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Biomedical subjects

M Baudry

Publications and source records attributed to M Baudry.

At least 163 records · Page 9Linked to original sources
PubMed1990

Regional distribution of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding sites in rat brain: effect of chemical modification of SH- groups in tissue sections.

Previous studies have shown that chemical modifications of sulfhydryl (SH-) groups with mercurial compounds in rat brain membrane preparations increase the binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid [(3H]AMPA), a ligand for the quisqualate/AMPA type of glutamate receptors. In the present study we investigated the regional distribution of SH- group modification by quantitative analysis of autoradiographic images of [3H]AMPA binding in tissue sections. We also compared the effect of SH- group modification to that of the chaotropic ion thiocyanate (SCN-) which has been generally utilized to study [3H]AMPA binding sites. Low levels of binding sites were observed in the absence of potassium thiocyanate (KSCN), with binding predominantly found in telencephalic structures. The presence of KSCN induced a relatively uniform and large (four- to fivefold) increase in binding throughout the different brain structures. Pretreatment of the tissue sections with the SH- group reagent p-chloromercuriphenylsulfonic acid produced a 0.5- to 1.5-fold increase in [3H]AMPA binding. The enhanced binding displayed a regional variation with the largest increase in binding observed in the outer layer of the parietal cortex whereas the lowest increase occurred in the striatum. These results indicate that SH- group modification of tissue sections produces an increase in [3H]AMPA binding similar to that observed in detergent-treated membrane preparations. Moreover they reveal that [3H]AMPA binding sites in different brain regions vary in their susceptibility to modification by SH- reagents, suggesting the existence in brain of a heterogeneous distribution of quisqualate/AMPA receptor subtypes.

4-Chloromercuribenzenesulfonate↗
PubMed1990

N-Methyl-D-aspartate and quisqualate/DL-alpha-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors: differential regulation by phospholipase C treatment.

The effect of phospholipase C (PLC) treatment of rat brain membranes on the binding properties of excitatory amino acid receptors was investigated using both a phosphsphatidylcholine-hydrolyzing PLC from Clostridium perfringens and a phosphatidylinositol-specific PLC from Bacillus thuringiensis. PLC from C. perfringens produced an increased affinity of the quisqualate/DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor for its ligand, whereas kainate receptor binding was not affected. Both kinetic analysis and equilibrium saturation experiments indicated that PLC treatment produced a decrease in affinity for [3H]N-(1-[thienyl]cyclohexyl)-piperidine [( 3H]TCP), a ligand for the N-methyl-D-aspartate (NMDA) receptor-associated ionic channel, when the channel was fully activated by high concentrations of glutamate and glycine but increased its binding under conditions in which the channel was presumably closed. This latter component of the binding was not due to an interaction of [3H]TCP with non-glutamate receptor sites, such as sigma opioid and histamine H3 receptors. Binding of [3H]glutamate and [3H] glycine to the NMDA receptors was not modified by PLC treatment, but there was a large decrease in the binding of the NMDA antagonist [3H]3-[(+/-)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. Stimulation by glycine of [3H]glutamate binding was also abolished following PLC treatment. In contrast to PLC from C. perfringens, phosphatidylinositol-specific PLC treatment did not detectably modify the binding properties of the quisqualate/AMPA receptor or the NMDA receptor channel. These data indicate that alterations in the lipid microenvironment of the glutamate receptors modulate both the conformation and the function of the receptors and suggest a possible role for phospholipases in the regulation of synaptic transmission at excitatory synapses.

Animals↗
PubMed1989

The protease inhibitor leupeptin interferes with the development of LTP in hippocampal slices.

The effect of leupeptin, an inhibitor of thiol-proteases, was tested on the induction of long-term potentiation (LTP) in field CA1 of hippocampal slices. Two h of drug application did not produce substantial changes while a greater than 3-h application caused a sizeable reduction in the degree of LTP induced. Leupeptin had no obvious effects on the facilitation of postsynaptic responses occurring within or between the short high frequency bursts used to induce LTP, suggesting that the drug acted on cellular chemistries occurring after the initial physiological events that normally trigger LTP. These results are consistent with the hypothesis that a calcium-activated thiol protease (calpain) is involved in the induction of LTP.

