Article

Recovery from calcium-induced damage in a neuroblastoma cell line

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Calcium may act as a second messenger in normal cellular signal transduction systems. However, an excessive influx of calcium into the cytoplasm is well known to be a final common pathway causing cell death under various pathological conditions. The purpose of this study was to investigate the effect of a transient treatment with the calcium ionophore A23187 on the recovery process of cell viability, energy metabolism, amino acid incorporation and calcium uptake in a neuroblastoma cell line. When neuroblastoma cells were treated with 20 microM of the calcium ionophore A23187 in combination with extracellular calcium, rapid energy failure and marked inhibition of amino acid incorporation by the cells occurred together with a massive influx of calcium, and finally resulted in cell death. Recovery from this calcium-induced damage with regards to energy metabolism and prognosis of cell viability was better after a 10-min treatment than after a 30-min treatment with A23187. After a 10-min treatment, the viability was higher in calcium-free medium than in calcium-containing medium in contrast with the cases after treatment for 30 min. The above difference in viability after treatment for 10 min had a very significant correlation with the degree of exclusion of excessive calcium and the recovery of CTP, indicating that the recovery of CTP and the rate of calcium exclusion may be final markers of the recovery of cells from calcium-induced damage rather than the recovery of ATP or amino acid incorporation. Amino acid incorporation was restricted to a level lower than that of the control long after the recovery of GTP and the GTP/GDP ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The problem of inducing an iso-!ated increase in cytoplasmic calcium activity in intact cells has hindered attempts to investigate the effect of calcium activity on protein synthesis up to now. For example, it has been shown that protein synthesis is severely inhibited in a neuronal cell line after incubation with the calcium ionophore A23187 (Abe and Kogure, 1987). However, the ionophore renders all cellular membranes permeable to Ca2~,leading to an influx of Ca2~from the extracellular space and a depletion of the ER Ca2~reserves. ...
Article
We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 µM caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with ∼5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.
... Of particular interest in the latter context are DC shift-related transient decreases of the extracellular calcium content (Kraig and Nicholson, 1978 ), which may be taken as indirect evidence of an increased intracellular calcium concentration. Because elevated calcium levels were shown to impair rates of protein synthesis in vitro (Abe and Kogure, 1987; De Haro et al., I983), transient derangement of intracellular calcium homeostasis may also play a role in the inhibition of cortical protein synthesis during repetitive spreading depression in vivo. Another mechanism, namely, a compartmental disturbance of energy-rich compound availability for tRNA aminoacylation during CSD, has been proposed to inhibit protein synthesis (Krivanek, 1978 ). ...
Article
The effect of cortical spreading depression (CSD) on cerebral protein synthesis (CPS) was examined. CSD was evoked in normal rats with KCl, and CPS was measured autoradiographically with [1-14C]leucine. Average rates (mean +/- SD) of CPS in layers I-IV of cortex decreased significantly from 10.7 +/- 0.6 (sham-operated controls; n = 4) to 6.7 +/- 0.7 nmol/g/min (n = 4; p < 0.01) and in layers V-VI from 10.9 +/- 0.5 to 9.4 +/- 0.4 nmol/g/min (p < 0.05) during 60 min of repetitive CSD. Spreading depression did not affect CPS rates in other subcortical brain regions. These results indicate that KCl-evoked CSD induces inhibition but not suppression of cortical protein synthesis.
... The mechanism by which metalloporphyrins protect neurons probably largely involves suppression of Ca 2+ influx in neurons, because ionomycin was not toxic in the absence of extracellular Ca 2+ . Ionomycin and A23187 act as plasma membrane Ca 2+ /H + exchangers; increases in [Ca 2+ ] i , intracellular alkalinization, mitochondrial depolarization and cell death strongly depend on Ca 2+ influx (Perney et al. 1984;Abe and Kogure 1987;Choi 1987;Mattson et al. 1991;Lukas and Jones 1994;OuYang et al. 1995;Shimohama et al. 1996;Castillo and Babson 1998;Yamamoto et al. 1998;Abramov and Duchen 2003). Ca 2+ ionophores are preferentially toxic to neurons (Choi 1987;Gwag et al. 1999), even in the presence of glutamate receptor antagonists (Sattler et al. 1998), consistent with [Ca 2+ ] i increases observed predominantly in neurons (Safran et al. 1996;Abramov and Duchen 2003), although glial death results under more intense conditions (Choi 1987;Safran et al. 1997), consistent with percentage PI uptake > 100% observed in the current study. ...
