Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzene sulfonate in tissues: a useful alternative to Immunohistochemistry for fluorescence microscopy imaging of protein nitration.

Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein- or peptide-bound 3-NT to a highly fluorescent benzoxazole product. In this study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum to: (i) test this method for imaging nitrated proteins in fixed brain tissue sections and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzene sulfonic acid (ABS) and K(3)Fe(CN)(6) at pH 9, after sodium dithionite reduction of 3-NT to 3-aminotyrosine, showed a very similar pattern of relative intensity of cell labeling and improved resolution compared with antibody labeling. Our data demonstrate that ABS derivatization may be either a useful alternative to or a complementary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular colocalization of nitrated proteins and any protein of interest.

Neuronal Glud1 (glutamate dehydrogenase 1) over-expressing mice: increased glutamate formation and synaptic release, loss of synaptic activity, and adaptive changes in genomic expression.

Glutamate dehydrogenase 1 (GLUD1) is a mitochondrial enzyme expressed in all tissues, including brain. Although this enzyme is expressed in glutamatergic pathways, its function as a regulator of glutamate neurotransmitter levels is still not well defined. In order to gain an understanding of the role of GLUD1 in the control of glutamate levels and synaptic release in mammalian brain, we generated transgenic (Tg) mice that over-express this enzyme in neurons of the central nervous system. The Tg mice have increased activity of GLUD, as well as elevated levels and increased synaptic and depolarization-induced release of glutamate. These mice suffer age-associated losses of dendritic spines, nerve terminals, and neurons. The neuronal losses and dendrite structural changes occur in select regions of the brain. At the transcriptional level in the hippocampus, cells respond by increasing the expression of genes related to neurite growth and synapse formation, indications of adaptive or compensatory responses to the effects of increases in the release and action of glutamate at synapses. Because these Tg mice live to a relatively old age they are a good model of the effects of a "hyperglutamatergic" state on the aging process in the nervous system. The mice are also useful in defining the molecular pathways affected by the over-activation of GLUD in glutamatergic neurons of the brain and spinal cord.

Photoelectrochemical characterisation and optimisation of electrodeposited ZnO thin films sensitised by porphyrins and phthalocyanines.

Hybrid thin films of crystalline ZnO modified by 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato zinc (TSTPPZn) and 2,9,16,23-tetrasulfophthalocyaninatozinc(II) (TSPcZn) were prepared by electrochemical deposition from aqueous zinc salt solutions. A "one-step" process with the sensitisers adsorbed during ZnO deposition represented the most simple approach. ZnO was also grown independently in the presence of Eosin Y as a structure-directing agent, which was then removed and the sensitisers were chemisorbed from solutions ("readsorption" method). The photoelectrochemical characteristics of the electrodes were studied by photocurrent spectra and by time-resolved photocurrent measurements in an acetonitrile-based solution containing I3(-)/I(-) as the redox electrolyte. In films with both sensitisers present, both sensitisers worked in parallel providing panchromatic sensitisation. Recombination of electrons injected into the conduction band of ZnO with remaining holes in the HOMO of the sensitisers was indicated for the one-step films, but was considerably suppressed for the films prepared by the readsorption method. Films prepared by the readsorption method showed a significantly increased efficiency by an increased surface area and suppressed recombination.

Superoxide modification and inactivation of a neuronal receptor-like complex.

Excessive superoxide (O(-)(2)) formation is toxic to cells and organisms. O(-)(2) reacts with either iron-sulfur centers or cysteines (Cys) of cytoplasmic proteins. Reactions with membrane proteins, however, have not been fully characterized. In the present studies, the reaction of O(-)(2) with a protein complex that has glutamate/N-methyl-D-aspartate (NMDA) receptor characteristics and with one of the subunits of this complex was examined. Exposure of the complex purified from neuronal membranes and the recombinant glutamate-binding protein (GBP) subunit of this complex to the O(-)(2)-generating system of xanthine (X) plus xanthine oxidase (XO) caused strong inhibition of L-[3H]glutamate binding. Inhibition of glutamate binding to the complex and GBP by O(-)(2) was greater than that produced by H(2)O(2), another product of the X plus XO reaction. Mutation of two cysteine (Cys) residues in recombinant GBP (Cys(190,191)) eliminated the effect of O(-)(2) on L-[3H]glutamate binding. Both S-thiolation reaction of GBP in synaptic membranes with [35S]cystine and reaction of Cys residues in GBP with [3H]NEM were significantly decreased after exposure of membranes to O(-)(2). Inhibition of cysteylation of membrane GBP by O(-)(2) was still observed after iron chelation by desferrioxamine, albeit diminished, and was not altered by the presence of catalase. Overall, the results indicated that GBP exposure to O(-)(2) modified Cys residues in this protein. The modification was not characterized but it was probably that of disulfide formation.

