Recovery of neuronal protein synthesis after irreversible inhibition of the endoplasmic reticulum calcium pump

In the physiological state, protein synthesis is controlled by calcium homeostasis in the endoplasmic reticulum (ER). Recently, evidence has been presented that dividing cells can adapt to an irreversible inhibition of the ER calcium pump (SERCA), although the mechanisms underlying this adaption have not yet been elucidated. Exposing primary neuronal cells to thapsigargin (Tg, a specific irreversible inhibition of SERCA) resulted in a complete suppression of protein synthesis and disaggregation of polyribosomes indicating inhibition of the initiation step of protein synthesis. Protein synthesis and ribosomal aggregation recovered to 50-70% of control when cells were cultured in medium supplemented with serum for 24 h, but recovery was significantly suppressed in a serum-free medium. Culturing cells in serum-free medium for 24 h already caused an almost 50% suppression of SERCA activity and protein synthesis. SERCA activity did not recover after Tg treatment, and a second exposure of cells to Tg, 24 h after the first, had no effect on protein synthesis. Acute exposure of neurons to Tg induced a depletion of ER calcium stores as indicated by an increase in cytoplasmic calcium activity, but this response was not elicited by the same treatment 24 h later. However, treatments known to deplete ER calcium stores (exposure to the ryanodine receptor agonists caffeine or 2-hydroxycarbazole, or incubating cells in calcium-free medium supplemented with EGTA) caused a second suppression of protein synthesis when applied 24 h after Tg treatment. The results suggest that after Tg exposure, restoration of protein synthesis was induced by recovery of the regulatory link between ER calcium homeostasis and protein synthesis, and not by renewed synthesis of SERCA protein or development of a new regulatory system for the control of protein synthesis. The effect of serum withdrawal on SERCA activity and protein synthesis points to a role of growth factors in maintaining ER calcium homeostasis, and suggests that the ER acts as a mediator of cell damage after interruption of growth factor supplies.     

Effect of nitric oxide on endoplasmic reticulum calcium homeostasis, protein synthesis and energy metabolism

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca2+ pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (+/-)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca(2+)-ATPase; the activity of ER Ca(2+)-ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca(2+)-ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NO-related effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca(2+)-ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.     

Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

内质网应激与心肌肥大

肌浆网是调控心肌细胞钙稳态、蛋白质合成和细胞凋亡的重要亚细胞器。内质网应激是指内质网理化环境改变和过负荷等因素导致未折叠/误折叠蛋白在内质网聚集和钙稳态失衡等内质网功能紊乱状态。适度的内质网应激有利于心肌细胞代偿,持续而严重的内质网应激则触发内质网应激相关细胞凋亡,造成肥大心肌由代偿转向衰竭,是影响心肌肥大发生、发展的重要因素。本文综述了内质网应激反应在心肌肥大发生、发展中的作用。     

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress

Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES). Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES). Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES) and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.     

Effects of Disrupting Calcium Homeostasis on Neuronal Maturation: Early Inhibition and Later Recovery

It has become increasingly clear that agents that disrupt calcium homeostasis may also be toxic to developing neurons. Using isolated primary neurons, we sought to understand the neurotoxicity of agents such as MK801 (which blocks ligand-gated calcium entry), BAPTA (which chelates intracellular calcium), and thapsigargin (TG; which inhibits the endoplasmic reticulum Ca²⁺-ATPase pump). Thus, E18 rat cortical neurons were grown for 1 day in vitro (DIV) and then exposed to vehicle (0.1% DMSO), MK801 (0.01–20 μM), BAPTA (0.1–20 μM), or TG (0.001–1 μM) for 24 h. We found that all three agents could profoundly influence early neuronal maturation (growth cone expansion, neurite length, neurite complexity), with the order of potency being MK801 < BAPTA < TG. We next asked if cultures exposed to these agents were able to re-establish their developmental program once the agent was removed. When we examined network maturity at 4 and 7 DIV, the order of recovery was MK801 > BAPTA > TG. Thus, mechanistically distinct ways of disrupting calcium homeostasis differentially influenced both short-term and long-term neuronal maturation. These observations suggest that agents that act by altering intracellular calcium and are used in obstetrics or neonatology may be quite harmful to the still-developing human brain.     

Measurement of hepatic protein fractional synthetic rate with stable isotope labeling technique in thapsigargin stressed HepG2 cells

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97 ± 0.02 and 0.99 ± 0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68 ± 0.03 and 0.60 ± 0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.     

