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E Aizenman

Publications and source records attributed to E Aizenman.

At least 73 records · Page 4Linked to original sources
PubMed1990

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site.

A novel modulatory site on the N-methyl-D-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.

Animals↗
PubMed1990

Reduction of NMDA receptors with dithiothreitol increases [3H]-MK-801 binding and NMDA-induced Ca2+ fluxes.

1. We have investigated the modulation of N-methyl-D-aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). 2. Increases in [3H]-MK-801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2mM) and diminished by DTNB (0.5 mM). 3. The inhibition of [3H]-MK-801 binding by CGS 19755 and 7-chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT. 4. NMDA-induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB-reversible fashion. In addition to augmenting the magnitude of NMDA-induced increase in intracellular free Ca2+, 10 mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl. 5. These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.

Animals↗
PubMed1989

Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli.

The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone. Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains. Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response. The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction.

Alleles↗
PubMed1989

Two pharmacological classes of quisqualate-induced electrical responses in rat retinal ganglion cells in vitro.

The pharmacological properties of steady state responses elicited by the excitatory amino acid agonists quisqualate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) have been examined in isolated rat retinal ganglion cells with patch electrodes. Based upon testing retinal ganglion cells with both agonists at saturating concentrations (30 microM for quisqualate and 60 microM for AMPA), these neurons can be grouped into three different categories: (a) type I cells, in which AMPA steady state responses were larger than those induced by quisqualate; (b) type II cells, in which quisqualate steady state responses were larger than or equal to those produced by AMPA; and (c) type III cells, in which neither AMPA nor quisqualate elicited responses. Regardless of cell type (I or II), AMPA responses were substantially inhibited by the antagonists kynurenate (750 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM). In contrast, the effects of these antagonists on quisqualate responses varied depending on the cell type, with both kynurenate and CNQX being more effective in type II than in type I cells. Based upon further pharmacological manipulations, it is proposed that quisqualate-induced steady state responses in retinal ganglion cells are divisible into two pharmacological classes that exist in varying proportions in these cells in vitro.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1989

Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex.

In cultures of rat cerebral cortex in which astrocyte proliferation was stringently suppressed, glutamate neurotoxicity occurred at glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. Concentrations of glutamate one hundred-fold higher were required to produce neurotoxicity in the presence of abundant astrocytes. This suggests that the sensitivity of central neurons to glutamate toxicity may be dependent upon astrocyte function.

2-Amino-5-phosphonovalerate↗
PubMed1989

Selective modulation of NMDA responses by reduction and oxidation.

Electrophysiological responses to the glutamate analog N-methyl-D-aspartate (NMDA) measured in three different central neuronal preparations are subject to a novel modulatory mechanism: they are substantially potentiated after exposure to the disulfide reducing agent dithiothreitol, while oxidation with 5-5-dithiobis-2-nitrobenzoic acid decreases the magnitude of the response. Modification of the NMDA response by either oxidation or reduction does not appear to affect the pharmacological properties of the receptor-channel complex. Since we observe that the redox state of the native receptor-channel complex varies widely among neurons, an in vivo mechanism that can strongly regulate NMDA-activated functions by either reduction or oxidation may exist. In addition, these results suggest that it may be possible to design specific redox agents for characterizing the NMDA receptor-channel complex.

Animals↗
PubMed1989

Characterization of nicotinic receptors in chick retina using a snake venom neurotoxin that blocks neuronal nicotinic receptor function.

Nicotinic receptor function has been described in the retinas of a variety of vertebrate species. Neuronal bungarotoxin (NBT, also known as bungarotoxin 3.1, toxin F, or kappa-bungarotoxin) blocks nicotinic receptors in several neuronal preparations, while the neuromuscular antagonist alpha-bungarotoxin (BGT) fails to block most of these receptors. NBT (100 nM), but not BGT (10 microM), substantially blocks nicotinic function on ganglion cells in intact chick retina. 125I-NBT binds to 2 sites in homogenates of chick retina; one site that is shared with BGT (Kd = 5-7 nM, Bmax approximately 500 fmol/retina) and one which is not (Kd = 2-3 nM, Bmax approximately 100 fmol/retina). 125I-NBT binding to the NBT-specific site (binding in the presence of 1 microM unlabeled BGT) is localized to 2 bands in the inner plexiform layer, corresponding to regions richly innervated by neurons containing immunoreactivity for choline acetyltransferase. Furthermore, this binding is blocked by competitive nicotinic agonists and antagonists, but nicotine or other nicotinic agonists do not displace 125I-NBT binding with very high affinity relative to the displacement of 3H-nicotine reported by others in brain. Thus, of the 2 NBT binding sites, the site not recognized by BGT most likely represents functional nicotinic receptors in the chick retina, but these receptors have relatively low affinity for nicotinic agonists, similar to nicotinic receptors found in autonomic ganglia.