Article
We evaluated whether both inert and catalytically active metalloporphyrin antioxidants, meso-substituted with either phenyl-based or N-alkylpyridinium-based groups, suppress Ca(2+)-dependent neurotoxicity in cell culture models of relevance to cerebral ischemia. Representatives from both metalloporphyrin classes, regardless of antioxidant strength, protected cultured cortical neurons or PC-12 cultures against the Ca(2+) ionophores ionomycin or A23187, by suppressing neurotoxic Ca(2+) influx. Some metalloporphyrins suppressed excitotoxic Ca(2+) influx indirectly induced by the Ca(2+) ionophores in cortical neurons. Metalloporphyrins did not quench intracellular fluorescence, suggesting localization to the plasma membrane interface and/or interference with Ca(2+) ionophores. Metalloporphyrins suppressed ionomycin-induced Mn(2+) influx, but did not protect cortical neurons against pyrithione, a Zn(2+) ionophore. In other Ca(2+)-dependent paradigms, Ca(2+) influx via plasma membrane depolarization, but not through reversal of plasmalemmal Na(+)/Ca(2+) exchangers, was modestly suppressed by Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP) or by an inert analog, Zn(II)TBAP. Mn(III)TBAP and Zn(II)TBAP potently protected cortical neurons against long-duration oxygen-glucose deprivation (OGD), performed in the presence of antagonists of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and L-type voltage-gated Ca(2+) channels, raising the possibility of an unconventional mode of blockade of transient receptor protein melastatin 7 channels by a metalloTBAP family of metalloporphyrins. The present study extends the range of Ca(2+)-dependent insults for which metalloporphyrins demonstrate unconventional neuroprotection. MetalloTBAPs appear capable of targeting an OGD temporal continuum.
Article
We used a gerbil model of cerebral ischemia to study the effects of ion channel blockers on neuronal death resulting from enhanced glutamate release and calcium ion influx. The common carotid arteries of gerbils were occluded for 5 minutes and injected intraperitoneally immediately after ischemia with an alkylene iminopropylene derivative (glutamate blocker) or a piperazinyl ethanol derivative (calcium blocker) given at high or low doses. Two vehicle groups received saline or 0.2% methyl cellulose solution. Seven days later, the gerbils were perfusion-fixed and their brains were processed for histologic study. The number of neurons per millimeter (neuronal density) of the CA1 region was calculated, and the neuronal density in each group was statistically compared using the Mann-Whitney U test. Compared with a control group not subjected to carotid ligation, neurons of the two vehicle groups and the low-dose calcium blocker group were almost nonexistent in the CA1 region. Neuronal densities of the glutamate blocker group and the high-dose calcium blocker group were similar and were found to be within normal limits by statistical analysis. Our study shows that detrimental membrane phenomena and the incidence of delayed neuronal death may be counteracted by the systemic administration of these ion channel blockers after ischemic insult.
Article
The CA 1 neurons in the gerbil hippocampus exhibiting necrosis with delayed onset following 5 min ischemia were reduced markedly by the systemic administration of dihydroergotoxine mesylate (Hydergine; HYG). Immediately after 5 min of forebrain ischemia, the animals were injected intraperitoneally with HYG. Seven days after ischemia, perfusion-fixed brains were processed by conventional histology. The number of neurons per millimeter in the CA 1 pyramidal cell layer were calculated and they were labelled neuronal density. In the control group, the neuronal density was 66.03 +/- 7.37 (mean +/- SEM), in the vehicle group, it was 11.25 +/- 4.93. The neuronal density in the HYG group was 69.19 +/- 6.49. The difference in the neuronal density between the HYG group and the control group was not statistically significant. These data indicate that HYG protects on the CA 1 neurons, and this suggest that the suppression of adrenoceptors by this drugs may be the main mechanism of action. This morphologic outcome may explain the functional amelioration of mental impairment by HYG.
Article
We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 microM caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with approximately 5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.