Activation of Excitatory Amino Acid Receptors Cannot Alone Account for Anoxia-Induced Impairment of Protein Synthesis in Rat Hippocampal Slices

We have investigated the contribution of excitatory amino acid receptor activation to the inhibition of protein synthesis observed after anoxia in rat hippocampal slices. Protein synthesis was assessed in normoxic medium by measuring the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. Protein synthesis was impaired after anoxia; the extent of inhibition was dependent on the duration of anoxia and on the time allowed for postanoxic recovery. There was a similar impairment under normoxic conditions when the N-methyl-D-aspartate (NMDA) receptor channel was activated by removing Mg2+ and adding NMDA. This was prevented by noncompetitive antagonists of the NMDA receptor channel (MK-801, phencyclidine, and N-allylnormetazocine). In contrast, incubation with the NMDA antagonists failed to prevent the protein synthesis inhibition caused by anoxia, although it moderately facilitated the postanoxic recovery. Protein synthesis was also impaired under normoxic conditions after incubation with quisqualate and kainate, agonists of non-NMDA glutamate receptors. This impairment was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of these receptors. Although 6-cyano-7-nitroquinoxaline-2,3-dione alone failed to prevent anoxic damage, when used in combination with an NMDA antagonist it did partially enhance the later recovery of protein synthesis. These results indicate that the activation of excitatory amino acid receptors cannot alone account for anoxia-induced impairment of protein synthesis in rat hippocampal slices.     

Loading neurons with BAPTA-AM activates xbp1 processing indicative of induction of endoplasmic reticulum stress

Loading cells with the calcium chelator BAPTA-AM is an analytical tool which has been used to suppress a rise in cytoplasmic calcium activity under various experimental conditions and thus, to evaluate the role of elevated cytoplasmic calcium levels in the process under investigation. BAPTA-AM may, however, not only have an isolated effect on cytoplasmic processes but also on functions of other subcellular compartments such as the endoplasmic reticulum (ER). Under conditions associated with ER dysfunction, the unfolded protein response is activated which is characterized by suppression of translation and processing of xbp1 mRNA, resulting in activation of the expression of genes coding for ER stress proteins. To investigate whether BAPTA-AM causes ER stress, primary neuronal cell cultures were loaded with varying amounts of BAPTA-AM. Exposure of cells to BAPTA-AM induced a marked rise in processed xbp1 mRNA levels, correlating with exposure times and BAPTA-AM concentrations in the medium used for loading. The increase in processed xbp1 mRNA was associated with suppression of protein synthesis and induction of cell injury. The results of this study indicate that loading primary neuronal cell cultures with BAPTA-AM activates xbp1 processing, implying that this calcium chelator does not have an isolated effect on cytoplasmic calcium activity but also an affect on ER function.     

Changes in the phosphorylation of initiation factor eIF-2alpha, elongation factor eEF-2 and p70 S6 kinase after transient focal cerebral ischaemia in mice

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1-6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2alpha, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2alpha phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2alpha was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2alpha phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2alpha phosphorylation. Dephosphorylation of eIF-2alpha was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2alpha from phosphorylation, even in the presence of active eIF-2alpha kinase. Phosphorylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short-lasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.     

Superoxide dismutase/catalase mimetics but not MAP kinase inhibitors are neuroprotective against oxygen/glucose deprivation-induced neuronal death in hippocampus

Although oxygen/glucose deprivation (OGD) has been widely used as a model of ischemic brain damage, the mechanisms underlying acute neuronal death in this model are not yet well understood. We used OGD in acute hippocampal slices to investigate the roles of reactive oxygen species and of the mitogen-activated protein kinases (MAPKs) in neuronal death. In particular, we tested the neuroprotective effects of two synthetic superoxide dismutase/catalase mimetics, EUK-189 and EUK-207. Acute hippocampal slices prepared from 2-month-old or postnatal day 10 rats were exposed to oxygen and glucose deprivation for 2 h followed by 2.5 h reoxygenation. Lactate dehydrogenase (LDH) release in the medium and propidium iodide (PI) uptake were used to evaluate cell viability. EUK-189 or EUK-207 applied during the OGD and reoxygenation periods decreased LDH release and PI uptake in slices from 2-month-old rats. EUK-189 or EUK-207 also partly blocked OGD-induced ATP depletion and extracellular signal-regulated kinases 1 and 2 (ERK1/2) dephosphorylation, and completely eliminated reactive oxygen species generation. The MEK inhibitor U0126 applied together with EUK-189 or EUK-207 completely blocked ERK1/2 activation, but had no effect on their protective effects against OGD-induced LDH release. U0126 alone had no effect on OGD-induced LDH release. EUK-207 had no effect on OGD-induced p38 or c-Jun N-terminal kinase dephosphorylation, and when the p38 inhibitor SB203580 was applied together with EUK-207, it had no effect on the protective effects of EUK-207. SB203580 alone had no effect on OGD-induced LDH release either. In slices from p10 rats, OGD also induced high-LDH release that was partly reversed by EUK-207; however, neither OGD nor EUK-207 produced significant changes in ERK1/2 and p38 phosphorylation. OGD-induced spectrin degradation was not modified by EUK-189 or EUK-207 in slices from p10 or 2-month-old rats, suggesting that their protective effects was not mediated through inhibition of calpain activation. Thus, both EUK-189 and EUK-207 provide neuroprotection in acute ischemic conditions, and this effect is related to elimination of free radical formation and partial reversal of ATP depletion, but not mediated by the activation or inhibition of the MEK/ERK or p38 pathways, or inhibition of calpain activation.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.