Animals↗
PubMed1988

The nucleotide sequence and characterization of the relA gene of Escherichia coli.

The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.

Bacterial Proteins↗
PubMed1988

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

Animals↗
PubMed1988

Central mammalian neurons normally resistant to glutamate toxicity are made sensitive by elevated extracellular Ca2+: toxicity is blocked by the N-methyl-D-aspartate antagonist MK-801.

It is widely held that a glutamate-like toxin that resembles N-methyl-D-aspartate may be responsible for the death of nerve cells seen after severe neurological insults including stroke, seizures, and degenerative disorders, such as Huntington disease, Alzheimer disease, and the amyotrophic lateral sclerosis-parkinsonism-dementia complex found on Guam. One puzzling fact about these maladies is the differential vulnerability of specific groups of neurons peculiar to each condition. We report here that an identified population of central neurons, rat retinal ganglion cells, are resistant to the neurotoxic effects of millimolar concentrations of glutamate under otherwise normal culture conditions. Patch-clamp experiments show that this resistance is associated with a very small ionic current response to N-methyl-D-aspartate. Varying the ionic milieu by increasing the extracellular Ca2+ concentration, however, results in a striking increase in glutamate-induced cell death in this population. Under these conditions, Mg2+ or the amino acid antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo-(alpha,gamma)-cyclohepten-5 ,10-imine maleate], blockers of N-methyl-D-aspartate receptor-coupled ion channels, completely abrogate the lethal effects of glutamate. These findings strongly suggest that Ca2+ entry through N-methyl-D-aspartate-activated channels is responsible for this type of neuronal death and suggest strategies that may be clinically useful in the treatment of various neurological disorders.

Animals↗
PubMed1988

Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells from rat.

1. The pharmacological properties of excitatory amino acid responses on ganglion cells dissociated from the rat retina were examined with the use of the whole-cell voltage-clamp technique. 2. L-Glutamate at a concentration of 50 microM produced inward non-desensitizing currents at negative holding potentials in nearly every cell tested (83%, n = 18) In physiological solutions, L-glutamate responses reversed at approximately -9 mV, and higher concentrations of this agonist introduced a desensitizing component to the response. 3. At negative holding potentials, kainate (25-125 microM) produced inward currents in all of the cells tested (n = 37). These currents never desensitized, even at high agonist concentrations, and reversed near -6 mV. Currents induced by 50 microM-kainate were reversibly antagonized by kynurenate (100-300 microM) but not by 100 microM-2-amino-5-phosphonovalerate (APV). 4. Quisqualate generated smaller, non-desensitizing currents in only 50% of the cells tested (n = 38). Quisqualate responses reversed in polarity near -4 mV and were maximal at an agonist dose of 25 microM, with higher concentrations introducing a rapidly desensitizing component without a detectable increase in amplitude. Currents produced by quisqualate at a concentration of 50 microM were not antagonized by either 750 microM-kynurenate or 100 microM-APV. 5. N-Methyl-D-aspartate (NMDA) produced inward currents at negative holding potentials in 68% of the cells tested (n = 31), but only when magnesium was excluded from the extracellular medium. NMDA currents were non-desensitizing at agonist concentrations of up to 200 microM, with higher concentrations introducing a rapidly desensitizing component. NMDA (200 microM) responses were blocked by APV (100 microM) and kynurenate (300 microM) and reversed near -1 mV. 6. Responses generated by kainate (50-125 microM) were antagonized by quisqualate (30-250 microM). This antagonism occurred even in cells having no measurable response to quisqualate alone, suggesting the possibility that quisqualate may be acting both as an agonist, in the 50% of the cells that have the quisqualate-specific receptor, and as an antagonist, at the kainate-specific site on all cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1988

The interaction of agonists and noncompetitive antagonists at the excitatory amino acid receptors in rat retinal ganglion cells in vitro.