Article
It is widely accepted that disturbances of calcium homeostasis play a key role in the development of cell damage produced by transient cerebral ischemia. It is believed that the sharp increase in cytosolic calcium activity during ischemia activates a cascade of calcium-dependent metabolic processes which ultimately destroy the integrity of the cell. However, it has never been taken into account that ischemic cell damage may, at least in part, be caused by a disturbance of calcium homeostasis within the endoplasmic reticulum after transient cerebral ischemia. In fact, depletion of the endoplasmic reticulum from calcium induces metabolic changes resembling, in many respects, those produced by transient cerebral ischemia: it causes an inhibition of the activity of the eucaryotic initiation factor elF-2 alpha (by phosphorylation), a disaggregation of polyribosomes and thus an inhibition of global protein synthesis, and an increased expression of certain genes such as transcription factors (c-fos and c-jun) and the glucose-related protein grp78. Finally, a depletion of calcium in the endoplasmic reticulum induces tissue damage within the brain and triggers apoptosis in neuronal and non-neuronal cells. It is therefore concluded that cell damage induced by transient ischemia may, at least in part, be caused by a disturbance of calcium homeostasis within the endoplasmic reticulum.
Article
1-Methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) serves as a valuable tool in animal models of Parkinson's disease. Primary cell cultures of mesencephalon from C57/Bl6 mice were used to investigate the effects of various dopaminergic neurotoxins on the intracellular calcium metabolism. MPP+ was compared to its precursor MPTP and a structural analogue paraquat (methylviologen). Direct addition of these neurotoxins (10 microM) to fura-2-labeled cells did not change intracellular calcium concentrations in the presence of 1 mM extracellular calcium. When mesencephalic neurons were exposed to the compounds for 24 hours, only MPP+ led to an increase in calcium concentration in the absence and presence of extracellular calcium (36%, p < 0.05 and 47%, p < 0.01 versus control group). Intracellular calcium concentrations in cortical cultures devoid of dopaminergic cells were not changed by the above neurotoxins. Thus MPP+ is shown to selectively increase intracellular calcium concentrations in mesencephalic cultures.
Article
In an attempt to study whether ischemic brain could express a foreign gene in vivo, a replication-defective adenoviral vector containing the Escherichia coli lacZ gene was directly injected into the ischemic or reperfused cerebral cortex of rat, and temporal and spatial profiles of the exogenous gene expression were compared with that of the control brain. Right middle cerebral artery (MCA) of rat was continuously occluded by an insertion of nylon thread for 2 days, or only transiently occluded for 90 min and then the blood flow was restored for 21 days. The adenoviral vector was administered just after the MCA occlusion or reperfusion in the case of continuous ischemia and reperfusion, respectively. Adenoviral vector was transferred into the continuous ischemic brain, and the lacZ gene was expressed until 2 days of the occlusion in the cerebral cortex of the occluded MCA territory with the number of expressing cells smaller and the staining just weaker than that of the control brain. In contrast, expression of the lacZ gene was not or only minimally observed in the reperfused brain until 2 days. However, the expression dramatically exploded at 7 days of reperfusion at a level similar to that of the control, and the expression diminished by 21 days. A few neurons in the ipsilateral thalamus, hypothalamus, and basal ganglia, and in the contralateral cerebral cortex expressed the lacZ gene at 7 days after reperfusion, a phenomenon similar to the case of the control. The majority of brain cells that expressed the lacZ gene were neurons, and a part (5-10%) were astroglial cells. Traumatic injury and immunological response in the brain were minimal both in the cases of control and ischemia/reperfusion. The present study shows an effective gene transfer and the expression in neural cells of ischemic and reperfused brains in vivo, and suggests a great potential of the gene therapy for ischemic stroke patients in the future.
