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1.
The effect of ammonia and calcium on the activity of monoamine oxidase (MAO) was studied. The enzyme activity in nonsynaptic brain mitochondria isolated from the rats treated with ammonium acetate was estimated from the release of H2O2using spectrophotometry. The effect of calcium on MAO was assayed directly after adding Ca2+to the nonsynaptic mitochondria isolated from the forebrain of control rats. Both ammonium acetate injectionin vivoand Ca2+additionin vitrostimulated the activity of MAO A but not that of MAO B in mitochondria. This is the first evidence for ammonia and Ca2+regulation of MAO A in the forebrain nonsynaptic mitochondria and for their contribution to oxidative stress in the neurons via MAO A activation.  相似文献   

2.
This investigation tested the importance of excitatory amino acids' effects on regional cerebral O2 consumption and the concomitant changes in cerebral blood flow (rCBF) in isoflurane anesthetized rats. In the glutamate or N-methyl-D-aspartate (NMDA) groups, 10–2 M glutamate or NMDA was topically applied to the right cortex and the left cortex was used as a control. One mg/kg dizocilpine maleate (MK-801), a non-competitive NMDA receptor antagonist, was administered (iv) to the MK-801 group and saline was given to the control group. Cortical rCBF was determined using 14C-iodoantipyrine and regional O2 extraction was measured microspectrophotometrically. Cerebral O2 consumption increased 77% after glutamate (contralateral cortex: 9.0 ± 1.1 ml O2/min/100 g, glutamate treated cortex: 15.9 ± 3.9), while a 46% increase was observed with the same concentration of NMDA (contralateral cortex: 9.8 ± 2.0, NMDA treated cortex: 14.3 ± 5.5). After MK-801, the O2 consumption decreased to 37% of the control value (control cortex: 7.0 ± 1.3, MK-801 treated cortex: 2.6 ± 3.9). MK-801 significantly decreased cerebral O2 extraction from 7.1 ± 1.3 ml O2/100 ml (control cortex) to 5.3 ± 0.6 (MK-801 treated cortex). However, there was no significant difference in cerebral O2 extraction between treated and contralateral cortex in either the glutamate or NMDA groups. The increase in O2 consumption caused by glutamate or NMDA was coupled with increased rCBF. Glutamate increased rCBF from 95 ± 5 ml/min/100 g (contralateral cortex) to 165 ± 31 (treated cortex), while NMDA increased rCBF from 114 ± 12 (contralateral cortex) to 178 ± 60 (treated cortex). MK-801 decreased O2 consumption with a lesser decrease of rCBF. The rCBF was 48 ± 9 in the MK-801 treated cortex and 99 ± 22 in the control cortex. Some substances produced by the activation of NMDA receptors may be related to the coupling of cerebral metabolism and blood flow, since after blockade of NMDA receptors with MK-801, this relationship is uncoupled. These findings suggest that glutamatergic processes have a major effect on cerebral O2 consumption and that this is at least partly due to NMDA receptors.  相似文献   

3.
In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca2+-independent phospholipase A2 (iPLA2), Ca2+-independent plasmalogen PLA2 or secretory PLA2 (sPLA2), but not by Ca2+-dependent cytosolic PLA2 (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca2+ into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca2+-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca2+ and of cPLA2. DHA signaling may be mediated by iPLA2, plasmalogen PLA2, or other enzymes insensitive to low concentrations of Ca2+. Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.  相似文献   

4.
Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (?Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ?Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ?Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges.  相似文献   

5.
Mitochondrial reactive oxygen species regulate many important biological processes. We studied H2O2 formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H2O2 generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca2+ (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5 mM). We have shown that the H2O2 formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H2O2 formation through mitochondrial respiratory chain complex II. The H2O2 formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.  相似文献   

6.
Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Mg2+ and Ca2+. Pertussis toxin treatment abolished this glutamate-induced [Ca2+]i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg2+, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca2+]i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg2+ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine2 (5-HT2) receptor signaling. EAAs inhibited 5-HT2 receptor-mediated intracellular Ca2+ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca2+. In addition, this inhibitory effect on 5-HT2 receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT2 receptor-mediated [Ca2+]i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca2+ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.  相似文献   

7.
Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ~70% by MK-801 (a noncompetitive NMDA-receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with NG-nitro-l -arginine methyl ester (l -NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O2 andH2O2. Concurrent inhibition of O2 and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A2 inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca2+-free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate-induced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with l -NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca2+-mediated process. Activation of NO synthase and phospholipaseA2 contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.  相似文献   

