thapsigargin and Ischemic-Attack--Transient

Transient global cerebral ischemia triggers suppression of the initiation step of protein synthesis, a process which is controlled by endoplasmic reticulum (ER) function. ER function has been shown to be disturbed after transient cerebral ischemia, as indicated by an activation of the ER-resident eIF2alpha kinase PERK. In this study, we investigated ischemia-induced changes in protein levels and phosphorylation states of the initiation factors eIF2alpha, eIF2B epsilon, and eIF4G1 and of p70 S6 kinase, proteins playing a central role in the control of the initiation of translation. Transient focal cerebral ischemia was induced in mice by occlusion of the left middle cerebral artery. Transient ischemia caused a long-lasting suppression of global protein synthesis. eIF2alpha was transiently phosphorylated after ischemia, peaking at 1-3 h of recovery. eIF2B epsilon and p70 S6 kinase were completely dephosphorylated during ischemia and phosphorylation did not recover completely following reperfusion. In addition, eIF2B epsilon, eIF4G1, and p70 S6 kinase protein levels decreased progressively with increasing recirculation time. Thus, several different processes contributed to ischemia-induced suppression of the initiation of protein synthesis: a long-lasting dephosphorylation of eIF2B epsilon and p70 S6K starting during ischemia, a transient phosphorylation of eIF2alpha during early reperfusion, and a marked decrease of eIF2B epsilon, eIF4G1, and p70 S6K protein levels starting during vascular occlusion (eIF4G1). Study of the mechanisms underlying ischemia-induced suppression of the initiation step of translation will help to elucidate the role of protein synthesis inhibition in the development of neuronal cell injury triggered by transient cerebral ischemia.

Topics: Animals; Blotting, Western; Brain; Brain Chemistry; Cells, Cultured; Disease Models, Animal; Disease Progression; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-2B; Eukaryotic Initiation Factor-4G; Ischemic Attack, Transient; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Neurons; Peptide Chain Initiation, Translational; Phosphorylation; Protein Subunits; Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Thapsigargin

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.

Topics: Animals; Cells, Cultured; Cerebral Cortex; Cerebrovascular Circulation; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Immunoblotting; Immunohistochemistry; Ischemic Attack, Transient; Laser-Doppler Flowmetry; Mice; Middle Cerebral Artery; Neurons; Peptide Elongation Factor 2; Phosphorylation; Protein Biosynthesis; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases; Thapsigargin

MyD116 is the murine homologue of growth arrest- and DNA damage-inducible genes (gadd34), a gene family implicated in growth arrest and apoptosis induced by endoplasmic reticulum dysfunction. The present study investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. MyD116 mRNA levels were measured by quantitative PCR. After 2 h of recovery following 30 min forebrain ischemia, MyD116 mRNA levels rose to about 550% of control both in the cortex and hippocampus. In the cortex, MyD116 mRNA levels gradually declined to 290% of control 24 h after ischemia, whereas in the hippocampus they remained high (538% of control after 24 h of recovery). To elucidate the possible mechanism underlying this activation process, MyD116 mRNA levels were also quantified in primary neuronal cell cultures under two different experimental conditions, both leading to a depletion of endoplasmic reticulum (ER) calcium pools. Changes in cytoplasmic calcium activity were assessed by fluorescence microscopy of fura-2-loaded cells, and protein synthesis (PS) was evaluated by measuring the incorporation of l-[4,5-3H]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase, produced a parallel increase in cytoplasmic calcium activity and a long-lasting suppression of PS, while the second, immersion in a calcium-free medium supplemented with the calcium chelator EGTA, caused a parallel decrease in cytoplasmic calcium levels and a short-lasting suppression of PS. Exposure of neurons to Tg induced a permanent increase in MyD116 mRNA levels. Exposure of cells to calcium-free medium supplemented with EGTA produced only a transient rise in MyD116 mRNA levels peaking after 6 h of recovery. The results demonstrate that depletion of ER calcium stores without any increase in cytoplasmic calcium activity is sufficient to activate MyD116 expression. A similar mechanism may be responsible for the increase in MyD116 mRNA levels observed after transient forebrain ischemia. It is concluded that those pathological disturbances triggering the activation of MyD116 expression after transient forebrain ischemia are only transient in the cerebral cortex but permanent in the hippocampus.