Article
Results from experiments performed with permanent non-neuronal cell lines suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concluded that disturbances in ER calcium homeostasis may contribute to the suppression of PS triggered by a severe metabolic stress (W. Paschen, Med. Hypoth., 47 (1996) 283-288). To elucidate how an emptying of ER calcium stores of these cells would effect PS and ribosomal aggregation of non-transformed fully differentiated cells, experiments were run on primary neuronal cell cultures. ER calcium stores were depleted by treating cells with thapsigargin (TG, a selective, irreversible inhibitor of ER Ca(2+)-ATPase), cyclopiazonic acid (CPA, a reversible inhibitor of ER Ca(2+)-ATPase), or caffeine (an agonist of ER ryanodine receptor). Changes in intracellular calcium activity were evaluated by fluorescence microscopy using fura-2-loaded cells. Protein synthesis was determined by measuring the incorporation of [3H]leucine into proteins. The degree of aggregation of ribosomes was evaluated by electron microscopy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused about 70% inhibition of PS, and PS recovered completely 60 min after treatment. Caffeine produced an inhibition of PS to about 50% of control. Loading cells with the calcium chelator BAPTA-AM (33.3 microM) alone suppressed PS without reversing TG- or caffeine-induced inhibition of PS, indicating that the suppression of PS was caused by a depletion of ER calcium stores and not by an increase in cytosolic calcium activity. TG-treatment of cells induced a complete disaggregation of polysomes which was not reversed within the 4 h recovery period following TG-treatment. After caffeine treatment of cells, we observed a heterogenous pattern of ribosomal aggregation: in some neurons ribosomes were almost completely aggregated while in other cells a significant portion of polyribosomes were disaggregated. The results indicate that a depletion of neuronal ER calcium stores disturbs protein synthesis in a similar way to the effects of transient forms of metabolic stress (ischemia, hypoglycemia or status epilepticus), thus implying that a disturbance in ER calcium homeostasis may contribute to the pathological process of stress-induced cell injury.
Article
A replication-defective adenoviral vector containing the E. coli lacZ gene was directly injected into normal and post-ischemic gerbil right hippocampus and lateral ventricle, and temporal profiles of the exogenous gene expression were compared. In case of ischemia, common carotid arteries (CCA) were transiently occluded for 5 min, and the adenoviral vector was administered just after the reperfusion. The animals were recovered for 8 h, 1, 3, 7 or 21 days. A small to moderate number of neural cells in the normal hippocampus expressed the gene from 1-3 days except for the cells around dentate gyrus (DG) and the needle route that began to express from 8 h of injection. Some normal hippocampal cells persisted the expression until 7 days. A moderate to large number of ventricular cells expressed the lacZ gene from 8 h to 7 days in the normal brain. On the other hand, no expression of the lacZ gene was observed in the post-ischemic hippocampus at 8 h including cells at DG and the needle route. Hippocampal CA1 neurons, that were selectively lost at 7 days of reperfusion, never expressed the gene throughout the post-ischemic course. The other hippocampal cells such as CA3 and dentate granule cells that survived ischemia expressed the gene only transiently at 1 day. A robust expression of the gene persisted in the ventricular cells from 8 h to 7 days. The majority of brain cells in the hippocampus that expressed the lacZ gene was not the pyramidal neurons, but small neurons at around the pyramidal layers of DG. Some astroglial, but no microglial, cells expressed the lacZ gene in the hippocampus. The present study shows that an expression of the lacZ gene was limited in the post-ischemic gerbil hippocampus especially at the vulnerable CA1 layer in contrast to the strong and persistent expression in the ventricular cells, and that the majority of beta-gal positive cells were not the pyramidal neurons but small neurons at around the cell layer both in the control and post-ischemic gerbil hippocampus.
The impact of an increase of intracellular Ca2+ i on the energy metabolism of trout hepatocytes was assessed by applying the Ca2+ ionophore A23187 and studying the consequences of the ensuing elevation of Ca2+ i on various metabolic parameters. After application of A23187 no loss of viability occurred for 2 h, but glutathione content decreased by 46%. A concomitant decrease of [ATP] as well as of Na,K-ATPase activity by over 50% could be prevented by incubating the cells in a Ca2+-free medium. Upon addition of the ionophore cellular oxygen consumption more than doubled in a strictly Ca2+-dependent manner, with half of this increase being sensitive to ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. This increase in oxygen consumption was transient in nature and at its peak it was similar in magnitude to that induced by 2,4-dinitrophenol. Similarly, oxygen consumption sensitive to the protein synthesis inhibitor cycloheximide was transiently increased by A23187, but returned to control levels within 30 min of incubation. These results suggest that elevation of intracellular Ca2+ leads to an energetic imbalance not related to stimulation of ATP consuming processes, but mainly due to impairment of mitochondrial function, possibly by the decoupling of oxidative phosphorylation and by inducing dissipative Ca2+ cycling.
