Dye Uptake Experiments Research Articles

P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro

Glioblastoma is the most aggressive and lethal primary brain malignancy with limited treatment options and poor prognosis. Self-renewing glioblastoma cancer stem cells (GSCs) facilitate tumour progression, resistance to conventional treatment and tumour recurrence. GSCs are resistant to standard treatments. There is a need for novel treatment alternatives that effectively target GSCs. The purinergic P2X receptor 7 (P2X7R) is expressed in glioblastomas and has been implicated in disease pathogenesis. However, the roles of P2X7R have not been comprehensively elucidated in conventional treatment-resistant GSCs. This study characterised P2X7R channel and pore function and investigated the effect of pharmacological P2X7R inhibition in GSCs. Immunofluorescence and live cell fluorescent dye uptake experiments revealed P2X7R expression, and channel and pore function in GSCs. Treatment of GSCs with the P2X7R antagonist, AZ10606120 (AZ), for 72 hours significantly reduced GSC numbers, compared to untreated cells. When compared with the effect of the first-line conventional chemotherapy, temozolomide (TMZ), GSCs treated with AZ had significantly lower cell numbers than TMZ-treated cultures, while TMZ treatment alone did not significantly deplete GSC numbers compared to the control. AZ treatment also induced significant lactate dehydrogenase release by GSCs, indicative of treatment-induced cytotoxic cell death. There were no significant differences in the expression of apoptotic markers, Annexin V and cleaved caspase-3, between AZ-treated cells and the control. Collectively, this study reveals for the first time functional P2X7R channel and pore in GSCs and significant GSC depletion following P2X7R inhibition by AZ. These results indicate that P2X7R inhibition may be a novel therapeutic alternative for glioblastoma, with effectiveness against GSCs resistant to conventional chemotherapy.

SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels.ResultsWe found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP.ConclusionsWe hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.

Does scion–rootstock compatibility modulate photoassimilate and hormone trafficking through the graft junction in melon–pumpkin graft combinations?

The effect of the rootstock on the acropetal and basipetal transport of photoassimilates and hormones was studied in the 'Kiran' (Ki) melon cultivar grafted onto pumpkin rootstocks with different degrees of compatibility. A complementary experiment was performed to compare the incompatible combination (as evidenced by plant collapse at the fruit ripening stage), designated Ki/r53, with self-grafted r53/r53 as a model compatible combination. Both experiments showed the accumulation of a number of amino acids, sugars, and sugar alcohols in the scion of the incompatible Ki/r53 grafts. Additionally, they showed a marked reduction of trans-zeatin-type cytokinins and an elevated content of cis-zeatin-type cytokinins in the rootstock, and the opposite pattern in the scion, hinting at the possible involvement of a hormonal signal for graft compatibility. There was no direct evidence of a blockage at the graft union, since hormone acropetal and basipetal trafficking was demonstrated for all combinations. Dye uptake experiments did not show xylem flow impairment. A possibly significant finding in the incompatible combination was the deposition of undifferentiated cells in the hollow space that replaces the pith region in melon and pumpkin. The link between the above findings and the collapse of the plants of the incompatible combination remains unclear.

Idioblasts and peltate hairs as distribution networks for water absorbed by xerophilous leaves.

Capparis odoratissima is a tree species native to semi-arid environments of South America where low soil water availability coexists with frequent night-time fog. A previous study showed that water applied to leaf surfaces enhanced leaf hydration, photosynthesis and growth, but the mechanisms of foliar water uptake are unknown. Here, we combine detailed anatomical evaluations with water and dye uptake experiments in the laboratory, and use immunolocalization of pectin and arabinogalactan protein epitopes to characterize water uptake pathways in leaves. Abaxially, the leaves of C. odoratissima are covered with peltate hairs, while the adaxial surfaces are glabrous. Both surfaces are able to absorb condensed water, but the abaxial surface has higher rates of water uptake. Thousands of idioblasts per cm2 , a higher density than stomata, connect the adaxial leaf surface and the abaxial peltate hairs, both of which contain hygroscopic substances such as arabinogalactan proteins and pectins. The highly specialized anatomy of the leaves of C odoratissima fulfils the dual function of minimizing water loss when stomata are closed, while maintaining the ability to absorb liquid water. Cell-wall related hygroscopic compounds in the peltate hairs and idioblasts create a network of microchannels that maintain leaf hydration and promote water uptake.

