The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself. 相似文献
A strong linkage between adrenergic and glutamatergic systems exists in the CNS but it is still unclear whether the excessive release of noradrenaline under ischemic conditions is modulated by excitatory amino acids. We studied the effect of selective glutamate receptor antagonists on the release of [3H]noradrenaline evoked by glucose and oxygen deprivation in hippocampal CA1, CA3 and dentate gyrus subregions. The release of glutamate, aspartate and GABA was measured by HPLC. Omission of oxygen and glucose increased the release of [3H]noradrenaline as well as the release of amino acids. Maximum effect on noradrenaline release was observed in CA1 region. The relative increase of the release after 30 min energy deprivation (R(2)) versus the basal release under normal conditions (R(1)), i.e. the R(2)/R(1) ratio was 7.1+/-1.0, 3.87+/-0.4 and 3.26+/-0.27 for CA1, CA3 and dentate gyrus, respectively. The [3H]noradrenaline outflow in response to glucose and oxygen deprivation was abolished at low temperature, but not by Ca(2+) removal, suggesting a cytoplasmic release process. In CA1 and CA3 [3H]noradrenaline release was significantly attenuated by MK-801, an NMDA receptor antagonist. The AMPA receptor antagonist GYKI-53784 had no effect in CA3, but partly reduced noradrenaline release in CA1.Our results suggest that ionotropic glutamate receptors seem to be implicated in the massive cytoplasmic release of noradrenaline in CA1 what may contribute to its selective vulnerability. 相似文献
Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hyposia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or non-lethal cellular injury was produced as reflected by a significant release of lactate dehydrogenase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per 1 and a normal atmosphere of 20% O2, they had levels of LDH leakage comparable to those of control cultures. 相似文献
Rat posterior eyecups containing the retina were prepared, loaded with [3H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [3H]glycine release, an effect that was found to be external Ca2+-independent. Whereas oxygen and glucose deprivation increased [3H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [3H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [3H]glycine release. Oxygen and glucose deprivation also evoked [3H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [3H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [3H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca2+-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na+–K+-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-d-glucose, led to increase of retinal [3H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-d-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures [3H]glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Müller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina. 相似文献
The intracellular phosphoribosyl diphosphate (prpp) levels of Ehrlich ascites tumor cells increased during glucose supplementation and decreased during glucose deprivation, while the numbers of glucose carriers as determined by glucose-reversible cytochalasin-B binding changed in an opposite manner relating to the extracellular glucose concentrations and the intracellular prpp levels of Ehrlich ascites tumor cells in vitro. Incubation of cells with hypoxanthine or 2,4-dinitrophenol lowered the intracellular prpp levels and resulted in an increase in numbers of glucose carriers. 相似文献
Summary A novel incubation unit is described that is highly suitable for thorough studies of oxygen deprivation states. Its application
with cultured heart cells is experimentally demonstrated. The release of enzymes, taken as a marker for cell damage, has clearly
shown that restriction of the volume of extracellular medium combined with oxygen plus glucose deprivation caused greatest
cellular damage. It may be considered as an experimental ischemia-like state. Furthermore, the onset of cellular damage followed
a time table very much like that occurring in vivo under similar conditions, more so than any other previously described studies.
A time lag between the release of cytoplasmic enzymes and lysosomal enzymes and other observations made in the present study
suggests a sequential order of events in which the release of cytoplasmic enzymes occurs at a stage of reversible damage due
to oxygen deprivation, whereas the release of lysosomal enzymes may point at irrepairable damage.
Supported by grants from The Chief Scientist, Ministry of Health, State of Israel; The Ministry of Education and Sciences,
State of Niedersachsen (FRG); and The Foundation for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France. This
study was done as partial fulfilment of Vemuri's Ph.D. thesis in biochemistry. 相似文献
Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically. 相似文献
The involvement of NMDA glutamate receptors in the effects of glucose/oxygen deprivation (in vitro ischaemia) on spontaneous endogenous acetylcholine and glutamate overflow from the guinea pig ileum was studied. Neurotransmitter overflow was measured by HPLC. Deprivation of glucose in the medium slightly reduced acetylcholine overflow, and did not significantly influence glutamate overflow. During oxygen deprivation and glucose/oxygen deprivation, acetylcholine overflow augmented with a biphasic modality: an early peak was followed by a long lasting increase, whereas glutamate overflow increased with a rapid and sustained modality. The effects of glucose/oxygen deprivation on both acetylcholine and glutamate overflow were abolished after reperfusion with normal oxygenated medium. Acetylcholine and glutamate overflow induced by glucose/oxygen deprivation were significantly reduced in the absence of external Ca(2+) as well as by the addition of the mitochondrial Na(+)-Ca(2+) exchanger blocker, CGP 37157, and of the endoplasmic reticulum Ca(2+)/ATPase blocker, thapsigargin. +/-AP5, an NMDA receptor antagonist, and 5,7-diCl-kynurenic acid, an antagonist of the glycine site associated to NMDA receptor, markedly depressed glucose/oxygen deprivation-induced acetylcholine and glutamate overflow as well. Our results suggest that in vitro simulated ischaemia evokes acetylcholine and glutamate overflow from the guinea pig ileum, which is partly linked to an increase in intracellular Ca(2+) concentration dependent on both Ca(2+) influx from the extracellular space and Ca(2+) mobilization from the endoplasmic reticulum and mitochondrial stores. During glucose/oxygen deprivation, ionotropic glutamate receptors of the NMDA type exert both a positive feedback modulation of glutamate output and contribute to increased acetylcholine overflow. 相似文献
Summary Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hypoxia and glucose deprivation
for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and
leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did
not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls.