Chapter
The occurrence of selective vulnerability of distinct neuronal structures has been described in gerbil and rat brain [3, 4] following transient forebrain ischemia. Neither cerebral blood flow nor glucose utilization limits the metabolic requirements of central nervous system cells throughout the first day of recirculation after 5-min ischemia induced by bilateral occlusion of common carotid arteries in the gerbil [5]. Two separate openings of the blood-brain barrier (BBB) have been reported [6], one occurring immediately after reperfusion in the CA4 sector, and a second one after several days in the CA1 structure. However, we have recently observed different intrinsic biochemical properties of CA1 neurons in this model of ischemia with respect to cerebral protein synthesis [2]. Although RNA synthesis was unaffected, CA1 neurons suddenly failed to reactivate their protein synthesis machinery to maintain normal protein turnover. Furthermore, susceptibility to protein synthesis reductions in the hippocampus was absent in infant animals at the age of 15–17 days [7], while it started to appear at around 20–22 days of life.
Article
Regional cerebral protein synthesis was investigated in anesthetized, mechanically ventilated rats during progressive insulin-induced hypoglycemia and the recovery period following glucose infusion. Polysome profiles from precomatose animals with slow wave/polyspike EEG revealed a slight reduction of polyribosomes and a concurrent increase in monoribosomes, but autoradiographs showed a pattern of L-[3-3H]tyrosine incorporation indistinguishable from that of control rats. During the initial 30 min of insulin-induced isoelectric EEG ("coma"), autoradiographs showed a selective inhibition of protein synthesis in neurons and glial cells of the hippocampus and cerebral cortex, i.e., regions with high susceptibility for the development of hypoglycemic brain damage. Basal ganglia were less affected and areas with low vulnerability (hypothalamus, brainstem, and cerebellum) exhibited a normal pattern of amino acid incorporation. Using a flooding dose of L-[1-14C]valine (7.5 mmol/kg; 15 microCi/mmol), the rate of incorporation in cerebral cortex and cerebellum was found to be reduced to 2% and 80% of control values, respectively. Inhibition of protein synthesis was paralleled by a breakdown of polyribosomes and a concomitant increase in ribosomal subunits, indicating a block in peptide chain initiation. After 90 min of isoelectric EEG all brain structures with the exception of hypothalamus and area postrema showed an almost complete lack of amino acid incorporation. Glucose infusion after a 30-min period of hypoglycemic coma led to a partial restoration of cortical and hippocampal protein synthesis. Within 70-90 min of recovery, L-[1-14C]valine incorporation into neocortical and cerebellar proteins amounted to 47% and 125% of fasted controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal γ-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 μM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.
Article
Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.
Article
Complete global cerebral ischemia was induced in dogs by temporary ligation of the ascending aorta for 10min. Prior to the ischemic period, half of the animals were given pentobarbital 30-38 mg/kg, a maneuver previously reported to prevent or attenuate cerebral damage in this same model. Cerebral blood flow (CBF) and cerebral metabolic rate (CMRO2) were followed from prior to the ischemic period to 6 h post-ischemia. At varying time intervals following ischemia, brain biopsies were obtained and analyzed for cerebral metabolites to determine the cerebral energy state. Only a few differences were observed between pentobarbital-treated and untreated animals. Post-ischemic CMRO2, stabilized at a significantly lower level in treated than in untreated animals. However, CBF was proportionately lower and thus O2 delivery relative to O2 needs in the two groups was comparable. Also in both groups, the CBF and CMRO2 stabilized at levels significantly below pre-ischemia controls. Cerebral energy stores in both groups were depleted after 10min of ischemia but were restored to near normal within 4min post-ischemia. Total restoration of the adenine nucleotide pool and ATP were delayed as was the return of brain lactate to normal. A 10min period of post-ischemic hyperemia was observed in all animals and in the initial 4min post-ischemia CMRO2 was also increased. The latter is probably accounted for by the O2 needs for restoration of cerebral energy and O2 stores. We conclude that cerebral protection as provided by barbiturates following complete global ischemia cannot be accounted for by any measurable effect on CBF, CMRO2, or the cerebral energy stores during the initial 6 h post-ischemia.
Article
Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol-bis-(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.
Article
A comparison of the affinities of eukaryotic initiation factor 2 and eukaryotic elongation factor 1 for GTP and GDP, and of the responses of initiation and elongation complex formation to various GTP mol fractions indicated that the initiation reaction was more sensitive to changes in the GTP: GDP ratio. In vitro regulation of the GTP: GDP ratio by the adenylate energy charge, a sensitive control parameter, also demonstrated a preference for regulation of formation of initiation complexes when compared to elongation complexes. These studies suggest that, based on the availability of energy, initiation is the rate-limiting step in the overall protein synthetic process.