Topics: Animals; Antigens, Differentiation; Calcium; Calcium-Transporting ATPases; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation; Homeostasis; Ischemic Attack, Transient; Neoplasm Proteins; Nerve Tissue Proteins; Neurons; Prosencephalon; Proto-Oncogene Proteins; Rats; Rats, Wistar; Thapsigargin

The expression of the gene encoding the C/EBP-homologous protein (CHOP), which is also known as growth arrest and DNA-damage-inducible gene 153 (gadd153), has been shown to be specifically activated under conditions that disturb the functioning of the endoplasmic reticulum (ER). To investigate a possible role of ER dysfunction in the pathological process of ischemic cell damage, we studied ischemia-induced changes in gadd153 expression using quantitative PCR. Transient cerebral ischemia was produced in rats by four-vessel occlusion. In the hippocampus, ischemia induced a pronounced increase in gadd153 mRNA levels, peaking at 8 h of recovery (6.4-fold increase, p<0.01), whereas changes in the cortex were less marked (non-significant increase). To elucidate the possible mechanism underlying this activation process, gadd153 mRNA levels were also evaluated in primary neuronal cell cultures under two different conditions, both leading to a depletion of ER calcium pools in the presence or absence of an increase in cytoplasmic calcium activity. The first procedure, exposure to thapsigargin, an irreversible inhibitor of ER Ca2+-ATPase, caused a marked increase in gadd153 mRNA levels both in cortical and hippocampal neurons, peaking at 12-18 h after treatment. The second procedure, immersion of cells in calcium free medium supplemented with EGTA, caused only a transient increase in gadd153 mRNA levels, peaking at 6 h of recovery, indicating that a depletion of ER calcium stores in the absence of an increase in cytoplasmic calcium activity is sufficient to activate neuronal gadd153 expression. The results imply that transient cerebral ischemia disturbs the functioning of the ER and that these pathological changes are more pronounced in the hippocampus compared to the cortex.

Topics: Animals; Calcium; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Cerebral Cortex; Chelating Agents; DNA-Binding Proteins; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression; Hippocampus; Homeostasis; Ischemic Attack, Transient; Male; Neurons; Nuclear Proteins; Rats; Rats, Wistar; RNA, Messenger; Thapsigargin; Transcription Factor CHOP; Transcription Factors

To evaluate whether the interferon system in the brain is activated by a severe form of metabolic stress, and to elucidate the possible mechanism underlying this activation, changes in the interferon regulatory factor-1 (irf-1) mRNA levels were evaluated after transient cerebral ischemia, and after exposure of primary neuronal cells to experimental conditions resulting in a depletion of ER calcium stores. Following 30 min ischemia and 2 h recovery, irf-1 mRNA levels rose significantly and stayed high for up to 24 h of recovery. Irf-1 mRNA levels were also significantly increased in neurons in vitro after exposing cells to conditions resulting in ER calcium store depletion with or without a parallel increase in cytoplasmic calcium activity. It is concluded that transient cerebral ischemia induces activation of the interferon system and that disturbances of ER calcium homeostasis may play a role in this process.

Topics: Animals; Brain Chemistry; Calcium; Cerebral Cortex; Cerebrovascular Circulation; Chelating Agents; DNA-Binding Proteins; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression; Hippocampus; Interferon Regulatory Factor-1; Interferons; Ischemic Attack, Transient; Male; Phosphoproteins; Rats; Rats, Wistar; RNA, Messenger; Thapsigargin; Transcription Factors

Endothelial cells are affected in the cerebral vasospasm that occurs at the time of erthyrocyte lysis in a subarachnoid clot. A red blood cell lysate was added to bovine pulmonary artery endothelial cells in vitro to determine whether hemolysate can trigger tyrosine kinase mediated cell signalling and if so, whether this signal is independent of the elevation of intracellular free calcium levels, [Ca2+]i induced by hemolysate. Hemolysate was found by Western blotting to induce a dose dependent increase in the level of tyrosine phosphorylation of two proteins, approximately 60 and 110 kD, that was maximal between 1 and 2 min. The biphasic increase in [Ca2+]i induced by hemolysate consists of a peak complete within 1 min which is the result of release of intracellular calcium stores and a plateau phase due to an influx of extracellular Ca2+. Addition of hemolysate to cells in the presence of EGTA indicated that an extracellular Ca2+ influx is not required for the increases in tyrosine phosphorylation. Release of intracellular Ca2+ stores by thapsigargin, a Ca(2+)-ATPase inhibitor, was, however, found to increase the phosphotyrosine content of the same 60 and 110 kD proteins. Endothelial cells pretreated with tyrosine kinase inhibitors, tyrphostin 25 or genistein, before exposure to hemolysate blocked the plateau phase of the [Ca2+]i response indicating that tyrosine kinase activity is required for the influx. Ca2+ and phosphotyrosine mediated cell signalling induced by hemolysate in endothelial cells may be activated by a single component but represent distinct targets for possible control of the cerebral vasospasm response.

Topics: Animals; Calcium; Cattle; Cells, Cultured; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Genistein; Hemolysis; Ischemic Attack, Transient; Isoflavones; Nitriles; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Pulmonary Artery; Signal Transduction; Subarachnoid Hemorrhage; Thapsigargin; Tyrphostins