The pharmacological properties of the interaction between the excitatory amino acid (EAA) analogs kainate and N-methyl-D-aspartate (NMDA) have been examined on the isolated rat retinal ganglion cell preparation. In addition, we have studied the effects on this interaction of 2 noncompetitive NMDA antagonists, the dissociative anesthetic phencyclidine (PCP) and the anticonvulsant MK-801. Electrophysiological measurements were performed with the whole-cell patch-clamp technique on cultured ganglion cells that had been back-labeled with a fluorescent dye. Whereas only 69% of the cells showed responses to NMDA (in the absence of extracellular Mg2+), every ganglion cell responded to kainate under the same conditions. When a given cell was voltage-clamped at -60 mV, the large inward currents elicited by 125 microM kainate generally exceeded the responses evoked by 200 microM NMDA, when present, by 1 or 2 orders of magnitude. There was a poor correlation between the magnitudes of the currents produced by both agonists for the population of cells tested. Furthermore, NMDA proved to be an antagonist for the kainate receptor binding site. Without influencing the kainate-activated currents, PCP (75 microM) and MK-801 (20 microM) completely and reversibly blocked the responses evoked by NMDA (200 microM), independent of the membrane holding potential. The degree of block produced by a submaximal concentration of either antagonist was accentuated by increasing the concentration of NMDA. The independence of NMDA and kainate currents was examined. In the presence of NMDA and PCP (or MK-801), kainate-induced responses were comparable in amplitude to those generated by the application of kainate and NMDA together. Thus, kainate continued to produce an increase in membrane conductance at a time when NMDA-activated currents were blocked by either antagonist. The NMDA antagonism of kainate-induced currents was shown to be constant and independent of PCP or MK-801. Our results suggest that the 2 EAA analogs might not share a common ionophore, but rather activate separate receptor-ion channel complexes in rat retinal ganglion cell membranes.

Animals↗
PubMed1987

Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells.

Using the patch-clamp technique, whole-cell recordings from solitary rat retinal ganglion cells in culture have established the nicotinic nature of the acetylcholine responses in these central neurons. Currents produced by acetylcholine (5-20 mumol/l) or nicotine (5-20 mumol/l) reversed in polarity near -5 mV and were unaffected by atropine (10 mumol/l). Agonist-induced currents were blocked by low doses (2-10 mumol/l) of the classical 'ganglionic' antagonists hexamethonium and mecamylamine, as well as by d-tubocurarine and dihydro-beta-erythroidine (the latter two do not discriminate clearly between ganglionic and neuromuscular junction receptors). Treatment with the potent neuromuscular blocking agent alpha-bungarotoxin (10 mumol/l) did not affect the cholinergic responses of these cells, while toxin F (0.2 mumol/l), a neural nicotinic receptor antagonist, readily abolished acetylcholine-induced currents. Thus, the experiments performed to date show that the nicotinic responses of retinal ganglion cells in the central nervous system share the pharmacology of autonomic ganglion cells in the peripheral nervous system. The ionic current carried by the nicotinic channels was selective for cations, similar to that described for nicotinic channels in other tissues. In addition, single-channel currents elicited by acetylcholine were observed in whole-cell recordings with seals greater than 5 G omega as well as in occasional outside-out patches of membrane. These acetylcholine-activated events, which had a unitary conductance of 48 pS and a reversal potential of 0 mV, represent the ion channels that mediate the neural nicotinic responses observed in these experiments on retinal ganglion cells.

Acetylcholine↗
PubMed1986

Botulinum toxin prevents stimulus-induced backfiring produced by neostigmine in the mouse phrenic nerve-diaphragm.

The origin of motor nerve antidromic activity (backfiring) induced by anticholinesterase treatment was examined in the mouse phrenic nerve-hemidiaphragm preparation. Botulinum toxin was used to determine whether backfiring is due to (a) a direct effect of the cholinesterase inhibitor on the nerve terminal, or (b) an indirect effect via the prolongation of the action of acetylcholine. In previously untreated control preparations, neostigmine produced spontaneous and stimulus-induced antidromic activity in the phrenic nerve when rapidly introduced into the diaphragm via its vasculature. This activity could be reversibly blocked by d-tubocurarine and decamethonium, but not by atropine. Neostigmine-induced backfiring did not occur in preparations in which transmitter release was blocked with botulinum toxin. Infusion of a small bolus of a high concentration of acetylcholine following neostigmine treatment resulted in a short-term increase in the incidence of antidromic activity, followed by block, in both controls and botulinum toxin-treated preparations. It is concluded that transmitter release is necessary for the production of backfiring following cholinesterase inhibition since neostigmine alone does not elicit antidromic activity in botulinum toxin-treated preparations at concentrations which are effective in controls. Our results support the hypothesis that the effects of neostigmine on the motoneurone terminal are mediated by the prolonged action of acetylcholine that occurs with inhibition of acetylcholinesterase.

Acetylcholine↗
PubMed1985

Axonal transport of alpha-bungarotoxin binding sites in rat sciatic nerve.