Article
Information obtained over the past 25 years indicates that the amino acid glutamate functions as a fast excitatory transmitter in the mammalian brain. Studies completed during the last 15 years have also demonstrated that glutamate is a powerful neurotoxin, capable of killing neurons in the central nervous system when its extracellular concentration is sufficiently high. Recent experiments in a variety of preparations have shown that either blockade of synaptic transmission or the specific antagonism of postsynaptic glutamate receptors greatly diminishes the sensitivity of central neurons to hypoxia and ischemia. These experiments suggest that glutamate plays a key role in ischemic brain damage, and that drugs which decrease the accumulation of glutamate or block its postsynaptic effects may be a rational therapy for stroke.
Article
Both generalized and focal seizures dissociate brain polyribosomes and severely inhibit brain protein synthesis. This effect is found in freely convulsing animals and in animals that have been paralyzed and oxygen-ventilated in order to prevent hypoxemia, cerebral hypoxia, and other systemic changes associated with convulsions. Recent autoradiographic studies have shown that generalized seizures can result in striking focal inhibition of brain protein synthesis in adult rats and newborn marmoset monkeys. Local cerebral glucose metabolism and local cerebral blood flow were also studied in newborn marmosets by autoradiography. Although flow and metabolism are closely matched in control marmosets, seizures result in large local increases in 2-deoxyglucose metabolism, with lesser or no increases in local cerebral blood flow resulting in a relative mismatch. Those regions in which protein synthesis was most severely inhibited were those in which the relative mismatch between blood flow and metabolism was most marked. The molecular mechanisms regulating protein biosynthesis are not known. Translational regulation during seizures appears to be exerted, in large part, at the initiation step. A likely mechanism is the inhibition of ternary complex formation, one of the early steps in the initiation process, by increases in the intracellular ratio of [GDP]:[GTP]. This ratio is related to the cells' energy charge. Reduced levels of ATP during seizures can lead to an increased ratio of [GDP]:[GTP] via of the enzyme nucleoside diphosphate kinase (E.C. 2.7.4.6) and to inhibition of protein synthesis initiation. Regulation of protein biosynthesis during seizures is likely to be complex and exerted at many sites; some of these possibilities are discussed.
Article
Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.
Article
Although chlorpromazine was shown to greatly inhibit a Ca2+-mediated cell death at favorable concentrations (10(-6)-10(-5) M), it caused a drastic decrease in cell viability at higher concentrations (10(-4)-10(-3) M) in a human neuroblastoma cell line. The toxic effect of chlorpromazine also occurred in Ca2+-free medium and was not parallel to the amount of thiobarbituric acid-reactive substances produced. These results indicate that chlorpromazine has biphasic effects on cell viability according to the concentrations added, i.e. a protective effect against cell damage caused by Ca2+, and a direct toxic effect independent of extracellular Ca2+ or of lipid peroxidation.
Article
In gerbils the hemispheric blood flow was interrupted for 5 min by bilateral carotid artery occlusion to produce delayed selective destruction of the CA 1 sector of hippocampus. The influence of hemodynamic factors was studied by evaluating the microcirculation before and at two times after ischemia (3 min and 7 days), using Evans blue as an intravital vascular tracer. The density of perfused capillaries and the fractional volume of circulating blood were determined by quantitative morphometry and the values for the vulnerable CA 1 sector compared with those for the resistent CA 3 sector and cerebral cortex. In control animals the number of perfused capillaries in the CA 1 sector was about 20% lower, and the volume of circulating blood about 30% lower, than in the CA 3 sector or cerebral cortex. This difference was markedly enhanced after 5-min ischemia. During the early recirculation phase, capillary perfusion improved in the cortex, whereas in the CA 1 sector (and to a lesser degree also in the CA 3 sector) it declined. After 7 days, the density of perfused capillaries and the volume of circulating blood had returned to control levels in the cerebral cortex and CA 3 sector of hippocampus. In the CA 1 sector, in contrast, the microcirculation had further deteriorated. The density of perfused capillaries was less than 30%, and the circulating blood volume even less than 50%, of that in the cerebral cortex. The results obtained indicate that the microcirculatory capacity of the selectively vulnerable CA 1 sector of hippocampus is distinctly lower than that of resistant areas of the brain and suggest that hemodynamic factors may contribute to the difference in vulnerability of these regions to short-lasting cerebral ischemia.
Article
The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.
