithelial (E)-cadherin mediated cell–cell adhesion impairs gap junction formation and facilitates hemichannel-mediated ATP release in the diabetic kidney. Linked to inflammation and fibrosis, we hypothesized that local increases in inter-cellular ATP activate P2X7 receptors on neighboring epithelial cells of the proximal tubule, to further impair cell–cell adhesion and ultimately exacerbate tubular injury. Immunoblotting confirmed changes in E-cadherin expression in human kidney cells treated with non-hydrolysable ATPγS ± the P2X7 antagonist, A438079. Atomic force microscopy based single-cell force spectroscopy quantified maximum unbinding force, tether rupture events, and work of detachment. Confocal microscopy assessed cytoskeletal reorganization. Our studies confirmed that ATPγS downregulated E-cadherin expression in proximal kidney cells, loss of which was paralleled by a reduction in intercellular ligation forces, decreased tether rupture events and cytoskeletal remodeling. Co-incubation with A438079 restored loss of adhesion, suggesting that elevated extracellular ATP mediates tubular injury through P2X7 induced loss of E-cadherin mediated adhesion.

Connexin43 hemichannel block protects against retinal pigment epithelial cell barrier breakdown.

The retinal pigment epithelium (RPE) is an important component of the outer blood-retinal barrier (BRB) that separates the choroid from the rest of the retina. Loss of RPE-mediated BRB integrity is a key feature of diabetic macular oedema (DME), a chronic pathology resulting from diabetic retinopathy (DR). Recent studies have shown that connexin43 hemichannel opening mediates key inflammatory pathways in DR, though its effect on the barrier properties of RPE cells remains unknown. Therefore, RPE breakdown was induced by exposing a monolayer of ARPE-19 cells to high glucose (HG) and 10ng/mL each of the pro-inflammatory cytokines IL-1β and TNF-α. The role of connexin43 hemichannels was assessed using a connexin43 hemichannel blocker, Peptide5. Transepithelial resistance (TEER) and FITC-dextran dye leak across the ARPE-19 monolayer were used to measure RPE layer permeability. Immunohistochemistry was used to assess changes in connexin43, collagen IV and ZO-1 expression. ATP and lactate dehydrogenase (LDH) release were measured using commercially available kits. Connexin43 hemichannel block with Peptide5 prevented TEER reduction and FITC-dextran dye leak induced by a combination of HG and inflammatory cytokines. Peptide5 also blocked LDH and ATP release induced by the addition of HG and inflammatory cytokines. ZO-1 and connexin43 disruption and internalisation as well as upregulated secretion of collagen IV following HG and inflammatory cytokine exposure were also prevented. The addition of exogenous ATP into the culture medium was able to reverse Peptide5 protection against LDH release and change in connexin43 localisation, indicating that the initiating pathway in RPE disruption is connexin43 hemichannel-mediated ATP release. These findings support the idea that connexin43 hemichannels may mediate RPE disruption (and its role within the BRB) that occurs in DME through an ATP release/inflammasome pathway activation dependent manner. Connexin43 hemichannels are therefore a potential therapeutic target for the treatment of DME.

Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes

Background/Aims: The atrium is exposed to high shear stress during heart failure and valvular diseases. We aimed to understand atrial shear-induced Ca²⁺ signaling and its underlying mechanisms. Methods: Pressurized micro-flow was applied to single rat atrial myocytes, and Ca²⁺ signal, membrane potential, and ATP release were assessed using confocal imaging, patch clamp technique, and luciferin-luciferase assay, respectively. Results: Shear stress (∼16 dyn/cm²) induced global Ca²⁺ waves (∼0.1 events/s) from the periphery to the center of cells in a transverse direction (“T-wave”; ∼145 μm/s). Pharmacological interventions and simultaneous recording of membrane potential and Ca²⁺ demonstrated that shear-induced T-waves resulted from action potential (AP)-triggered Ca²⁺ release from the sarcoplasmic reticulum. T-waves were not sensitive to inhibitors of known shear signaling mechanisms except connexin hemichannels and ATP release. Shear stress caused ATP release from these myocytes (∼1.1x10^-17 moles/unit membrane, µm²); ATP release was increased by enhancement of connexin hemichannels and suppressed by inhibition of the hemichannels, but not affected by inhibitors of other ATP release pathways. Blockade of P2X receptor, but not pannexin or the Na⁺-Ca²⁺ exchanger, eliminated shear-induced T-wave initiation. Conclusion: Our data suggest that shear stress triggers APs and concomitant Ca²⁺ signaling via activation of P2X receptors by connexin hemichannel-mediated ATP release in atrial myocytes.

