EtBr Uptake Research Articles

Human coronavirus OC43 infection remodels connexin 43-mediated gap junction intercellular communication in vitro.

β-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human β-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding β-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human β-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also shows a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post-OC43 infection in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Impairment of Cx43 trafficking and the ability to form hemichannels and functional GJIC are hampered by virus-induced Golgi apparatus disruption. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human β-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human β-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human β-coronavirus OC43 can be considered an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the changes in Cx43-mediated cell-to-cell communication during human β-coronavirus OC43 infection. In vitro studies showed downregulation of the gap junction protein Cx43 and upregulation of the endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, HCoV-OC43 infection causes reduced Cx43 trafficking, causing impairment of functional hemichannel and GJIC formation by virus-mediated Golgi apparatus disruption. Overall, this study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.

Elevated calcineurin activity in primary astrocytes leads to the dephosphorylation of connexin 43 in conjunction with increased membrane permeability.

Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.

Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1M NaCl), hydrogen peroxide (40mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

Membrane Disruption Potential of Doped Cuprous Oxide Nanoparticles Against blaNDM-1 and mcr-1 Positive Colistin Resistant E. coli

Bacterial resistance against the last-resort antibiotic “colistin” is a growing problem worldwide. Novel strategies in the field of nanotechnology for the development of suitable antibacterial drug with minimal toxicities are need of the time. Here we synthesized, Cu2O nanoparticles (Cu2O-NPs) doped with 5% Zn metal ions by adopting solution-phase method using hydrazine reduction in aqueous medium. Antibacterial assays showed a promising activity against colistin-resistant Escherichia coli isolates. Leakage assay, propidium iodide uptake assay, EtBr uptake assay, and LIVE/DEAD® BacLight™ assay showed membrane damaging activity of Cu2O-NPs. Imaging by scanning electron microscopy of NP-treated bacterial cells confirmed membrane damaging potential of used Cu2O-NPs. Toxicity assessment by cell viability assay showed no cytotoxic activity to the HEK cell line. In conclusion, our results suggest that Cu2O-NPs doped with 5% Zn may exert their antimicrobial action by disruption of the bacterial cell membrane and probably generating reactive oxygen species (ROS). We suggest that Cu2O-NPs doped with 5% Zn can be used as effective antibacterial agent.

Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy.

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels—both candidates for non-vesicular ATP-release—by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

BackgroundGlia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown.MethodsPrimary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry.ResultsWe found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane’s Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane.ConclusionsWe propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

The chemoconvulsant pilocarpine induces astrocytic connexin43 hemichannel opening, which might contribute to its seizurogenic effect

The chemoconvulsant pilocarpine induces astrocytic connexin43 hemichannel opening, which might contribute to its seizurogenic effect

Connexin-based channels contribute to metabolic pathways in the oligodendroglial lineage.

Oligodendrocyte precursor cells (OPCs) undergo a series of energy-consuming developmental events; however, the uptake and trafficking pathways for their energy metabolites remain unknown. In the present study, we found that 2-NBDG, a fluorescent glucose analog, can be delivered between astrocytes and oligodendrocytes through connexin-based gap junction channels but cannot be transferred between astrocytes and OPCs. Instead, connexin hemichannel-mediated glucose uptake supports OPC proliferation, and ethidium bromide uptake or increase of 2-NBDG uptake rate is correlated with intracellular Ca(2+) elevation in OPCs, indicating a Ca(2+)-dependent activation of connexin hemichannels. Interestingly, deletion of connexin 43 (Cx43, also known as GJA1) in astrocytes inhibits OPC proliferation by decreasing matrix glucose levels without impacting on OPC hemichannel properties, a process that also occurs in corpus callosum from acute brain slices. Thus, dual functions of connexin-based channels contribute to glucose supply in oligodendroglial lineage, which might pave a new way for energy-metabolism-directed oligodendroglial-targeted therapies.