Article
In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70-kilodalton stress protein, identified by two-dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70-kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.
Article
Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.
Article
BSA, Bovine serum albumin; EDTA, Ethylenediaminetetraacetate; FFA, Free fatty acids; GABA, γ-Aminobutyric acid; GSH and GSSG, Reduced and oxidized glutathione, respectively; P/O and ADP/O ratios, Ratios between Pi or ADP consumed, respectively, and of oxygen (atoms) utilized; RCR, Respiratory control ratios
Article
Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono- and polyunsaturated FFAs increased approximately 12-fold in total at the end of a 30-min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic acid.
Article
Mechanisms involved in the postischemic delay in neuronal recovery or death in rat hippocampus were evaluated by light and electron microscopy at 3, 15, 30, and 120 min and 24, 36, 48, and 72 h following severe cerebral ischemia that was produced by permanent occlusion of the vertebral arteries and 30-min occlusion of the common carotid arteries. During the early postischemic period, neurons in the Ca1 and Ca3 regions both showed transient mitochondrial swelling followed by the disaggregation of polyribosomes, decrease in rough endoplasmic reticulum (RER), loss of Golgi apparatus (GA) cisterns, and decrease in GA vesicles . Recovery of these organelles in Ca3 neurons was first noted between 24 and 36 h and was accompanied by a marked proliferation of smooth endoplasmic reticulum (SER). Many Ca1 neurons initially recovered between 24 and 36 h, but subsequent cell death at 48-72 h was often preceded by peripheral chromatolysis, constriction and shrinkage of the proximal dendrites, and cytoplasmic dilatation that was continuous with focal expansion of RER cisterns. Because SER accumulates in resistant Ca3 neurons and proximal neuronal processes are damaged in vulnerable Ca1 neurons, we hypothesize that delayed cell recovery or death in vulnerable and resistant postischemic hippocampal neurons is related to abnormalities in neuronal processes.
Article
The cytotoxic effects of ionophore A23187 were studied in parallel with its action on calcium uptake in isolated mouse thymocytes. Under conditions where the cells were preincubated in a calcium-containing medium prior to ionophore treatment a close relationship could be observed between the extent of cell lysis and the stimulation of calcium uptake in the presence of A23187. In addition, increasing concentrations of calcium ions in the incubation medium lead to a pronounced decrease of cell viability and to a stimulation of calcium uptake suggesting that calcium is critical for cell survival.
Article
At the physiological concentration of Ca2+, the presumed calcium ionophore A23187 produced dose-related increases in 45Ca uptake by rat thymocytes and decreases in cell viability, effects that displayed a strong linear correlation. In media containing a very low concentration of Ca2+ (7.10(-6) M), in contrast, ionophore A23187 had no specific effect on either 45Ca uptake or cell viability. The calcium-dependent cytotoxicity of ionophore A23187 resembles that of other agents that are not ionophores, but that are known to perturb the plasma membrane. Consequently, we suggest that, in the rat thymocyte, ionophore, ionophore A23187 may not act as a true ionophore, but may perturb the cell membrane, allowing Ca2+ to pass freely through the membrane along its electrochemical gradient.
Article
Cell death is frequently encountered in human disease. Ischemia, chemicals, viruses, radiation and toxins are among its varied causes. The resulting pathology, however, is very uniform. The common pattern of altered morphology, coagulative necrosis, implies that, at some point, the diverse causes share common mechanisms. Recent evidence suggests that coagulative necrosis may ultimately reflect an alteration in the control of intracellular calcium homeostasis. Studies in intact animals and in cell culture suggest than an ultimate influx of calcium ions across injured plasma membranes and along a steep concentrations gradient converts potentially reversible alterations into the irreversible injury of cell death. The structural alterations in cellular constituents that characterize coagulative necrosis are themselves very likely the direct result of the action of an elevated calcium concentration on cellular macromolecules. The continuing need to identify the membrane alterations induced by the various causes of cell death in order to assess their potential reversibility in the absence of irreversible calcium accumulation is emphasized.