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release

BackgroundConnexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease. MethodsRetinal pigment epithelial cells were exposed to HG, 10ng/mL pro-inflammatory cytokines IL-1β and TNF-α, or a combination of both. Quantitative Cytometric Bead Array analysis was used to measure the release of inflammatory cytokines IL-6, IL-8, MCP-1, and sICAM-1, as well as VEGF and ATP. To determine the role of connexin43 hemichannels in the disease process, changes in cytokine and ATP release were evaluated following treatment with Peptide5, a connexin43 hemichannel blocker. Immunohistochemistry was used to compare NLRP3 inflammasome assembly under control and treatment conditions. ResultsCo-application of HG and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF and ATP, to significantly higher levels compared to cytokines alone. Peptide5 prevented cytokine release and prevented the increase in ATP release following co-application of HG and cytokines. Adding exogenous ATP negated Peptide5-mediated protection against inflammatory cytokine release in injury conditions. ConclusionsOur findings show that connexin43 hemichannels play an important role in the amplification and perpetuation of inflammation by mediating an ATP autocrine feedback loop in the inflammasome/inflammation cycle. General significanceTargeting connexin43 hemichannels offers a potential therapeutic strategy to break the inflammatory cycle in diseases such as DR, but also other chronic inflammatory indications.

Characterizing the mode of action of extracellular Connexin43 channel blocking mimetic peptides in an in vitro ischemia injury model

BackgroundNon-selective Connexin43 hemichannels contribute to secondary lesion spread. The hemichannel blocking peptidomimetic Peptide5, derived from the second extracellular loop of the human Connexin43 protein, prevents lesion spread and reduces vascular permeability in preclinical models of central nervous system injury. The molecular mode of action of Peptide5, however, was unknown and is described here. MethodsHuman cerebral microvascular endothelial cells and APRE-19 cells were used. Scrape loading was used to assess gap junction function and hypoxic, acidic ion-shifted Ringer solution induced ATP release used to assess hemichannel function. Peptide modifications, including amino acid substitutions and truncations, and competition assays were used to demonstrate Peptide5 functional specificity and site of action respectively. ResultsPeptide5 inhibits Connexin43 hemichannel-mediated ATP release by acting on extracellular loop two of Connexin43, adjacent to its matching sequence within the protein. Precise sequence specificity is important for hemichannel block, but less so for uncoupling of gap junction channels (seen only at high concentrations). The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels. Both the SRPTEKT motif and Peptide5 reduce gap junction communication, but neither uncoupling below 50%. ConclusionsReduced gap junction coupling at high peptide concentrations appears to be relatively non-specific. However, Peptide5 at low concentrations acts upon extracellular loop two of Connexin43 to block hemichannels in a precise, sequence specific manner. General significanceThe concentration dependent and sequence specific action of Peptide5 supports its development for the treatment of retinal injury and chronic disease, as well as other central nervous system injury and disease conditions.

Shear stress induces a longitudinal Ca(2+) wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes.

Atrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca(2+) wave. Here, we investigated the cellular mechanisms underlying the shear stress-mediated Ca(2+) wave, using two-dimensional confocal Ca(2+) imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ∼16 dyn cm(-2) for 8 s induced ∼1.2 aperiodic longitudinal Ca(2+) waves (∼79 μm s(-1)) with a delay of 0.2-3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP3 Rs) abolished shear stress-induced Ca(2+) wave generation. Furthermore, in atrial myocytes from type 2 IP3R (IP3R2) knock-out mice, shear stress failed to induce longitudinal Ca(2+) waves. The phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the shear-induced longitudinal Ca(2+) wave. However, pretreating atrial cells with blockers for stretch-activated channels, Na(+)-Ca(2+) exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y1 receptor antagonist MRS 2179, both suppressed the Ca(2+) wave, whereas the P2X receptor antagonist, iso-PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP-metabolizing apyrase inhibited the wave. Removal of external Ca(2+) to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca(2+) release through RyRs is triggered by P2Y1-PLC-IP3R2 signalling that is activated by gap junction hemichannel-mediated ATP release in atrial myocytes under shear stress.

Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response.

In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction. In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis. JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle. These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital aspect of this work is that it demonstrates that targeted molecular therapeutics, such as JM2, provide the capacity to regulate inflammation in a clinically relevant system.

Mechanosensitive activation of ATP‐sensitive K+ channels in native pulmonary epithelium depends on pannexin and connexin hemichannel mediated ATP release

ObjectiveThe pulmonary epithelium (PE) is permanently exposed to mechanical forces (shear force, stretch). There is evidence that distention of PE leads to an activation of ATP‐sensitive K+ (KATP) channels. The aim of this study was to clarify the mechanisms of KATP channel activation and the contribution of hemichannels in particular.MethodsA modified Ussing chamber that enabled the application of hydrostatic pressure (HP) was used to mechanically stimulate native PE derived from Xenopus laevis while the short circuit current (ISC) was recorded. Activation of KATP channels was ascertained by the application of the specific inhibitor HMR 1098. Contribution of hemichannels was investigated by application of connexin (meclofenamic acid) and pannexin (probenecide) inhibitors. Extracellular ATP was determined via a luminescence ATP detection assay.ResultsApical application of HP led to a pressure‐induced current decrease (17.6 ± 2%, P&lt;0.001). This effect was prevented by HMR 1098. The HP‐induced effect was also reversed by the hemichannel inhibitors. Under control conditions (without pharmaca) the HP‐induced effect was accompanied by an increased ATP release which was also inhibited by the hemichannel inhibitors.ConclusionKATP channel activation in response to HP is caused by ATP release through a mechanosensitive activation of connexin and pannexin hemichannels.

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

492 CAVEOLAE-MEDIATED REGULATION OF PURINERGIC SIGNALING IN THE BLADDER SUPPRESSES SPONTANEOUS ACTIVITY

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2012492 CAVEOLAE-MEDIATED REGULATION OF PURINERGIC SIGNALING IN THE BLADDER SUPPRESSES SPONTANEOUS ACTIVITY Vivian Cristofaro, Subbarao Yalla, and Maryrose Sullivan Vivian CristofaroVivian Cristofaro Boston, MA More articles by this author , Subbarao YallaSubbarao Yalla Boston, MA More articles by this author , and Maryrose SullivanMaryrose Sullivan Boston, MA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.562AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder smooth muscle (BSM) caveolae, membrane domains that modulate responses to physiologic stimuli, are reduced in animal models of DO. These animals exhibit increased bladder spontaneous activity (SA) and altered purinergic signaling. In this study, we investigated whether caveolae-mediated regulation of the purinergic system plays a role in maintaining the level of SA in the bladder. METHODS Rat bladder strips without mucosa were mounted in organ bath. SA and responses induced by αβmeATP were measured before and after caveolae depletion (achieved by methyl-β-cyclodextrin, mβCD). Changes in SA were also determined after P2X receptor inhibition (NF449, NF279, PPADS, or after αβmeATP-desensitization) and in the presence of ivermectin (IVC, a P2X4 positive modulator) or BDBD (P2X4 antagonist), or after the connexin (Cx) hemichannel blocker, carbenoxolone (CBX). Differences among these treatments in levels of ATP released either spontaneously or upon stimulation (induced by KCl in BSM tissue and hypotonic Krebs exposure in BSM cells) were determined by luciferase assay. P2X4 receptor expression in BSM tissue and the interaction of caveolin proteins with P2X4 receptor and Cx43 were determined by western blot and co-immunoprecipitation. RESULTS Depletion of caveolae inhibited P2X-mediated contractile responses and augmented SA in BSM tissue, and increased basal ATP release from BSM cells. SA and ATP levels in BSM tissue were also increased by inhibiting P2X receptors and by exposure to IVC. BDBD prevented the augmentation in SA caused by P2X receptor inhibition and the KCl-induced ATP release from BSM tissue. Similarly, CBX attenuated the increase in ATP induced by mβCD or by hypotonic Krebs in BSM cells, and prevented the KCl-induced ATP release from BSM tissue. P2X4 and Cx43 were expressed in BSM tissue and co-precipitated with caveolin proteins. CONCLUSIONS The impaired regulation of purinergic signaling and the ATP-induced increase in SA after mβCD is consistent with enhanced SA in animal models of DO in which caveolae are reduced, suggesting a role for caveolae in restraining SA. Loss of caveolae exposed an excitatory component, potentially mediated by P2X4 receptor activation, which was prevented by CBX, suggesting that SA levels are modulated by the effects of endogenous ATP released from BSM cells through Cx hemichannels. The molecular interaction of Cx43 with caveolin proteins supports the concept that hemichannel-mediated ATP release is suppressed by caveolae. Therefore, loss of BSM caveolae may cause dysregulation of this mechanism and lead to DO. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e201 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vivian Cristofaro Boston, MA More articles by this author Subbarao Yalla Boston, MA More articles by this author Maryrose Sullivan Boston, MA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