Structure–activity relationship studies of pyrimidine-2,4-dione derivatives as potent P2X7 receptor antagonists

As an optimization strategy, the flexible structure of KN-62, a known P2X7 receptor antagonist, was converted into conformationally constrained derivatives using pyrimidine-2,4-dione as the core skeleton. Various modifications at the 4-position of the piperazine moiety of the new lead compound were performed to improve P2X7 receptor antagonistic activities, which were evaluated in HEK293 cells stably expressing the human P2X7 receptor (EtBr uptake assay) and in THP-1 cells (IL-1β ELISA assay). According to the results, polycycloalkyl acyl or di-halogenated benzoyl substituents were much more favorable than the original phenyl group of KN-62. Among these compounds, the trifluoromethyl-chloro benzoyl derivative 18m and adamantyl carbonyl derivatives 19g–19i and 19k showed potent antagonistic effects, with IC50 values ranging from 10 to 30 nM. In addition, the in vitro adsorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of 18m was determined to be in acceptable ranges in terms of metabolic stability and cytotoxicity. These results suggest that pyrimidine-2,4-dione derivatives may be promising novel P2X7 receptor antagonists for the development of anti-inflammatory drugs.

P2X7 receptor as predictor gene for glioma radiosensitivity and median survival

Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival.

Allium sativum produces terpenes with fungistatic properties in response to infection with Sclerotium cepivorum

This study investigated terpene biosynthesis in different tissues (root, protobulb, leaf sheath and blade) of in vitro-grown garlic plants either infected or not (control) with Sclerotium cepivorum, the causative agent of Allium White Rot disease. The terpenes identified by gas chromatography–electron impact mass spectrometry (GC-EIMS) in infected plants were nerolidol, phytol, squalene, α-pinene, terpinolene, limonene, 1,8-cineole and γ-terpinene, whose levels significantly increased when exposed to the fungus. Consistent with this, an increase in terpene synthase (TPS) activity was measured in infected plants. Among the terpenes identified, nerolidol, α-pinene and terpinolene were the most abundant with antifungal activity against S. cepivorum being assessed in vitro by mycelium growth inhibition. Nerolidol and terpinolene significantly reduced sclerotia production, while α-pinene stimulated it in a concentration-dependent manner. Parallel to fungal growth inhibition, electron microscopy observations established morphological alterations in the hyphae exposed to terpinolene and nerolidol. Differences in hyphal EtBr uptake suggested that one of the antifungal mechanisms of nerolidol and terpinolene might be disruption of fungal membrane integrity.

Abstract 3786: Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration.

Abstract Connexin 43 (Cx43) can form both gap junction channels and halves of gap junctions known as hemichannels. Hemichannels allow communication between cells and the extracellular environment. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation through fluid flow shear stress (FFSS) as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. Here, we explore the role of Cx43 hemichannels in osteocytes and how it affects cancer cell migratory behavior. We confirm the opening of Cx43 hemichannels in the presence of AD by measuring uptake of ethidium bromide (EtBr) in osteocytic MLO-Y4 cells. As expected, an increase in EtBr uptake was observed after AD treatment. To ensure the specificity of Cx43 hemichannels, we treated the MLO-Y4 cells with Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity. This prevented the uptake of EtBr by the MLO-Y4 cells. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell migration, we stimulated the opening of Cx43 hemichannels on MLO-Y4 osteocytes with either AD or FFSS. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays. The CM collected from MLO-Y4 osteocytes treated with AD or by FFSS reduced cancer cell migration in a dose-dependent manner. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody. Treatment with this antibody significantly attenuated the inhibitory effect of the CM on the migration of MDA-MB-231 and PC3 cells. Interestingly, the inhibitory effects on cancer cell migration were not observed when we used CM collected from osteoblasts, whose hemichannels were not shown to open by AD treatment. In order to determine whether ATP released from the Cx43 hemichannels in osteocytes would have an effect on cancer cell migration, we depleted ATP from the CM collected from MLO-Y4 cells using apyrase, an ATP hydrolyzing enzyme. The addition of apyrase increased MDA-MB-231 cell migration. To test the effect of purinergic signaling on breast cancer cell migration, we treated the CM with oxidized ATP (oATP), a potent inhibitor of ATP P2X receptors. The addition of oATP attenuated the inhibitory effect of CM on MDA-MB-231 cell migration. Then we stimulated the purinergic receptors with a nonhydrolyzable ATP analogue, ATPγS, the migration of MDA-MB-231 cells decreased. Together, our results point to the role of adenosine nucleotides released from Cx43 hemichannels in tumor migration and metastasis. Citation Format: Jade Z. Zhou, Jean X. Jiang. Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2013-3786