Topological Strain-Induced Regioselective Linker Elimination in a Chiral Zr(IV)-Based Metal-Organic Framework

Summary Zr-carboxylate metal-organic frameworks (MOFs) are structurally robust materials, in part due to their strong coordination bonds. The regioselective Zr–O bond cleavage and formation between 3D architectures are thus challenging and are heretofore unexplored. In this work, by introducing highly flexible 18-crown-6-ether functionalities into a homochiral Zr-MOF, we report an unprecedented topology transition in which a 4,10-connected framework undergoes a rapid solid-state transition into a thermodynamically more stable 4,8-connected analog by a regioselective-linker-elimination under ambient conditions. The transition process was unambiguously unraveled by single-crystal and powder X-ray diffraction studies, and we proposed a possible transition mechanism based on various control experiments and theoretical calculations. The excellent chemical stability and substantially expanded porosity and pore apertures endowed the transformed chiral MOF with an exceptional capacity for the enantioadsorptive and solid-phase extractive separation of the racemic drug molecule of lansoprazole with 98% ee and 93% ee, respectively.

Cx43 channels and signaling via IP3/Ca2+, ATP, and ROS/NO propagate radiation-induced DNA damage to non-irradiated brain microvascular endothelial cells

Radiotherapeutic treatment consists of targeted application of radiation beams to a tumor but exposure of surrounding healthy tissue is inevitable. In the brain, ionizing radiation induces breakdown of the blood–brain barrier by effects on brain microvascular endothelial cells. Damage from directly irradiated cells can be transferred to surrounding non-exposed bystander cells, known as the radiation-induced bystander effect. We investigated involvement of connexin channels and paracrine signaling in radiation-induced bystander DNA damage in brain microvascular endothelial cells exposed to focused X-rays. Irradiation caused DNA damage in the directly exposed area, which propagated over several millimeters in the bystander area. DNA damage was significantly reduced by the connexin channel-targeting peptide Gap26 and the Cx43 hemichannel blocker TAT-Gap19. ATP release, dye uptake, and patch clamp experiments showed that hemichannels opened within 5 min post irradiation in both irradiated and bystander areas. Bystander signaling involved cellular Ca2+ dynamics and IP3, ATP, ROS, and NO signaling, with Ca2+, IP3, and ROS as crucial propagators of DNA damage. We conclude that bystander effects are communicated by a concerted cascade involving connexin channels, and IP3/Ca2+, ATP, ROS, and NO as major contributors of regenerative signal expansion.

Analysis of the dominant mutation N188T of human connexin46 (hCx46) using concatenation and molecular dynamics simulation.

Connexins (Cx) are proteins that form cell‐to‐cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co‐expressed wild‐type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt‐hCx46N188T and hCx46N188T‐hCx46wt to analyze how hCx46N188T affected co‐expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt‐hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt‐hCx46wt, while the hCx46N188T‐hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co‐expressing the variants.

Creating Aligned Nanopores by Magnetic Field Processing of Block Copolymer/Homopolymer Blends.

We describe the phase behavior of a cylinder-forming block copolymer(BCP)/homopolymer blend and the generation of aligned nanopores by a combination of magnetic field alignment and selective removal of the minority-block-miscible homopolymer. Alignment is achieved by cooling through the order-disorder transition temperature (Todt) in a 6 T field. The system is a blend of poly(styrene-block-4-vinylpyridine) (PS-b-P4VP) and poly(ethylene glycol) (PEG). PEG is miscible with P4VP and partitions preferentially into the cylindrical microdomains. Calorimetry and X-ray scattering show that Todt decreases linearly with PEG concentration until the onset of macrophase separation, inferred by PEG crystallization. Beyond this point, Todt is invariant with PEG content. Increasing PEG molar mass decreases the concentration at which macrophase separation is observed. Nanopore formation is confirmed by dye uptake experiments that show a clear dependence of dye uptake on PEG content before removal. We anticipate that this strategy can be extended to other BCP/homopolymer blends to produce nanoporous materials with reliable control of pore alignment and effective pore dimensions.

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.

Enzyme-like Click Catalysis by a Copper-Containing Single-Chain Nanoparticle.

A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the micromolar range. We report that copper cross-linked single-chain nanoparticles (SCNPs) are able to significantly increase the efficiency of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions at low substrate concentration in aqueous buffer by promoting substrate binding. Using a fluorogenic click reaction and dye uptake experiments, a structure-activity study is performed with SCNPs of different size and copper content and substrates of varying charge and hydrophobicity. The high catalytic efficiency and selectivity are attributed to a mechanism that involves an enzyme-like substratebinding process. Saturation-transfer difference (STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentrations, and computational simulations are consistent with a Michaelis-Menten, two-substrate, random-sequential enzyme-like kinetic profile. This general approach may prove useful for developing more-sustainable catalysts and agents for biomedicine and chemical biology.

Cues to Opening Mechanisms From in Silico Electric Field Excitation of Cx26 Hemichannel and in Vitro Mutagenesis Studies in HeLa Transfectans

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family.

Inhibition of astroglial connexin43 hemichannels with TAT-Gap19 exerts anticonvulsant effects in rodents.

Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT-Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT-Gap19. In vivo, pilocarpine-induced seizures as well as the accompanying increase in D-serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT-Gap19 was reversed by exogenous D-serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D-serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment.

Restraint stress increases hemichannel activity in hippocampal glial cells and neurons.

Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions, we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression.

Prenatal nicotine exposure enhances Cx43 and Panx1 unopposed channel activity in brain cells of adult offspring mice fed a high-fat/cholesterol diet.

Nicotine, the most important neuroteratogen of tobacco smoke, can reproduce brain and cognitive disturbances per se when administered prenatally. However, it is still unknown if paracrine signaling among brain cells participates in prenatal nicotine-induced brain impairment of adult offspring. Paracrine signaling is partly mediated by unopposed channels formed by connexins hemichannels (HCs) and pannexins serving as aqueous pores permeable to ions and small signaling molecules, allowing exchange between the intra- and extracellular milieus. Our aim was to address whether prenatal nicotine exposure changes the activity of those channels in adult mice offspring under control conditions or subjected to a second challenge during young ages: high-fat/cholesterol (HFC) diet. To induce prenatal exposure to nicotine, osmotic minipumps were implanted in CF1 pregnant mice at gestational day 5 to deliver nicotine bitartrate or saline (control) solutions. After weaning, offspring of nicotine-treated or untreated pregnant mice were fed ad libitum with chow or HFC diets for 8 weeks. The functional state of connexin 43 (Cx43) and pannexin 1 (Panx1) unopposed channels was evaluated by dye uptake experiments in hippocampal slices from 11-week-old mice. We found that prenatal nicotine increased the opening of Cx43 HCs in astrocytes, and Panx1 channels in microglia and neurons only if offspring mice were fed with HFC diet. Blockade of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2) and prostaglandin E receptor 1 (EP1), ionotropic ATP receptor type 7 (P2X7) and NMDA receptors, showed differential inhibition of prenatal nicotine-induced channel opening in glial cells and neurons. Importantly, inhibition of the above mentioned enzymes and receptors, or blockade of Cx43 and Panx1 unopposed channels greatly reduced adenosine triphosphate (ATP) and glutamate release from hippocampal slices of prenatally nicotine-exposed offspring. We propose that unregulated gliotransmitter release through Cx43 and Panx1 unopposed channels may participate in brain alterations observed in offspring of mothers exposed to tobacco smoke during pregnancy.

The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

Gas exchange is constrained by the whole-plant hydraulic conductance (K plant). Leaves account for an important fraction of K plant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (K leaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, K leaf recovered only 2 hours after plants were rewatered. Recovery of K leaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in K leaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in K leaf.

P2X7 receptors are a potential novel target for anti-glioma therapies

Human gliomas pose significant morbidity and mortality to those afflicted by them, and currently there are no curative treatment modalities available for these highly invasive tumours. With the approval from the human ethics committee, patients diagnosed with brain tumour (glioma) were recruited for this study. At the time of surgical resection, freshly resected tumour as well as ‘peri-tumour’ tissue were taken directly from theatre to the laboratory and were successfully cultured. Confocal fluorescence microscopy techniques and immunohistochemistry were used for characterization of human glioma cultures. Dye uptake experiments and confocal microscopy were utilized for P2X7 receptor (P2X7R) pore activity. We reveal human glioma cultures to contain microglia in close association with glioma (tumour) cells. Both glioma cells and microglia were found to express the purinergic, ATP sensing, P2X7R. P2X7R protein expression was increased in microglia derived from tumour when compared to ‘peri-tumour’ tissue. The pore capacity of P2X7R in tumour-associated microglia was functional, as evidenced by dye uptake experiments. Importantly, inhibition of P2X7R with the synthetic antagonist, brilliant blue G (BBG) resulted in a significant decrease in the number of glioma cells in culture. P2X7R was found to be over-expressed in grade IV human gliomas and its pore capacity was functional. Antagonism of P2X7R with BBG resulted in a decrease in tumour cell number. This identifies P2X7R as a promising therapeutic target to combat human glioma proliferation.

Relationship between temperature-induced changes in internal microscopic structures of poly(N-isopropylacrylamide) microgels and organic dye uptake behavior.