TRPV4 in Porcine Lens Epithelium Regulates Hyposmotic Stress‐Induced ATP Release and Na, K‐ATPase Activity

In several tissues TRPV4 channels are involved in the response to hyposmotic challenge. Our objective was to detect TRPV4 protein in porcine lens epithelium and its role in hyposmotic stress‐induced ATP release.The Western blot analysis showed presence of TRPV4 in the lens epithelial homogenate. The TRPV4 antagonist RN 1734 (10μM) completely prevented ATP release by lenses exposed to hyposmotic (200 mOsm) solution. Lenses exposed to a TRPV4 agonist, GSK1016790A (GSK), or to hyposmotic solution displayed ATP release and an increased ability of propidium iodide (PI) (MW 668) to enter the epithelium. The increases in PI uptake and ATP release both were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18á‐glycyrrhetinic acid (AGA, 100μM) and probenecid (1mM). An increase in Na, K‐ATPase activity was detected in the epithelium of lenses exposed to GSK in isosmotic solution and the response was prevented by AGA + probenecid. TRPV4 antagonists prevented the activation of Src family kinase and increase of Na, K‐ATPase activity that occurred in the epithelium of lenses exposed to hyposmotic solution.The findings are consistent with hyposmotic shock‐induced TRPV4 channel activation which triggers hemichannel‐mediated ATP release. The results point to a pivotal role for TRPV4 activation as an initial event in a multi‐step response of the lens to hyposmotic stress.

Key role of Pannexin‐based hemichannels in flow‐induced nitric oxide production in resistance arteries

Nitric oxide (NO) production and connexin‐based channels play a key role in the control and coordination of vascular function. Blood flow induces NO production by the activation of endothelial NO synthase phosphorylation at serine 1177 (P‐eNOSS1177) through the PI3K/Akt pathway. The participation of connexin‐ or pannexin‐formed hemichannels in the response to flow has not been evaluated. We used the rat arterial mesenteric bed to study the involvement of hemichannels in flow‐induced NO production. Mesenteric vessels were stimulated with flow by increasing the perfusion rate from 2 to 10 mL/min and hemichannel activity was assessed by ethidium or Lucifer Yellow uptake. Flow increased NO production, Akt‐dependent P‐eNOSS1177, ATP release and endothelial hemichannel activity. Treatment with 18‐beta‐glycyrrhetinic acid or probenecid, but not La3+, blocked the hemichannel activation and Akt dependent‐P‐eNOSS1177 induced by flow. The increase in P‐eNOSS1177 was prevented by the specific inhibition of P2Y1 receptors with MRS2179 or global inhibition of purinergic receptors with PPADS, but not by the P2X receptor blocker iso‐PPADS. These results suggest that pannexin hemichannel‐mediated ATP release is involved in the activation of NO production in response to flow, which may be involved in the development of cardiovascular diseases.FONDECYT 1100850, 1090757, 1111033, Anillos ACT‐71.

The ‘connexin’ between astrocytes, ATP and central respiratory chemoreception

The ‘connexin’ between astrocytes, ATP and central respiratory chemoreception