The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29)

The potential gap junction forming mouse connexin29 (Cx29) protein is concomitantly expressed with connexin32 (Cx32) in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47) in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harboring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29-mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

Abstract P3-13-06: Osteocytic Connexin 43 Hemichannels in Prevention of Bone Metastasis

Abstract Bone metastases are major, potentially fatal complications in patients with advanced cancers including breast, prostate and lung cancers. The gap junction-forming protein connexin 43 (Cx43) has been known to inhibit primary tumor cell growth. In addition to gap junctions, Cx43 also forms halves of gap junctions known as hemichannels which allow communication between the intracellular and extracellular environments. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. To confirm the opening of Cx43 hemichannels in the presence of AD, we measured uptake of ethidium bromide (EtBr) in MLO-Y4 cells after the addition of AD. As expected, an increase in Cx43 hemichannels EtBr uptake was observed after AD treatment. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell growth and migration, we treated MLO-Y4 osteocytes with AD. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays, and cell proliferation was measured using the soft agarose anchorage-independent growth assay. The CM collected from MLO-Y4 osteocytes treated with AD reduced cancer cell migration and proliferation in a dose-dependent manner. However, direct treatment with AD had no effect on the cell migration or proliferation of the MDA-MB-231 cells. These data validated that the decrease in migration and proliferation was not a result of direct action by AD on the cancer cells. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody, a specific antibody which blocks hemichannel activity but not gap junction channels formed by Cx43. The inhibitory effect of the CM on the migration and proliferation of MDA-MB-231 and PC3 cells was diminished. Interestingly, the inhibitory effects of osteocytes on cancer cell migration were not observed when using osteoblasts. Together, these results suggest the functional importance of osteocytic Cx43 hemichannels in attenuating cancer cell migration and growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-13-06.

Structure–Activity Relationships and Optimization of 3,5-Dichloropyridine Derivatives As Novel P2X7 Receptor Antagonists

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X(7) receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R(2)) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X(7) antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R(2) position optimized antagonistic activity. In the EtBr uptake assay in hP2X(7)-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC(50) values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC(50) = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X(7) receptor targeted anti-inflammatory agents.

Neuroinflammation Leads to Region-Dependent Alterations in Astrocyte Gap Junction Communication and Hemichannel Activity

Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

Induction of Mitochondrial Permeability Transition by the DNA-intercalating Cationic Dye Ethidium Bromide

This work shows that the DNA cationic probe, ethidium bromide (EtBr), induces the transition from selective to non-selective mitochondrial permeability. This statement is based on the findings, indicating: (i) EtBr induced the release of accumulated Ca(2+) through a mechanism sensitive to cyclosporin A and octylguanidine; (ii) EtBr induced the release of cytochrome c and (iii) EtBr induced mitochondrial swelling. Interestingly, mersalyl inhibited, in a non-competitive fashion, EtBr uptake, which would indicate that the uptake may be carried out through a protein membrane system. This work also shows that the effect of the dye on permeability transition was stimulated by carboxyatractyloside. Taking into account the facts that EtBr inhibited the ADP exchange reaction and increased the binding of the fluorescent probe eosin-5-maleimide to adenine nucleotide translocase, it is tempting to assume a possible interaction between EtBr and the ADP/ATP carrier.

Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP-activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co-transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co-expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration-response curves in Na+-containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co-expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 microM, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mM. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.

A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase(Erk1/2) module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase(Erk1/2) and non-selective pore opening in P2X7R-induced thymocyte death.

Pharmacological characterization of P2X7 receptors in rat peritoneal cells

P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.