Action Potentials↗
PubMed1989

Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain).

The present study tested the hypothesis that calpain is responsible for the limited proteolytic conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). We compared the effects of various proteases on the activity and molecular weight of a purified preparation of xanthine dehydrogenase from rat liver. In agreement with previous reports, trypsin treatment produced a complete conversion of XD to XO accompanied by a limited proteolysis of XDH from an Mr of 140 kD to an Mr of 90 kD. Treatment with calpain I or calpain II did not produce a conversion from XD to XO nor did it result in partial proteolysis of the enzyme. Similarly, trypsin treatment partially degraded a reversibly oxidized form of xanthine dehydrogenase while calpain I or calpain II were ineffective. The possibility that an endogenous inhibitor prevented the proteolysis of XDH by calpain I or II was excluded by verifying that brain spectrin, a known calpain substrate, was degraded under the same incubation conditions. The results indicate that calpain is not likely to be responsible for the in vivo conversion of XD to XO under pathological conditions.

Animals↗
PubMed1989

Quinoxaline derivatives are high-affinity antagonists of the NMDA receptor-associated glycine sites.

Membranes from rat telencephalon contain strychnine-insensitive glycine binding sites associated with NMDA receptors. Three quinoxaline derivatives, among them the high-affinity AMPA receptor antagonists CNQX and DNQX, were found to inhibit [3H]glycine binding to these sites with micromolar affinities. Binding of these compounds to the glycine site also inhibited glutamate-stimulated association and dissociation of [3H]TCP. This suggests that these AMPA antagonists, like the structurally related compound kynurenate, act as glycine site antagonists.

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

Enhancement of mitogen-induced lymphocyte proliferation by some inhibitors of alkaline phosphatase and diamine oxidase.

We selected various compounds [bromolevamisole, levamisole, cimetidine, L-homoarginine, 2,3,5,6-tetrahydroimidazo-(2,1-b)thiazole (IT), imidazole, theophylline] previously reported as inhibitors of alkaline phosphatase (ALP) and/or diamine oxidase (DAO) and studied their activity on concanavalin A (ConA)-induced mouse spleen cell lymphocyte proliferation. According to the Ki values, the decreasing order of potency for ALP inhibition was: bromolevamisole, levamisole, theophylline, cimetidine, IT, imidazole and L-homoarginine. The order of potency was different for DAO inhibition. Cimetidine was the most potent inhibitor of DAO, followed by bromolevamisole, levamisole, IT, imidazole and L-homoarginine. Theophylline had no inhibitory effect on DAO. We show that these compounds, except theophylline, enhance ConA-induced lymphocyte proliferation. Similarly, all the compounds except imidazole and theophylline, significantly inhibited ALP at concentrations which enhanced lymphocyte proliferation as measured by (3H)-thymidine uptake. DAO inhibition correlated with DNA synthesis only for IT and cimetidine. These observations suggest that ALP and DAO play a negative role in the proliferation process; however, the degree of enhancement of ConA-induced proliferation did not correlate strictly with the degree of ALP and DAO inhibition.

Alkaline Phosphatase↗
PubMed1989

Intrahippocampal colchicine injection results in spectrin proteolysis.

