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Intracellular Application (intracellular + application)
Selected AbstractsElectrophysiological and morphological characterization of dentate astrocytes in the hippocampusDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2005Masako Isokawa Abstract We studied electrophysiological and morphological properties of astrocytes in the dentate gyrus of the rat hippocampus in slices. Intracellular application of Lucifer yellow revealed two types of morphology: one with a long process extruding from the cell body, and the other with numerous short processes surrounding the cell body. Their electrophysiological properties were either passive, that is, no detectable voltage-dependent conductance, or complex, with Na+/K+ currents similar to those reported in the Ammon's horn astrocytes. We did not find any morphological correlate to the types of electrophysiological profile or dye coupling. Chelation of cytoplasmic calcium ([Ca2+]i) by BAPTA increased the incidence of detecting a low Na+ conductance and transient outward K+ currents. However, an inwardly rectifying K+ current (Kir), a hallmark of differentiated CA1/3 astrocytes, was not a representative K+ -current in the complex dentate astrocytes, suggesting that these astrocytes could retain an immature form of K-currents. Dentate astrocytes may possess a distinct current profile that is different from those in CA1/3 Ammon's horn. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Molecular mechanisms of cross-inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK-293 cellsNEUROGASTROENTEROLOGY & MOTILITY, Issue 8 2010D. A. Decker Abstract Background, P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and ,3,4subunit-containing nAChRs. Methods, The function of P2X2 and ,3,4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Key Results, In HEK-293 cells expressing ,3,4 nAChRs and P2X2 receptors, co-application of ATP and acetylcholine caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing ,3,4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing ,3,4 nAChRs transfected with P2X2TR. Currents caused by low (,30 ,mol L,1), but not high (,100 ,mol L,1) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with ,3,4 nAChRs. Conclusions & Inferences, The C-terminal tail of P2X2 receptors mediates cross-inhibition between ,3,4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross-inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. [source] Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2003Dominique Chesnoy-Marchais Abstract Dideoxyforskolin, a forskolin analogue unable to stimulate adenylate cyclase, and tamoxifen, an antioestrogen widely used against breast cancer, are both known to block some Cl, channels. Their effects on Cl, responses to glycine or GABA have been tested here by using whole-cell recording from cultured spinal neurons. Dideoxyforskolin (4 or 16 µm) and tamoxifen (0.2,5 µm) both potentiate responses to low glycine concentrations. They also induce blocking effects, predominant at high glycine concentrations. At 5 µm, tamoxifen increased responses to 15 µm glycine by a factor >4.5, reaching 20 in some neurons. Potentiation by extracellular dideoxyforskolin or tamoxifen persisted after intracellular application of the modulator and was not due to Zn2+ contamination. Potentiation by tamoxifen also persisted in a Ca2+ -free extracellular solution, after intracellular Ca2+ buffering and protein kinase C blockade. Thus, the critical sites of action are not intracellular. The EC50 for glycine was lowered 6.6-fold by 5 µm tamoxifen. The kinetics and voltage-dependence of the effects of tamoxifen on glycine responses support the idea that this hydrophobic drug may act from a site located within the membrane. Tamoxifen (5 µm) also increased responses to 2 µm GABA by a factor of 3.5, but barely affected peak responses to 20 µm GABA. The demonstration that tamoxifen affects some of the main inhibitory receptors should be useful for better evaluating its neurological effects. Furthermore, the results identify a new class of molecules that potentiate glycine receptor function. [source] A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000J. Russell Burley Abstract The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+ -dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+ -channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2B,3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 ,m FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+ -dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 m m BAPTA or 1 ,m FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels. [source] Noradrenaline inhibits exocytosis via the G protein ,, subunit and refilling of the readily releasable granule pool via the ,i1/2 subunitTHE JOURNAL OF PHYSIOLOGY, Issue 18 2010Ying Zhao The molecular mechanisms responsible for the ,distal' effect by which noradrenaline (NA) blocks exocytosis in the ,-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 ,-cells. NA inhibited Ca2+ -evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca2+ influx, by intracellular application of antibodies against the G protein subunit G, and was mimicked by the myristoylated ,,-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca2+]i and is mediated by an interaction between G,, and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a G,i1/2 blocking peptide demonstrating that the effect is mediated via activation of G,i1 and/or G,i2. [source] Conversion of the Synthetic Catalase Mimic Precursor TAA-1 into the Active Catalase Mimic in Isolated HepatocytesCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2009Ursula Rauen In previous studies we reported on the catalase-like activity and antioxidative properties of a non-heme Fe(III)-tetraaza[14]annulene complex, 5,4-didehydro-5,9,14,18-tetraaza-di(2,2-dimethyl-[5,6]benzo[1,3]dioxolo)[a,h]cyclotetradecene,Fe(III) chloride (TAA-1/Fe). We proposed that intracellular application of the parent, iron-free tetraaza[14]annulene ligand, TAA-1, as precursor would allow antioxidative defense along two lines, i.e. by chelation of potentially toxic cellular iron ions and, subsequently, by catalase-mimic activity. We here set out to establish whether the active catalase mimic is indeed formed intracellularly when cells are loaded with the ligand. When isolated rat hepatocytes were preloaded with TAA-1, they were protected against iron-induced cell injury and oxidative stress elicited by exposure to the membrane-permeable iron complex Fe(III)/8-hydroxyquinoline. After lysis of the cells, followed by ultrafiltration to remove endogenous catalase, the lysate exhibited catalase-like activity, while lysates of control cells not treated with TAA-1 showed no catalase-like activity. By comparison with authentic TAA-1/Fe, an intracellular formation of 2.0 ± 0.3 ,m of the active catalase mimic in native hepatocytes exposed to TAA-1 and of 6.5 ± 1.0 ,m in hepatocytes exposed to both TAA-1 and iron ions was estimated. The intracellular formation of the active catalase mimic thus renders TAA-1 an attractive compound for protection against iron- and/or hydrogen peroxide-dependent cell injuries. [source] Disulfide Bond Substitution by Directed Evolution in an Engineered Binding ProteinCHEMBIOCHEM, Issue 8 2009Antoine Drevelle Dr. Abstract Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications. The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques. [source] | ||||||||||