However, reversible or nonlethal cellular injury was produced as reflected by a significant release of lactate dehydro-genase
(LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures
were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not
glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose
deprivation in complete medium containing 1000 mg glucose per l and a normal atmosphere of 20% O2, they had levels of LDH leakage comparable to those of control cultures.
This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano
Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was
provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute. 相似文献
Summary An in vitro model of myocardial ischemia has been established with primary monolayer cultures of postnatal rat myocardial
cells. Ischemic conditions were simulated in vitro by subjecting the myocardial cell cultures to various levels of oxygen
and glucose deprivation. The experimental protocol consisted of treatment with 20% or 0% O2 and 1000, 500 or 0 mg glucose per 1 of medium for 4 or 24 hr. Control cultures were treated with 20% O2 and 1000 mg glucose. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for signs of injury,
i.e. leakage of cytoplasmic enzymes into the culture medium. Differences were found in leakage of lactate dehydrogenase (LDH)
and creatine phosphokinase (CPK) from the cultures that were exposed to partial ischemia of glucose deprivation and from those
cultures that were exposed to total ischemia of oxygen and glucose deprivation. Glucose deprivation alone resulted in a slight-to-moderate
loss of LDH and CPK from the cells, whereas total ischemia resulted in a significant release of the two cytoplasmic enzymes.
When the cultures were allowed to recover after ischemic treatment in complete medium (1000 mg glucose) and a normal atmosphere
of 20% O2, they had levels of LDH leakage comparable to those of control cultures. Cell viability and total protein content of the
ischemic cultures did not differ significantly from controls.
This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute. 相似文献
Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders. 相似文献
Brain ischemia causes neuronal cell death by several mechanisms involving necrotic and apoptotic processes. The contributions of each process depend on conditions such as the severity and duration of ischemia, and the availability of ATP. We examined whether glucose affected the development of apoptosis after transient ischemia, and whether this was sensitive to caspase inhibition. Retinoic acid-differentiated SH-SY5Y human neuroblastoma cells were subjected to oxygen and glucose deprivation for 15 h followed by various periods of reoxygenation in either the presence or absence of glucose. Oxygen and glucose deprivation induced cell death in the hours following reoxygenation, as detected by propidium iodide staining. At the end of the period of oxygen and glucose deprivation, both cytochrome c and apoptosis-inducing factor translocated from mitochondria to cytosol. Reoxygenation in the presence of glucose accelerated cell death, and enhanced caspase-3 activity and apoptosis. The glucose-dependent increase in apoptosis was prevented by treatment with the caspase inhibitor zVAD-fmk, but not with calpeptin, a calpain inhibitor. Nevertheless, both zVAD-fmk and calpeptin decreased cell death in the glucose-treated group. ATP levels dropped dramatically after oxygen and glucose deprivation, but recovered steadily thereafter, and were significantly higher at 6 h of reoxygenation in the glucose-treated group. This indicates that energy recovery may promote the glucose-dependent cell death. We conclude that glucose favours the development of caspase-dependent apoptosis during reoxygenation following oxygen and glucose deprivation. 相似文献
The effect of food deprivation on the pulsatile release of LH was examined in the normal cycling and the ovariectomized (OVX) adult female rat. In the cycling animals, there were significant decreases in the mean plasma LH levels as well as the frequency and amplitude of the LH pulse 48 h after the onset of food deprivation. On the other hand, food deprivation for up to 72 h did not cause any changes in pulsatile LH release in the OVX animals. No difference in the changes in body weight and blood glucose concentration were found between the cycling and OVX rats throughout the period of food deprivation for up to 96 h. These findings suggest that ovarian factors play an important role in the early manifestation of the effect of food deprivation on pulsatile LH release and that metabolic changes as expressed by decreases in body weight and blood glucose level per se were not the direct causes in the decrease of LH release during the period of food deprivation. 相似文献
