Chitosan-Alginate Gels for Sorption of Hazardous Materials: The Effect of Chemical Composition and Physical State.

Chitosan, alginate, and chitosan-alginate (50:50) mixed hydrogels were prepared by freeze casting, freeze-drying, and subsequent physical cross-linking. Chitosan was cross-linked with citrate and alginate with calcium ions, while the mixed gels were cross-linked with both cross-linking agents. Both cryogels and xerogels were obtained by lyophilization and drying of the hydrogels. We investigated the effect of the chemical composition and the physical state of gels on the gel structure and sorption of model dyes. Alginate and mixed gels cross-linked with Ca2+ ions sorbed 80-95% of cationic dye from the solutions. The chitosan gels are primarily capable of adsorbing anionic dyes, but at near-neutral pH, their capacity is lower than that of alginate gels, showing 50-60% dye sorption. In the case of alginate gels, the dye sorption capacity of xerogels, cryogels, and hydrogels was the same, but for chitosan gels, the hydrogels adsorbed slightly less dye than the dried gels.

Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

Synthesis and characterizations of super adsorbent hydrogel based on biopolymer, Guar Gum-grafted-Poly (hydroxyethyl methacrylate) (Gg-g-Poly (HEMA)) for the removal of Bismarck brown Y dye from aqueous solution.

Chemical modification of guar gum was done by graft copolymerization of monomer hydroxyethyl methacrylate (HEMA) using azobisisobutyronitrile (AIBN) as initiator. Optimal reaction parameters were settled by varying one reaction condition and keeping the other constant. The optimum reaction conditions worked out were solvent system: binary, [H2O] = 15.00 mL, [acetone] = 5.00 mL, [HEMA] = 82.217× 10-2 mol/L, [AIBN] = 3.333 × 10-2 mol/L, reaction time = 3 h, reaction temperature = 60 °C on to 1.00 g guar gum with Pg = 1694.6 and %GE = 68,704.152. Pure guar gum polymer and grafts were analyzed by several physicochemical investigation techniques like FTIR, SEM, XRD, EDX, and swelling studies. Percent swelling of the guar gum polymer and grafts was investigated at pH 2.2, 7.0, 7.4 and 9.4 concerning time. The finest yield of Ps was recorded at pH 9.4 with time 24 h for graft copolymer. Guar gum and grafted samples were explored for the sorption of toxic dye Bismarck brown Y from the aqueous solution with respect to variable contact time, pH, temperature and dye concentration so as to investigate the stimuli responsive sorption behaviour. Graft copolymers showed better results than guar gum with percent dye uptake (Du) of 97.588 % in 24 h contact time, 35 °C temperature, 9.4 pH at 150.00 ppm dye feed concentration as compared to Guar gum which only showed 85.260 % dye uptake at alike dye fed concentration. The kinetic behaviour of the polymeric samples was evaluated by applying many adsorption isotherms and kinetic models. The value of 1/n was between 0 â†’ 1 showing that there was physisorption of the BB dye that took place on the surface of the polymers. Thermodynamics of BB Y adsorption onto hydrogels was investigated concerning the Van't Hoff equation. -∆G° values obtained from the curve proved the spontanity of the process. Within the context of adsorption efficiency, an investigation was conducted to examine the process of sorption of Bismarck brown Y dye from aqueous solutions. The graft copolymers demonstrated remarkable adsorption abilities, achieving a dye uptake (Du) of 97.588 % over a 24-h period at a temperature of 35 °C, pH level of 9.4, and a dye concentration of 150.00 ppm. The raised adsorption capacity was additionally corroborated by the application of several adsorption isotherms and kinetic models, which indicated that physisorption is the prevailing process/mechanism. Additionally, the thermodynamic research, utilising the Van't Hoff equation, validated the spontaneity of the adsorption phenomenon, as evidenced by the presence of a negative ∆G° values. The thermodynamic analysis revealed herein establishes a strong scientific foundation for the effectiveness of adsorbent composed of graft copolymers based on guar gum. The research conclude the efficiency of the guar gum based grafted copolymers for the water remediation as efficient adsorbents. The captured dye can be re-utilised and the hydrogels can be used for the same purpose in number of cycles.

Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

Identification of residues in the first transmembrane domain of the P2X7 that regulates receptor trafficking, sensitization, and dye uptake function.

P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.

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

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

Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors.

Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-ß-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.

Determination of hepatic clearance by derivations of the indocyanine green retention test in cirrhosis.