[125I]alpha-Bungarotoxin (alpha-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [125I]alpha-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [125I]alpha-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature. D-Tubocurarine, nicotine, decamethonium and atropine displaced [125]alpha-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [125I]alpha-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.

Animals↗
PubMed1984

Selective retrograde axonal transport of free glycine in identified neurons of Aplysia.

The specific retrograde axonal transport of free glycine within the identified neurons R3-14 of Aplysia californica was studied. The soma of the R3-14 neurons are located in the parietovisceral ganglion and their axons project down the branchial nerve to end in a large peripheral field. Using a double-chambered apparatus, the peripheral tissue was incubated in medium containing a 3H-amino acid for 4-48 hr, while the nerve and ganglion were isolated and perfused with plain or chemically altered medium. The nerve and ganglion were then either rapidly frozen for scintillation counting or fixed for autoradiography. When 3H-glycine was used, radioactivity entered the nerve rapidly, reached the ganglion in 3 hr, and was transported largely (greater than 80%) in the free amino acid form [trichloroacetic acid (TCA) soluble]. The right parietovisceral hemiganglion accumulated up to nine times more radioactivity than the left hemiganglion, reflecting the presence of the R3-14 axons and soma. Two phases of radioactivity were observed, a fast component moving at about 3 mm/hr and a slower (but larger) component moving at about 0.4 mm/hr. Light microscope autoradiography on nerves containing 3H-glycine revealed that the R3-14 axons accounted for more than 30% of the total label in the nerve but occupied less than 7% of the total cross-sectional area of the axonal core. Electron microscope autoradiography showed a close association of silver grains and dense core vesicles in the R3-14 axons. Retrograde axonal transport of free glycine was inhibited by (in decreasing order of effectiveness) mercuric chloride, vinblastine, colchicine, Nocodazole, and 2,4-dinitrophenol (2,4-DNP). Comparative studies of other amino acids [3H-leucine, 3H-serine, 3H-glutamic acid, 3H-gamma-aminobutyric acid (3H-GABA), and 3H-alanine] showed that 3H-glycine is the only amino acid that is rapidly axonally transported in large quantities within the R3-14 axons. This work demonstrates, for the first time, that a free amino acid, glycine, is transported in the retrograde direction within a select group of axons. The significance of this transport of glycine is discussed in relation to its use as a neural messenger by neurons R3-14.

2,4-Dinitrophenol↗
PubMed1984

Neuromuscular function and organotin compounds.

Mammalian exposure to toxic levels of the trialkyltin compounds, triethyltin (TET) and trimethyltin, results in pathological manifestations largely restricted to the nervous system. The remarkable features of TET toxicity are cerebral edema and muscular weakness. Rats exposed orally to TET (10-30mg TET Br/l of drinking water) progress through an increasingly compromised state beginning with mild ataxia and hindlimb weakness after one week, spastic paresis of the hindlimbs by two weeks; and hindlimb paraplegia and sensory changes by 3 weeks. Histopathological studies of chronic TET-exposed rats report minimal ultrastructural damage to distal peripheral nerves, myelin, and muscle. Chromatolytic reactions are observed in some alpha motor neurons; intramyelinic vacuolization in the ventral roots and horn is substantial by 3 weeks. Myelin vacuolization and degeneration are observed to a lesser extent in the dorsal roots of the spinal cord. Wet weights and myofiber diameters of EDL and soleus muscles are reduced during chronic TET intoxication, but no histopathology is evident using light microscopy. Conduction of compound action potentials in vivo along distal sensory fibers, ventral roots and distal motor fibers (in sciatic n.) is normal in 3 week TET rats as compared to control; however, nerve conduction velocity is decreased in the segment of the H-reflex arc involving the dorsal roots. Earlier studies by Stoner and coworkers led to suggestions that the neuromuscular junction may be preferentially affected by TET and could contribute, in part, to the symptoms of muscular weakness. In support of this hypothesis preliminary studies from our laboratory and others indicate that neurotransmission is functionally depressed at the myoneural junction following chronic TET treatment in vivo or when applied in vitro to isolated muscle preparations. Stimulated, but not unstimulated, release of acetylcholine from the vascular perfused rat phrenic nerve-hemidiaphragm preparation is decreased by TET especially at higher stimulation rates (20 Hz). In vitro administration of TET Br (10(-6)M) results in an irreversible decrease in the amplitude of evoked endplate potentials; chronic in vivo exposure to TET causes a decrease in the resting membrane potential of soleus muscle (in situ recordings) and provokes a peculiar post-stimulus (200Hz bursts) elevation of spontaneous miniature endplate potentials in isolated cut diaphragm preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