Neurons in the hippocampal formation vary markedly in their susceptibility to colchicine toxicity. The present study was directed at evaluating the effects of colchicine on the proteolytic breakdown of the cytoskeletal protein spectrin within the hippocampus in the rat. Quantified by immunoblot analysis of spectrin breakdown products, the extent of proteolysis was found to correlate with the relative vulnerability of different hippocampal subfields to colchicine toxicity. Levels of breakdown products increased dramatically between 1 and 2 days after colchicine injection, peaked between 2 and 4 days, and remained detectably elevated for at least 35 days. Two days after colchicine injection, the spectrin breakdown products were significantly more concentrated in the molecular layer than in the granule cell/hilar region of the dentate gyrus. The colchicine-induced increase in spectrin breakdown products was significantly reduced by pretreatment with the protease inhibitor leupeptin and was significantly elevated by pretreatment with the lysosomal inhibitor chloroquine. Immunohistochemical analyses of the hippocampus at various times after colchicine injection revealed changes in the distribution of spectrin-like immunoreactivity that paralleled the changes observed by Western blot analysis. Thus increased staining was observed in the molecular layer of the dentate gyrus at 2 and 4 days after the injection, while staining in CA3 was only slightly increased. In addition, abnormal staining of reactive astrocytes was prominent at 2 days. The mechanism whereby colchicine results in neuronal death is as yet unknown. However, the results presented here demonstrate that extensive proteolysis of a cytoskeletal protein occurs in response to the drug, suggesting a plausible mechanism for its neurotoxicity. The protease responsible for the effect is likely to be calpain since the process is non-lysosomal, leupeptin-sensitive and produces spectrin breakdown products indistinguishable from those generated by calpain treatment in vitro. These data support the hypothesis that calpain-mediated degradation of cytoskeletal elements is a common and early response to neurodegenerative events and serves as a trigger in the development of various neuropathologies.

Animals↗
PubMed1989

Dissociation between changes in glutamate receptor binding sites and their coupling to phosphatidylinositol metabolism following intrahippocampal colchicine injection.

Intrahippocampal colchicine injection produces a rapid death of granule cells and pyramidal neurons in the hippocampus in the rat. Under the appropriate assay conditions, [3H]glutamate labels the N-methyl-D-aspartate type of glutamate receptors while [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate labels the quisqualate type. Unilateral injection of colchicine (15 micrograms) in the dorsal hippocampus did not produce any change in [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding in membrane fractions from the dentate gyrus or CA1 field contralateral to the injection side, at least up to 12 days after the injection. However, it produced a progressive decrease in the binding of both ligands in dentate gyrus and CA1 of the injected hippocampus. In the dentate gyrus the changes in binding as a function of time after the injection were biphasic with a rapid exponential decrease (t1/2 about 8 days for both [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) until 12 days after the injection followed by a much slower decrease afterwards. A similar pattern was observed in CA1 although the changes in binding were smaller and delayed by about three days as compared to the dentate gyrus. Kinetic analyses of the binding at equilibrium were performed seven days after the injection and indicated that the changes in [3H]glutamate binding were due to a change in the maximum number of sites but not in affinity for the ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A glycine site associated with N-methyl-D-aspartic acid receptors: characterization and identification of a new class of antagonists.

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.

Animals↗
PubMed1989

Allosteric activation of brain mitochondrial Ca2+ uptake by spermine and by Ca2+: developmental changes.

Kinetic analysis of 45Ca2+ uptake by rat brain mitochondria in Ca2+ - 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that spermine both increased the apparent affinity for Ca2+ and decreased the cooperativity of uptake. Both effects are consistent with an allosteric activation of uptake by spermine. The stimulating effect of spermine on 45Ca2+ uptake was maximal with mitochondria from postnatal day 10 animals and then steadily decreased with increasing age to reach adult values by approximately 30 postnatal days; this was observed independently of the substrates used to fuel mitochondria. Mitochondrial Ca2+ buffering was also analyzed by use of a Ca2+-selective electrode. Addition of a large bolus of Ca2+ produced a decrease in the subsequent equilibrium extramitochondrial Ca2+ concentration (or a "rebound overshoot") under some conditions. It is proposed that this effect is the result of an allosteric activation of Ca2+ uptake by Ca2+. This effect was slowly reversible, or hysteretic, and was blocked by spermine. The overshoot was increased in the presence of higher concentrations of Mg2+ and was absent when mitochondria were incubated with 0.3 mM Mg2+. It was maximal in mitochondria prepared from early postnatal brain, and changes in the magnitude of the effect during development paralleled those obtained with spermine stimulation of 45Ca2+ uptake. The data suggest that spermine produces an allosteric activation of Ca2+ uptake by binding to the same regulatory sites that are involved in the Ca2+-induced activation. The results as a whole suggest that spermine could modulate mitochondrial buffering of the intracellular Ca2+ concentration in brain, particularly during the early postnatal period.