Temperature-induced changes in the internal structures of poly(N-isopropylacrylamide) (pNIPAm) microgels were evaluated by small-angle X-ray scattering (SAXS), and the results were used to explain organic dye uptake by the microgels. The dye uptake experiments were conducted using two organic dyes: cationic rhodamine 6G (R6G) and anionic erythrosine. In the SAXS investigation, the internal structures of the microgels were characterized in terms of the correlation length, ξ, and the distance, d*, which originated from the local packing of the isopropyl groups of two neighboring chains. With increasing temperature up to the volume phase transition temperature (VPTT) of the microgels, the correlation length, ξ, was increased and the distance, d*, was decreased. At the same time, the amounts of the dyes taken up by the pNIPAm microgels were increased, despite a decrease in the volume of the microgels. The results indicated that the pNIPAm chains were closer to each other due to the hydrophobic association of isopropyl groups, which resulted in the growth of the hydrophobic domains. Thus, the hydrophobic interactions between the dyes and pNIPAm were probably accompanied by the domain formation. With a further increase of temperature above the VPTT, the correlation length, ξ, was decreased and then not defined because the Ornstein-Zernike type contribution disappeared, and the distance, d*, was not largely changed. At the same time, the uptake amounts of the dyes per unit volume of the microgels were also not largely changed, which behaved similar to the distance, d*. It was probably due to the fact that the internal structures of the microgels were not largely changed because the isopropyl groups were in contact with each other. The view was supported by the result of the uptake study of the nonthermoresponsive microgels which did not have the hydrophobic isopropyl groups.

Astrocytes inhibit nitric oxide-dependent Ca2+dynamics in activated microglia: Involvement of ATP released via pannexin 1 channels

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca(2+) and produce high amounts of nitric oxide (NO). However, whether NO, Ca(2+) dynamics, and gliotransmitter release are reciprocally modulated is not fully understood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO-dependent Ca(2+) dynamics and ATP release in LPS-stimulated microglia. Griess assays and Fura-2AM time-lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca(2+) ]i that depended on the sequential activation of iNOS, COXs, and EP1 receptor. TGFβ1 released by astrocytes inhibited the abovementioned responses and also abolished LPS-induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS-stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS-stimulated microglia, exogenous ATP triggered activation of purinergic P2Y1 receptors resulting in Ca(2+) release from intracellular stores. Interestingly, TGFβ1 released by astrocytes inhibited ATP-induced Ca(2+) response in LPS-stimulated microglia to that observed in control microglia. Finally, COX/EP1 receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS-stimulated microglia. Thus, Ca(2+) dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies.

Is pannexin the pore associated with the P2X7 receptor?

The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes. Activation of P2X7R has been implicated in the formation of a cytolytic pore (i.e., a large conductance channel) that allows the passage of molecules up to 900 Da in macrophages. At least two hypotheses have been presented to explain the conversion of a nonselective cation channel to a cytolytic pore. One hypothesis suggests that the pore is a separate molecular structure activated by P2X7R, and the second asserts that this is an intrinsic property of P2X7R (pore dilation). Based on connexin knockout and hemichannel antagonist studies, some groups have concluded that connexins and pannexins, the hemichannel-forming proteins in vertebrates, are fundamental components of the large conductance channel associated with P2X7R. Dye uptake and electrophysiology experiments were used to evaluate the efficacy and specificity of some hemichannel antagonists under conditions known to open the large conductance channel associated with P2X7R. Hemichannel antagonists and interference RNA (RNAi) targeting pannexin-1 did not affect P2X7R macroscopic currents [ATP, 1,570±189 pA; ATP+100 μM carbenoxolone (CBX), 1,498±100 pA; ATP+1 mM probenecid (Prob), 1,522±9 pA] or dye uptake in a FACS assay (ATP, 63±5%; ATP+100 μM CBX, 51.51±8.4%; ATP+1 mM Prob, 57.7±4.3%) in mouse macrophages. These findings strongly suggest that the high-permeability pore evident after prolonged P2X7R activation does not occur through connexin or pannexin hemichannels in murine macrophages. Another membrane protein may be involved in P2X7R pore formation.

Effects of Plasma Treatment of Wool on the Uptake of Sulfonated Dyes with Different Hydrophobic Properties

A wool fabric has been subjected to an atmospheric-pressure treatment with a helium plasma for 30 seconds. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry confirmed removal of the covalently-bound fatty acid layer (F-layer) from the surface of the wool fibers, resulting in exposure of the underlying, hydrophilic protein material. Dye uptake experiments were carried out at 50°C to evaluate the effects of plasma on the rate of dye uptake by the fiber surface, as well as give an indication of the adsorption characteristics in the early stages of a typical dyeing cycle. The dyes used were typical, sulfonated wool dyes with a range of hydrophobic characteristics, as determined by their partitioning behavior between water and n-butanol. No significant effects of plasma on the rate of dye adsorption were observed with relatively hydrophobic dyes. In contrast, the relatively hydrophilic dyes were adsorbed more rapidly (and uniformly) by the plasma-treated fabric. It was concluded that adsorption of hydrophobic dyes on plasma-treated wool was influenced by hydrophobic interactions, whereas electrostatic effects predominated for dyes of more hydrophilic character. On heating the dyebath to 90°C in order to achieve fiber penetration, no significant effect of the plasma treatment on the extent of uptake or levelness of a relatively hydrophilic dye was observed as equilibrium conditions were approached.