Article
In the CA1 subfield of the gerbil hippocampus, an unusual series of changes were noticed after ischemia. Mongolian gerbils were subjected to bilateral carotid occlusion for 5 min. Perfusion fixation was performed 3, 6 and 12 h or 1, 2, 4, 7 and 21 days afterwards. Specimens obtained from the dorsal hippocampus were processed for light and electron microscopy. Three different types of changes were observed in the CA4, CA2 and CA1 subfields. In CA4, the change was rapid and corresponded to ischemic cell change. The alteration in CA2 was relatively slow, and identical to what has been called reactive change. On the contrary, the change in the CA1 pyramidal cells was very slow, only becoming apparent by light microscopy 2 days following ischemia. The CA1 subfield was selected for electron microscopic observation. The lamellar alignment of proliferated cisterns of the endoplasmic reticulum was the most conspicuous finding in these cells. Four days following ischemia, almost all of the pyramidal cells in CA1 were destroyed. In the CA1 neuropil, numerous presynaptic terminals remained without being apposed to normal postsynaptic sites. These changes in CA1, called here 'delayed neuronal death', may differ from those thought to be typical of ischemic neuronal damage. It was unlikely that the disturbance of local blood vessels was the cause of these changes.
Article
This study examined the temporal profile of ischemic neuronal damage following transient bilateral forebrain ischemia in the rat model of four-vessel occlusion. Wistar rats were subjected to transient but severe forebrain ischemia by permanently occluding the vertebral arteries and 24 hours later temporarily occluding the common carotid arteries for 10, 20, or 30 minutes. Carotid artery blood flow was restored and the rats were killed by perfusion-fixation after 3, 6, 24, and 72 hours. Rats with postischemic convulsions were discarded. Ischemic neuronal damage was graded in accordance with conventional neuropathological criteria. Ten minutes of four-vessel occlusion produced scattered ischemic cell change in the cerebral hemispheres of most rats. The time to onset of visible neuronal damage varied among brain regions and in some regions progressively worsened with time. After 30 minutes of ischemia, small to medium-sized striatal neurons were damaged early while the initiation of visible damage to hippocampal neurons in the h1 zone was delayed for 3 to 6 hours. The number of damaged neurons in neocortex (layer 3, layers 5 and 6, or both) and hippocampus (h1, h3-5, paramedian zone) increased significantly (p less than 0.01) between 24 and 72 hours. The unique delay in onset of ischemic cell change and the protracted increase in its incidence between 24 and 72 hours could reflect either delayed appearance of ischemic change in previously killed neurons or a delayed insult that continued to jeopardize compromised but otherwise viable neurons during the postischemic period.
Article
Cerebral ischemia was induced in anesthetized rats by occluding both common carotid arteries; the vertebral arteries had been permanently occluded one day earlier. The levels of lipid-soluble antioxidants (alpha-tocopherol, reduced ubiquinones), free fatty acids and energy metabolites were measured in forebrain tissue after 30 min of ischemia, and after 15 min or 30 min of postischemic recirculation.Alpha-tocopherol decreased by 7% at the end of ischemia and decreased further during postischemic recirculation, indicating that free radical reactions were initiated during ischemia. All measured saturated, mono- and polyunsaturated free fatty acids accumulated markedly during ischemia. However, particularly rapid decrements were observed in polyunsaturated free fatty acids (arachidonic and docosahexaenoic acids) during an initial 15-min period of recirculation, while other free fatty acids gradually returned toward their preischemic levels after 30 min of recirculation. Both reduced and oxidized ubiquinone-9 tended to increase at the end of ischemia. During recirculation, reduced ubiquinone-9 declined below control by 20–24%,accompanied by a rise in the oxidized form. Profiles of free fatty acids and the changes in ubiquinones were compatible with the postischemic occurrence of lipid peroxidation. High energy phosphates were nearly depleted by ischemia, and recovery of adenosine triphosphate was limited to 73% of control after 30 min of recirculation.The results are compatible with the view that free radical reactions are initiated during ischemia, and that overt peroxidative processes become manifest during reflow when cerebral tissue is reoxygenated. Lipid peroxidation by free radical reactions may be a factor restricting postischemic recovery of energy metabolism, and lipid-soluble antioxidants may act to mitigate the extent of eventual brain damage.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
No-reflow state following cerebral ischemia
  • J G Wade
  • O Amtorp
  • S C Sprensen
Wade, J.G., Amtorp, O. and Sprensen, S.C.. No-reflow state following cerebral ischemia, Arch. Neurol., 32 (1975) 381-384.
Selective neuronal vulnerability to cerebral protein- and RNA synthesis in the hippocampus of the gerbil brain
  • Bodsh
No-reflow state following cerebral ischemia
  • Wade
Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments
  • Hagberg