8.
Oxidative stress plays a significant role in the neurotoxicity of a variety of agents that interact with the N-methyl-D-aspartate (NMDA) receptors. Here we investigated in a comparative way the pro-oxidative effects of quinolinic acid (QA) and malonate, two neurotoxic substances that act through distinct primary molecular mechanisms on the production of thiobarbituric acid reactive species (TBARS) by brain homogenates. In fact, QA is thought to activate directly the NMDA receptor, whereas malonate seems to act primarily by inhibiting oxidative metabolism. The malonate-induced TBARS formation was not modified by cyanide (CN) or 2,4-dinitrophenol. MK-801 did not reduce basal or malonate induced-TBARS production in fresh tissues preparations. However, in heat-treated preparations a significant effect of MK-801 against basal TBARS production was observed, but not on the malonate induced-TBARS production. QA induced-TBARS production was significantly prevented by MK-801 either in fresh or heat-treated preparations. The antioxidant effect of MK-801 on basal and QA-induced TBARS production increased as the temperatures used to treat S1 were increased. Succinate dehydrogenase (SDH) was inhibited by malonate but not by QA. Malonate was able to chelate iron(II) and the malonate-iron complex(es) is(are) active as measured by its(their) activity on deoxyribose degradation assay. These findings indicate that direct interactions of malonate with NMDA receptors are not involved in malonate pro-oxidative activity in vitro. QA pro-oxidative activity in vitro was related, at least in part, to its capability in stimulate NMDA receptors. Taken together, these findings indicated that malonate pro-oxidative activity in vitro could be attributed to its capability of changing the ratio Fe2+/ Fe3+, which is essential to TBARS production.  相似文献   

9.
The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [3H]MK-801 labeled a uniform population of sites (Kd = 3.2 ± 0.3 nM) in a saturable manner (Bmax = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [3H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [3H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.  相似文献   

10.
Spermine and spermidine enhance the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([3H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [3H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [3H]MK-801 is observed. However, the rates of both association and dissociation of [3H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.  相似文献   

11.
Abstract: Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [3H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [3H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [3H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the KD for [3H]MK-801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the Emax value in the ventromedial caudate putamen without changing the EC50. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.  相似文献   

12.
Using dizocilpine (MK-801), we tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors are important controllers of cerebral O2 supply/consumption balance in newborn piglets both during normoxia and hypoxia. Twenty-five 2 to 7-day-old piglets were anesthetized and divided into four groups: (1) Normoxia (n = 6), (2) Normoxia + MK-801 (n = 6), (3) Hypoxia (n = 6), and (4) Hypoxia + MK-801 (n = 7). Regional cerebral blood flow (rCBF) in ml/min/100 g was measured using 14C-iodoantipyrine, and we determined arterial and venous O2 saturations by microspectrophotometry, calculating cerebral O2 consumption (VO2) in ml O2/min/100 g in the cortex, hypothalamus and pons. MK-801 did not significantly affect regional VO2 or rCBF in normoxic piglets. Hypoxia resulted in an increase in local rCBF compared to controls: from 41 ± 6 to 103 ± 18 in the cortex; 34 ± 7 to 101 ± 20 in the hypothalamus; and 45 ± 10 to 95 ± 11 in the pons. Pretreatment with MK-801 abolished this hypoxic flow effect in the cortex (51 ± 2) and hypothalamus (49 ± 5), but not in the pons (91 ± 17). Similar results were observed for VO2 with control values of 1.9 ± 0.3, 1.6 ± 0.2 and 2.1 ± 0.3 for the cortex, hypothalamus and pons respectively. Hypoxia resulted in an increase in the VO2 to 3.9 ± 0.4 (cortex), 3.8 ± 0.6 (hypothalamus) and 3.9 ± 0.8 (pons). Pretreatment with MK-801 prior to hypoxia abolished these effects in the cortex (2.1 ± 0.2) and hypothalamus (2.1 ± 0.2), but not in the pons (2.9 ± 0.2). These findings suggest that NMDA receptors may play a role in the control of cerebral metabolism during hypoxia in this immature porcine model.  相似文献   