Aging↗
PubMed1989

Allosteric activation of brain mitochondrial Ca2+ uptake by spermine and by Ca2+: brain regional differences.

Analysis of the initial rates of 45Ca2+ uptake by rat brain mitochondria in Ca2+-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that nontelencephalic mitochondria exhibited both a much less pronounced stimulatory effect of spermine and significantly more hyperbolic kinetics of Ca2+ uptake than telencephalic mitochondria. Nontelencephalic mitochondria were also markedly less susceptible to a Ca2+-induced hysteretic allosteric activation of the Ca2+ uniporter. A new Ca2+ loading procedure, which strikingly illustrates differences in mitochondrial Ca2+ buffering characteristics, is also described. In this procedure, low concentrations of Ca2+ (1, 2, or 5 microM) were repetitively added to mitochondria every 30 s while changes in free Ca2+ concentration were recorded. Spermine induced a marked attenuation of the rise in free Ca2+ level under these conditions. Steady-state rates of Ca2+ uptake were determined by a quantitative analysis of the buffering of repetitive Ca2+ additions, and, again, brain regional differences were qualitatively similar to those observed in the initial rate kinetics; Ca2+ uptake by nontelencephalic mitochondria in the steady state was markedly less responsive to stimulation by spermine and appeared to have a more hyperbolic dependence on Ca2+ in the absence of spermine. These results also suggest that there is a lag time in the activation of the uniporter by Ca2+, in addition to the hysteresis that has previously been observed in the deactivation of the uniporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

2-Amino-5-phosphonovalerate↗
PubMed1988

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies.

Lesions of the rat entorhinal cortex cause extensive synaptic restructuring and perturbation of calcium regulation in the dentate gyrus of hippocampus. Calpain is a calcium-activated protease which has been implicated in degenerative phenomena in muscles and in peripheral nerves. In addition, calpain degrades several major structural neuronal proteins and has been proposed to play a critical role in the morphological changes observed following deafferentation. In this report we present evidence that lesions of the entorhinal cortex produce a marked increase in the breakdown of brain spectrin, a substrate for calpain, in the dentate gyrus. Two lines of evidence indicate that this effect is due to calpain activation: (i) the spectrin breakdown products observed following the lesion are indistinguishable from calpain-generated spectrin fragments in vitro; and (ii) their appearance can be reduced by prior intraventricular in fusion of leupeptin, a calpain inhibitor. Levels of spectrin breakdown products are increased as early as 4 h post-lesion, reach maximal values at 2 days, and remain above normal to some degree for at least 27 days. In addition, a small but significant increase in spectrin proteolysis is also observed in the hippocampus contralateral to the lesioned side in the first week postlesion. At 2 days postlesion the total spectrin immunoreactivity (native polypeptide plus breakdown products) increases by 40%, suggesting that denervation of the dentate gyrus produces not only an increased rate of spectrin degradation but also an increased rate of spectrin synthesis. These results indicate that calpain activation and spectrin degradation are early biochemical events following deafferentation and might well participate in the remodelling of postsynaptic structures. Finally, the magnitude of the observed effects as well as the stable nature of the breakdown products provide a sensitive assay for neuronal pathology.

Animals↗
PubMed1988

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies.

Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Chronic administration of a thiol-proteinase inhibitor blocks long-term potentiation of synaptic responses.