BACKGROUND AND AIM: The study was undertaken in order to compare single injection indocyanine green (ICG)-clearances with the steady-state ICG-clearance (ICGCl ) in patients with cirrhosis in order to assess the most accurate estimate for ICG-clearance and to relate the ICG-clearances to established indicators of liver dysfunction. METHODS: Thirty-eight patients (male 29) with cirrhosis (Child-Turcotte class A 8, class B 21, and class C 9) were studied during a hemodynamic investigation. A single injection of ICG was followed by blood samples for 5, 10, 15, and 20 min. The dose/plasma area clearance (ClA ) and plasma volume · initial slope clearance (ClPV ) were determined and compared with the steady-state infusion/plasma concentration ratio clearance (ICGCl ). RESULTS: The ClA (310; 214; 502 mL/min) and ClPV (294; 164; 481 mL/min) correlated closely with ICGCl (243; 120; 383 mL/min [median; interquartile range], R = 0.95-0.98, P < 0.000), but were significantly higher than ICGCl (P < 0.001). All three clearance measures correlated significantly with biochemical and hemodynamic variables of liver dysfunction (P < 0.05-0.000). All three ICG-clearances showed significantly lower values in patients with ascites compared to those without, and lower ICG-clearance values were present in patients with esophageal varices compared to those without (P < 0.05-0.002). CONCLUSION: Single injection markers (ClA and ClPV ) of the steady-state ICG-clearance as derived from the ICG-retention curve and the plasma volume correlate with ICGCl and established variables of portal hypertension and liver cell bile excretory dysfunction. Therefore, these markers can safely replace the more costly ICGCl .

A Solid-State Pathway towards the Tunable Carboxylation of Cellulosic Fabrics: Controlling the Surface's Acidity.

We report on a tunable solid-state approach to modify the acidity of cotton substrates using citric, oxalic, and fumaric acids. The first stage of the method involves soaking the cotton swatches in an ethanolic saturated solution of the corresponding acid. After drying, the carboxylation reaction proceeds at high temperature (T > 100 °C) and in solid state. We quantified the effect of temperature and reaction time on the solid-state carboxylation reaction, which allowed us to tune the carboxylation degree and the acidity of the surface. We characterized the modified cotton by performing adsorption isotherms and by determining the kinetics of adsorption of a cationic dye: methylene blue (MB). We found that the MB uptake kinetics varied as a function of the acidic strength of the surface, which is closely related to the strength of the acid used for surface modification. The proposed solid-state cotton carboxylation procedure allows us to achieve sustainable cotton modification, which constitutes a starting point for several applications using cotton as the substrate.

A method for assessing ionic and molecular permeation in connexin hemichannels.

Connexin hemichannels are permeable to both atomic ions and small molecules. Yet, they have different selectivity for ions and signaling molecules critical for biological functions. Activity of connexin hemichannels in living cells is commonly evaluated by methods that include electrophysiology and fluorescence-based approaches. Although less common, luminescence and radioactivity-based uptake/release assays have been also successfully used to determine selectivity and permeability to different molecules. The current methods, however, have important technical and quantitative limitations that make them unsuitable for simultaneously evaluating ionic and molecular permeability using different stimuli that control channel gating (e.g., voltage or extracellular Ca2+). To address this, we have recently designed a novel methodology that combines two-electrode voltage clamp (TEVC) and dye uptake assays in translucent Xenopus oocytes. This method allows for the evaluation of molecular transport kinetics in connexin hemichannels, and its utility can also be extended to other large pore channels, such as those formed by pannexin and CALHM. In this article, we describe step by step the protocol to perform the TEVC/Dye uptake assay.

Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells.

Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased hemichannel activity/function reflects increased loading. The technique relies on the uptake of a membrane-impermeable fluorescent tracer through open hemichannels, and can be used to compare channel activity between cell monolayers cultured under different conditions, e.g. control versus disease. Other techniques, such as biotinylation and electrophysiology can measure cell surface expression and hemichannel open probability respectively, however, carboxyfluorescein uptake provides a simple, rapid and cost-effective method to determine hemichannel activity in vitro in multiple cell types. Graphic abstract: Using dye uptake to measure hemichannel activity.

Synthetic 1,4-Naphthoquinones inhibit P2X7 receptors in murine neuroblastoma cells.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel and potential therapeutic target for new drug development. In this study, we synthesized a series of new 1,4-naphthoquinone (1,4-NQ) derivatives and investigated their antagonistic effects against mouse P2X7R. We explored the ability of the tested substances to block ATP-induced Ca2+ influx into mouse Neuro-2a cells and selected the four most effective substances: the 1,4-naphthoquinone thioglucosides U-548 and U-557 and their tetracyclic conjugates U-286 and U-556. Biological analysis of these compounds revealed significant in vitro inhibition of murine P2X7R. This inhibition resulted in marked blockade of ethidium bromide (EtBr) and YO-PRO-1 fluorescent dye uptake, pronounced decreases in ROS and NO production and protection of neuronal cell viability against the toxic action of high ATP concentrations. In silico analysis indicated favorable molecular docking results of these 1,4-NQs, pointing to their potential to bind in an allosteric site located in the extracellular region of P2X7R. These findings suggest compounds U-286, U-548, U-556 and U-557 as potential scaffolds for the design of new P2X7R blockers and drugs effective against neuropathic pain and neurodegenerative diseases.

Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation.

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity-measured by changes in DAPI uptake-of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water-lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

Arylboronic acids inhibit P2X7 receptor function and the acute inflammatory response.

The P2X7 receptor (P2X7R) is an ion channel which is activated by interactions with the extracellular ATP molecules. The molecular complex P2X7R/ATP induces conformational changes in the protein subunits, opening a pore in the ion channel macromolecular structure. Currently, the P2X7R has been studied as a potential therapeutic target of anti-inflammatory drugs. Based on this, a series of eight boronic acids (NO) analogs were evaluated on the biologic effect of this pharmacophoric group on the human and murine P2X7R. The boronic acids derivatives NO-01 and NO-12 inhibited in vitro human and murine P2X7R function. These analogs compounds showed effect better than compound BBG and similar to inhibitor A740003 for inhibiting dye uptake, in vitro IL-1ß release and ATP-induced paw edema in vivo. In both, in vitro and in vivo assays the compound NO-01 showed to be the hit compound in the present series of the arylboronic acids analogs. The molecular docking suggests that the NO derivatives bind into the upper body domain of the P2X7 pore and that the main intermolecular interaction with the two most active NO derivatives occur with the residues Phe 95, 103 and 293 by hydrophobic interactions and with Leu97, Gln98 and Ser101 by hydrogen bonds.. These results indicate that the boronic acid derivative NO-01 shows the lead compound characteristics to be used as a scaffold structure to the development of new P2X7R inhibitors with anti-inflammatory action.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of New Thiadiazole Derivatives as Potent P2X7 Receptor Inhibitors.

Twenty new 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs were synthetized to develop P2X7 receptor (P2X7R) inhibitors. P2X7R inhibition in vitro was evaluated in mouse peritoneal macrophages, HEK-293 cells transfected with hP2X7R (dye uptake assay), and THP-1 cells (IL-1ß release assay). The 1-(5-phenyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-5-amine derivatives 9b, 9c, and 9f, and 2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-(4-fluorophenyl)-1,3,4-thiadiazole (11c) showed inhibitory effects with IC50 values ranging from 16 to 122 nM for reduced P2X7R-mediated dye uptake and 20 to 300 nM for IL-1ß release. In addition, the in vitro ADMET profile of the four most potent derivatives was determined to be in acceptable ranges concerning metabolic stability and cytotoxicity. Molecular docking and molecular dynamics simulation studies of the molecular complexes human P2X7R/9f and murine P2X7R/9f indicated the putative intermolecular interactions. Compound 9f showed affinity mainly for the Arg268, Lys377, and Asn266 residues. These results suggest that 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs may be promising novel P2X7R inhibitors with therapeutic potential.

Interactive effects of As, Cd and Zn on their uptake and oxidative stress in As-hyperaccumulator Pteris vittata.

The effects of arsenic (As), cadmium (Cd) and zinc (Zn) on each other's uptake and oxidative stress in As-hyperaccumulator Pteris vittata were investigated. P. vittata plants were exposed to 50 µM As, Cd and/or Zn for 15 d in 0.2-strength Hoagland solution. When applied alone, P. vittata accumulated 185 mg kg-1 As, 164 mg kg-1 Cd and 327 mg kg-1 Zn in the fronds. While Cd and Zn did not impact each other's uptake, As affected Cd and Zn uptake. Whereas As decreased Zn uptake, Zn affected As speciation in P. vittata fronds, with more arsenate (AsV) than arsenite (AsIII) being present. At 50 µM As, 75 µM Zn increased As accumulation in P. vittata fronds by 10 folds to 2363 mg kg-1 compared to 50 µM Zn. Although AsV was the predominant As species in all tissues, Cd enhanced AsIII levels in the fronds but increased AsV in the roots. Co-exposure of Cd + Zn elevated oxidative stress basing on thiobarbituric acid reactive substances, H2O2 content, Evans blue dye uptake, membrane injury index and reactive oxygen species (ROS) relative to single metal. By lowering Cd and Zn concentrations in P. vittata fronds, As reduced the associated stress comparative to Cd or Zn treatment. The results enhance our understanding of the mechanisms underlying the interactions between As, Cd and Zn in As-hyperaccumulator P. vittata.

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages.

Extracellular adenosine 5'-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1ß cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100 µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24 h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.

A central role for P2X7 receptors in human microglia.

BACKGROUND: The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1ß release. However, the role of the P2X7R in primary adult human microglia is poorly understood. METHODS: We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels. RESULTS: We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl- channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl- channel antagonists. CONCLUSIONS: Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.