13.
Acute ammonia intoxication diminishes the activities of antioxidant enzymes and increases superoxide formation in brain. These effects could play a role in the mechanism of ammonia toxicity. It has been shown that ammonia toxicity is mediated by activation of NMDA receptors. The aim of this work was to assess whether ammonia-induced changes in antioxidant enzymes and in superoxide formation are mediated by activation of NMDA receptors. It is shown that MK-801, an antagonist of NMDA receptors prevents ammonia-induced changes in superoxide dismutase, glutathione peroxidase and catalase. Ammonia intoxication also induces a depletion of glutathione and an increase in lipid peroxidation. Both effects, as well as ammonia-induced increase in superoxide formation are prevented by MK-801. These results indicate that ammonia-induced oxidative stress in brain is mediated by excessive activation of NMDA receptors and support the idea that oxidative stress can play a role in the mechanism of ammonia toxicity.  相似文献   

14.
Enhancement of NMDA-mediated responses by cyanide   总被引:2,自引:0,他引:2  
The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate  相似文献   

15.
Oxidative stress remodels Ca2+ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca2+/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca2+ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca2+ current (ICa,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H2O2 induced an increase in ICa,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H2O2-induced LTF of ICa,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca2+ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H2O2 via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.  相似文献   

16.
17.
Three-hour incubation of rat cerebellar granule cells with 0.1 μM ouabain increases intracellular levels of Ca2+ ions and reactive oxygen species (ROS) resulting in pronounced activation of Mitogen-Activated Protein Kinase (MAPK). Higher concentrations of ouabain induce further increases in MAPK activity. The activating effect of ouabain is attenuated by the NMDA-receptor antagonists MK-801 and D-AP5. The data obtained suggest that similar to NMDA receptors ouabain-sensitive and ouabain-resistant isoforms of Na+,K+-ATPase are actively involved in intracellular signaling cascades controlling proliferative activity of neuronal cells.  相似文献   

18.
The effects of glutamate agonists and their selective antagonists on the Ca2+-dependent and independent releases of [3H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca2+-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca2+-free medium. The release evoked by QA in Ca2+-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca2+-free medium. Verapamil inhibited the QA-activated release in both Ca2+-containing and Ca2+-free media. The effect of QA but not that of AMPA was blocked in Ca2+-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca2+ from intracellular stores.  相似文献   

19.
Summary Converging lines of evidence suggest that theN-methyl-d-aspartic acid (NMDA) antagonist properties of conantokin G (ConG) are mediated through a novel polyamine-associated site. Moreover, structural modification of the heptadecapeptide yields peptides that can either mimic the NMDA antagonist properties of the parent peptide or produce polyamine-like actions at NMDA receptors. We synthesized a panel of ConG fragments and evaluated their effects using a neurochemical assay that predicts pharmacological actions at NMDA receptors. While the C-terminal tetrapeptide elicited a polyamine-like activation of [3H]MK-801 binding with a potency comparable to spermine, the N-terminal pentapeptide produced a marginal inhibition of spermine-enhanced [3H]MK-801 binding. These observations suggest that the parent peptide interacts with two distinct sites on NMDA receptors. In contrast, amino acid replacements in the middle region of ConG resulted in analogues that were of comparable or greater potency than the parent peptide. The Ala7, Tyr10 derivative is of particular interest since it is a potent inhibitor (IC50≈80 nM) of spermine-enhanced [3H]MK-801 binding, and may thus serve as a precursor for studies designed to125I-label putative ConG binding sites. Our observations are also consistent with the hypothesis that the termini of ConG are essential for an interaction with NMDA receptors, while the middle region of this peptide serves as a spacer unit. This hypothesis is consonant with spectroscopic evidence that ConG possesses a central rigid helical backbone with flexible N- and C-terminal regions. Nonetheless, ConG variants in which the termini were connected with conformationally stabilized α-or 310-helical spacers grew progressively less potent as NMDA antagonists as the structural stability of these peptides increased. Thus, the middle region of ConG appears to possess functions other than providing conformational stability. These newly synthesized ConG derivatives may serve as a basis for the design of novel peptide or peptidomimetic agents.  相似文献   

20.
Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca2+]i observed under hypoxic conditions is the result of an NMDA-mediated Ca2+ influx. Changes of [Ca2+]i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca2+]i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca2+]i was not observed in a Ca2+ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca2+]i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca2+ via the altered NMDA receptor with subsequent release of Ca2+ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.  相似文献   

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