It has been proposed that activation of a calcium-sensitive protease (calpain) is a crucial step in the induction of long-term potentiation (LTP). To test this hypothesis, we used chronic recording techniques to measure the effects of intraventricular infusion of leupeptin, a calpain inhibitor, on LTP in the hippocampus. Rats implanted bilaterally with stimulating electrodes in the Schaffer-commissural system and one recording electrode in the apical dendrites of field CA1 were fitted with osmotic mini-pumps delivering either leupeptin (20 mg/ml) or saline at a rate of 0.5 microliter/h into the lateral ventricle. Short bursts of high-frequency stimulation with the bursts delivered at 5/s were used to induce LTP in those animals which had stable responses for several days. Rats in the saline group (n = 11) exhibited an immediate LTP effect that remained in place over successive days of testing, while only 3 of 13 leupeptin treated animals showed evidence of LTP 24 h after high-frequency stimulation, and in only one of those was a sizeable effect recorded over several days. The average change in responses at the 24-h test point was +33% for the controls and +4% for the leupeptin group (P less than 0.01). The block of LTP induction was reversible, since high-frequency stimulation applied after disconnecting the pumps led to a robust LTP effect that lasted for several days in 6 of 7 animals tested. There were no detectable differences in baseline responses in the presence and absence of leupeptin.

Animals↗
PubMed1988

The ultrastructural localization of calcium-activated protease "calpain" in rat brain.

Calpain I, a calcium-activated neutral protease which degrades a number of cytoskeletal proteins, has been implicated in the rapid turnover of structural proteins that may participate in synaptic plasticity. In the present study, an antibody raised against purified erythrocyte calpain I was biochemically characterized and demonstrated to specifically bind the Mr = 80,000 subunit of both rat erythrocyte and brain calpain I. This antibody was used to examine the cellular distribution of calpain I at the electron microscopic level in rat brain and spinal cord using the avidin-biotin immunocytochemical technique. Reaction product was observed throughout neuronal perikarya, within both axonal and dendritic processes, and within spine heads and necks. Postsynaptic densities in both shaft and spine synapses were also immunoreactive. Glial cell bodies and processes were densely stained. In both neurons and glia, the reaction product was deposited along cytoskeletal elements. The localization of calpain I immunoreactivity to glial processes suggests this degradative enzyme may play a role in the glial hypertrophy and process retraction seen in brain. The presence of the enzyme in spines and postsynaptic densities is consistent with the hypothesis that it is involved in the turnover of synaptic cytoskeleton, thus providing a means through which transient physiological events effect lasting changes in the chemistry and morphology of spines.

Animals↗
PubMed1988

Induction of ornithine decarboxylase in adult rat hippocampal slices.

Several factors involved in the regulation of ornithine decarboxylase (ODC) activity in adult rat brain tissue have been identified by using the in vitro hippocampal slice preparation. The same amino acids that have previously been reported to induce ODC in tissue culture, i.e., asparagine and glutamine, were found to produce a concentration- and time-dependent increase in ODC activity that reached a 100 fold the control value after 6 h of incubation. The effect of asparagine was totally blocked by inhibition of either protein or RNA synthesis, suggesting that the inducing amino acids increase ODC activity by stimulating the transcription of genes directly or indirectly regulating ODC activity. The effect of the inducing amino acids was potentiated by a variety of factors which by themselves did not modify ODC activity. In particular, opioid peptides markedly potentiated the effect of asparagine. Although the opiate antagonists naloxone and naltrexone totally blocked the effects of the opioid peptides on ODC induction, they also produced an inhibition of the asparagine-mediated increase in ODC activity. Other factors like dibutyryl cyclic AMP and insulin also potentiated the effects of asparagine on ODC activity. These results provide the first description of ODC induction in an in vitro preparation of adult brain tissue and indicate that the hippocampal slice preparation could be used to study the molecular mechanisms which regulate the expression and activity of ODC in the adult central nervous system. Moreover the data suggest possible mechanisms which may be involved in the induction of ODC in hippocampus by seizure activity.

Animals↗