Activity-dependent nitric oxide concentration dynamics in the laterodorsal tegmental nucleus in vitro.

The behavioral-state related firing of mesopontine cholinergic neurons of the laterodorsal tegmental nucleus appears pivotal for generating both arousal and rapid-eye-movement sleep. Since these neurons express high levels of nitric oxide synthase, we investigated whether their firing increases local extracellular nitric oxide levels. We measured nitric oxide in the laterodorsal tegmental nucleus with a selective electrochemical microprobe (35 microm diam) in brain slices. Local electrical stimulation at 10 or 100 Hz produced electrochemical responses that were attributable to nitric oxide. Stimulus trains (100 Hz; 1 s) produced biphasic increases in nitric oxide that reached a mean peak concentration of 33 +/- 2 (SE) nM at 4.8 +/- 0.4 s after train onset and decayed to a plateau concentration of 8 +/- 1 nM that lasted an average of 157 +/- 23.4 s (n = 14). These responses were inhibited by N(G)-nitro-L-arginine-methyl-ester (1 mM; 92% reduction of peak; n = 3) and depended on extracellular Ca(2+). Chemically reduced hemoglobin attenuated both the electrically evoked responses and those produced by authentic nitric oxide. Application of the precursor, L-arginine (5 mM) augmented the duration of the electrically evoked response, while tetrodotoxin (1 microM) abolished it. Analysis of the stimulus-evoked field potentials indicated that electrically evoked nitric oxide production resulted from a direct, rather than synaptic, activation of laterodorsal tegmental neurons because neither nitric oxide production nor the field potentials were blocked by ionotropic glutamate receptor inhibitors. Nevertheless, application of N-methyl-D-aspartate also increased local nitric oxide concentration by 39 +/- 14 nM (n = 8). Collectively, these data demonstrate that laterodorsal tegmental neuron activity elevates extracellular nitric oxide concentration probably via somatodendritic nitric oxide production. These data support the hypothesis that nitric oxide can function as a local paracrine signal during the states of arousal and rapid-eye-movement sleep when the firing of mesopontine cholinergic neurons are highest.

Chronic ethanol exposure increases gene transcription of subunits of an N-methyl-D-aspartate receptor-like complex in cortical neurons in culture.

Chronic exposure to ethanol leads to adaptive responses in neurons, including enhanced activity of N-methyl-D-aspartate (NMDA) receptors. Chronic treatment of neurons with ethanol also leads to increases in the protein levels of three subunits of an NMDA receptor-like complex. In the present study, we explored whether the increases in subunit protein levels are the result of either increased transcription or diminished protein turnover. We found that a 72-h exposure of cortical neurons in culture to 100 mM ethanol caused enhanced transcription of the genes for the glutamate-binding (GBP) and glycine-binding protein subunits of the receptor-like complex. This treatment had no effect on protein turnover of either GBP or of the NMDA receptor subunit NR1, suggesting that the increases in protein expression are the result of increased DNA transcription.

Calcium influx through NMDA receptors, chronic receptor inhibition by ethanol and 2-amino-5-phosponopentanoic acid, and receptor protein expression.

Chronic treatment of neurons with either ethanol or competitive and noncompetitive antagonists of NMDA receptors leads to enhanced expression of NMDA receptor density and function in neurons. The signal transduction pathways for such receptor up-regulation are not known. The focus of the present study was on the role of Ca2+ entry into neurons, either through receptor or voltage-gated channels, in the expression of the NMDA receptor subunit NR1 and the 71-kDa glutamate-binding protein (GBP) of a glutamate/NMDA receptor-like complex. Chronic inhibition of NMDA receptors in cortical neurons in primary cultures by either 100 mM ethanol or 100 microM 2-amino-5-phosphonopentanoic acid (2-AP5) increased the expression of NR1 and GBP. The effect of 2-AP5 on the expression of the two proteins was not additive with that of ethanol when neuronal cultures were treated with both agents at the same time. However, the effects of ethanol on NR1 and GBP expression were blocked by the simultaneous treatment with NMDA (50 microM). Activation or inhibition of other glutamate ionotropic receptors had no effect on the expression of NR1 and GBP. The inhibition of L- or N-type voltage-sensitive Ca2+ channels and voltage-gated Na+ channels also had little effect on the expression of either protein; neither did exposure of neurons to elevated extracellular Ca2+ concentrations (3 or 5 mM). On the other hand, treatment of neurons for 48 h with the intracellular Ca2+ chelator BAPTA-AM as well as partial chelation of extracellular Ca2+ with EGTA caused an up-regulation in NR1 and GBP expression. The enhanced expression of NR1 in neurons treated for 48 h with either ethanol or EGTA was correlated with increases in the activity of NMDA receptors demonstrated as a doubling of the NMDA-stimulated rise in intracellular free Ca2+ concentration. The effects of chronic administration of EGTA on both NR1 expression as well as NMDA receptor function were probably related to an acute inhibition by EGTA of NMDA-induced Ca2+ influx into neurons. It appears that the expression of both the NR1 subunit of NMDA receptors and the GBP of a receptor-like complex is regulated by intracellular Ca2+, especially that entering through NMDA receptor ion channels.