The sandwich system was recently developed as an in vitro tumor analog. Like spheroids, sandwiches are organized, multicellular systems in which the interplay between diffusion and consumption leads to the formation of spatial gradients; a necrotic center and a viable cell border subsequently develop. Using sandwiches of the 9L and V79 cell lines, the effects of oxygen and glucose deprivation on the onset and formation of necrosis were investigated. The data indicate that in sandwiches necrosis is a result of a shortage of both substances. Complementary cell monolayer experiments to determine a number of consumption parameters were performed. On the basis of the data, we propose a joint oxygen-glucose deprivation model for V79 cell necrosis. It is assumed a cell dies when oxygen deprivation in conjunction with glucose deprivation lowers the cell's ATP production rate below a critical value. Interactions of the concentrations and consumptions of oxygen and glucose are analyzed theoretically; concentration profiles are obtained by numerically solving coupled non-linear integral equations arising from the diffusion equation. The predicted viable border widths are in good agreement with the observed values. 相似文献
In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells. 相似文献
The role of physical exercise as a neuroprotective agent against ischemic injury has been extensively discussed. Nevertheless, the mechanisms underlying the effects of physical exercise on cerebral ischemia remain poorly understood. Here, we investigate the hypothesis that physical exercise increases ischemic tolerance by decreasing the induction of cellular apoptosis and glutamate release. Rats (n = 50) were submitted to a swimming exercise protocol for 8 weeks. Hippocampal slices were then submitted to oxygen and glucose deprivation. Cellular viability, pro‐apoptotic markers (Caspase 8, Caspase 9, Caspase 3, and apoptosis‐inducing factor), and glutamate release were analyzed. The percentage of cell death, the amount of glutamate release, and the expression of the apoptotic markers were all decreased in the exercise group when compared to the sedentary group after oxygen and glucose deprivation. Our results suggest that physical exercise protects hippocampal slices from the effects of oxygen and glucose deprivation, probably by a mechanism involving both the decrease of glutamatergic excitotoxicity and apoptosis induction.
Suspensions enriched in isolated rabbit proximal tubules were subjected to varying degrees of oxygen deprivation-induced injury by incubating them under hypoxic conditions at pH 7.4 or pH 6.6 or under high density pelleted conditions and adenine nucleotide degradation was characterized. The major metabolite was hypoxanthine. Its levels increased with the extent of irreversible injury. It was not further degraded or salvaged. Recovery of cell ATP during reoxygenation was predominantly from the remaining cell nucleotides. Allopurinol did not alter the pattern of purine metabolism or the extent of cell injury. These observations provide information on the intrinsic purine metabolic capacity of renal tubule cells during oxygen deprivation which is relevant to understanding both the salvage mechanisms available in these cells as well as the contribution of purine metabolism to the pathogenesis of oxygen deprivation-induced tubule cell injury. 相似文献
Mammalian cells have multiple responses to low or zero oxygen concentrations. In the complete absence of oxygen, cells undergo cell death through apoptosis, and not necrosis. Apoptotic signaling during oxygen deprivation occurs through the release of cytochrome c and apaf-1 mediated caspase-9 activation. The upstream regulators of cytochrome c release are the Bcl-2 family members. Pro-apoptotic Bcl-2 family members such as bax or bak are clearly required to initiate cytochrome c/apaf-1/caspase-9 mediated cell death during oxygen deprivation. Here we review what is currently known oxygen deprivation induced cell death and speculate about initiating mechanisms resulting in the activation of pro-apoptotic Bcl-2 family members. 相似文献
Abstract: l -Glutamate stimulates the liberation of arachidonic acid from mouse striatal neurons via the activation of N -methyl- d -aspartic acid (NMDA) receptors and by the joint stimulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic receptors. In this study, we investigated whether starving cultured mouse striatal neurons of glucose would modify glutamatergic receptor-mediated arachidonic acid release. Glucose deprivation for 30 min led to enhancement of the NMDA-evoked release of arachidonic acid, compared with that observed in the presence of glucose. This enhanced response depended on both the concentration of glucose and the length of time of glucose deprivation. The enhanced NMDA response appeared to result from both a release of glutamate and the subsequent additional release of arachidonic acid due to the activation of AMPA and metabotropic receptors. Indeed, the increased NMDA response was completely reversed when extracellular glutamate was enzymatically removed. Moreover, glucose deprivation potentiated the combined AMPA/metabotropic receptor-evoked release of arachidonic acid, even in the absence of extracellular glutamate. However, removing glucose did not improve the calcium rise induced by AMPA or NMDA. The ATP-evoked release of arachidonic acid from striatal astrocytes was not altered by glucose starvation. In summary, glucose deprivation affected two properties of striatal neurons: (a) it induced an NMDA-evoked release of glutamate from striatal neurons and (b) it selectively potentiated the AMPA/(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid-evoked release of [3H]arachidonic acid without altering the authentic NMDA-mediated response. 相似文献