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-D-aspartate receptor-like properties.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging.

Forty years of research into the function of L-glutamic acid as a neurotransmitter in the vertebrate central nervous system (CNS) have uncovered a tremendous complexity in the actions of this excitatory neurotransmitter and an equally great complexity in the molecular structures of the receptors activated by L-glutamate. L-Glutamate is the most widespread excitatory transmitter system in the vertebrate CNS and in addition to its actions as a synaptic transmitter it produces long-lasting changes in neuronal excitability, synaptic structure and function, neuronal migration during development, and neuronal viability. These effects are produced through the activation of two general classes of receptors, those that form ion channels or "ionotropic" and those that are linked to G-proteins or "metabotropic". The pharmacological and physiological characterization of these various forms over the past two decades has led to the definition of three forms of ionotropic receptors, the kainate (KA), AMPA, and NMDA receptors, and three groups of metabotropic receptors. Twenty-seven genes are now identified for specific subunits of these receptors and another five proteins are likely to function as receptor subunits or receptor associated proteins. The regulation of expression of these protein subunits, their localization in neuronal and glial membranes, and their role in determining the physiological properties of glutamate receptors is a fertile field of current investigations into the cell and molecular biology of these receptors. Both ionotropic and metabotropic receptors are linked to multiple intracellular messengers, such as Ca2+, cyclic AMP, reactive oxygen species, and initiate multiple signaling cascades that determine neuronal growth, differentiation and survival. These cascades of complex molecular events are presented in this review.

Cloning of a brain N-methyl-D-aspartate- and D, L-epsilon-2-amino-4-propyl-5-phosphono-3-pentanoic acid (CGP 39653)-binding protein.

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.

Effects of chronic ethanol treatment on the expression of calcium transport carriers and NMDA/glutamate receptor proteins in brain synaptic membranes.

Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca2+. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36-72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca2+, Mg2+-ATPases. The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.

Protein half-lives of calmodulin and the plasma membrane Ca-ATPase in rat brain.

We report the half-lives for two proteins involved in the regulation of intracellular calcium in the brain: the plasma membrane Ca-ATPase and its regulatory protein, calmodulin. [14C]-labeled leucine was injected into seven month old adult Fischer 344 rats and the time-dependent appearance and loss of radioactivity was monitored in both the serum and proteins from the brains of rats sacrificed from 4 hours to 13 days after injection. Experimental data obtained for calmodulin and the plasma membrane Ca-ATPase are best described by theoretical curves accounting for leucine reutilization that assume apparent half-lives of 18 (+/-2) hours and 12 (+/-1) days, respectively.

Protein half-lives of two subunits of an NMDA receptor-like complex, the 71-kDa glutamate-binding and the 80-kDa CPP-binding protein.

We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997, Biochem. Biophys. Res. Commun. 237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (+/- 2.4) and 18 (+/- 1.2) hours for CBP and GBP, respectively.

A synaptic membrane glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein: isolation and immunochemical characterization.

Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive L-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 microM) glycine binding sites. Glycine binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one(R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both L-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55-60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.

Ion channel properties of a protein complex with characteristics of a glutamate/N-methyl-D-aspartate receptor.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

The 71 kDa glutamate-binding protein is increased in cerebellar granule cells after chronic ethanol treatment.

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

Ethanol-induced inhibition of [3H]thienylcyclohexylpiperidine (TCP) binding to NMDA receptors in brain synaptic membranes and to a purified protein complex.

N-Methyl-D-aspartate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol-insensitive, but dizocilpine (MK-801)-sensitive, binding of N-[1-(2-[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration-dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by L-Glu when L-Glu was present at concentrations < 100 microM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of L-Glu (200-400 microM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has L-Glu-, Gly-, and [3H]TCP-binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either L-Glu or Gly. Activation constants (Kact) for L-Glu and Gly acting on the purified complex were 12 and 28 microM, respectively. Ethanol had no significant effect on the Kact of L-Glu but caused an increase in Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both L-Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.