Increase In Cytoplasmic Calcium Activity Research Articles

Role of calcium in neuronal cell injury: which subcellular compartment is involved?

It is widely believed that calcium plays a primary role in the development of neuronal cell injury in different pathological states of the brain. Disturbances of calcium homeostasis may be induced in three different subcellular compartments, the cytoplasm, mitochondria or the endoplasmic reticulum (ER). The traditional calcium hypothesis holds that neuronal cell injury is induced by a marked increase in cytoplasmic calcium activity during stress (e.g., cerebral ischemia). Recently, this hypothesis has been modified, taking into account that under different experimental conditions the extent of cell injury does not correlate closely with calcium load or total calcium influx into the cell, and that neuronal cell injury has been found to be associated with both increases and decreases of cytoplasmic calcium activity. The mitochondrial calcium hypothesis is based on the observation that after a severe form of stress there is a massive influx of calcium ions into mitochondria, which may lead to production of free radicals, opening of the mitochondrial permeability transition (MPT) pore and disturbances of energy metabolism. However, it has still to be established whether drugs such as cyclosporin A are neuroprotective through their effect on MPT or through the blocking of processes upstream of MPT. The ER calcium hypothesis arose from the observation that ER calcium stores are depleted after severe forms of stress, and that the response of cells to disturbances of ER calcium homeostasis (activation of the expression of genes coding for ER resident stress proteins and suppression of the initiation of protein synthesis) resembles their response to a severe form of stress (e.g., transient ischemia) implying common underlying mechanisms. Elucidating the exact mechanisms of calcium toxicity and identifying the subcellular compartment playing the most important role in this pathological process will help to evaluate strategies for specific therapeutic intervention.

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 Ca2+-ATPase; the activity of ER Ca2+-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 Ca2+-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 Ca2+-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.

Effect of 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8) on neuronal calcium homeostasis, protein synthesis, and energy metabolism.

It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 microM) induced a small transient increase in cytoplasmic calcium activity ([Ca2+]i), whereas a second dose of TMB-8 (200 microM) produced a marked and sustained rise in [Ca2+]i. The increase in [Ca2+]i evoked by blocking endoplasmic reticulum Ca(2+)-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.

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.

Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis

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- 3 H ]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca 2+-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.

Disturbances of the functioning of endoplasmic reticulum: a key mechanism underlying neuronal cell injury?

Cerebral ischemia leads to a massive increase in cytoplasmic calcium activity resulting from an influx of calcium ions into cells and a release of calcium from mitochondria and endoplasmic reticulum (ER). It is widely believed that this increase in cytoplasmic calcium activity plays a major role in ischemic cell injury in neurons. Recently, this concept was modified, taking into account that disturbances occurring during ischemia are potentially reversible: it then was proposed that after reversible ischemia, calcium ions are taken up by mitochondria, leading to disturbances of oxidative phosphorylation, formation of free radicals, and deterioration of mitochondrial functions. The current review focuses on the possible role of disturbances of ER calcium homeostasis in the pathologic process culminating in ischemic cell injury. The ER is a subcellular compartment that fulfills important functions such as the folding and processing of proteins, all of which are strictly calcium dependent. ER calcium activity is therefore relatively high, lying in the lower millimolar range (i.e., close to that of the extracellular space). Depletion of ER calcium stores is a severe form of stress to which cells react with a highly conserved stress response, the most important changes being a suppression of global protein synthesis and activation of stress gene expression. The response of cells to disturbances of ER calcium homeostasis is almost identical to their response to transient ischemia, implying common underlying mechanisms. Many observations from experimental studies indicate that disturbances of ER calcium homeostasis are involved in the pathologic process leading to ischemic cell injury. Evidence also has been presented that depletion of ER calcium stores alone is sufficient to activate the process of programmed cell death. Furthermore, it has been shown that activation of the ER-resident stress response system by a sublethal form of stress affords tolerance to other, potentially lethal insults. Also, disturbances of ER function have been implicated in the development of degenerative disorders such as prion disease and Alzheimer's disease. Thus, disturbances of the functioning of the ER may be a common denominator of neuronal cell injury in a wide variety of acute and chronic pathologic states of the brain. Finally, there is evidence that ER calcium homeostasis plays a key role in maintaining cells in their physiologic state, since depletion of ER calcium stores causes growth arrest and cell death, whereas cells in which the regulatory link between ER calcium homeostasis and protein synthesis has been blocked enter a state of uncontrolled proliferation.

Changes in interferon-regulatory factor-1 mRNA levels after transient ischemia in rat brain.

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.

Activation of gadd153 expression through transient cerebral ischemia: evidence that ischemia causes endoplasmic reticulum dysfunction

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 Ca 2+-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.

Role of calcium in the activation of erp72 and heme oxygenase-1 expression on depletion of endoplasmic reticulum calcium stores in rat neuronal cell culture

Endoplasmic reticulum (ER) calcium pool depletion was induced by 30 min exposure of primary neuronal cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca 2+-ATPase. Twelve hours later, erp72 and heme oxygenase-1 (HO-1) mRNA levels were quantified by PCR. Protein synthesis was also measured. Transient Tg exposure of neurons induced a marked rise in mRNA levels (7-fold and a 21-fold increase in erp72 and HO-1 mRNA levels; P<0.001). Loading of neurons with the calcium chelator 1,2-bis(o-Aminophenoxy)ethane- N, N, N′, N′-tetra(acetoxymethyl)ester (BAPTA-AM) prior to thapsigargin treatment had only a minor effect on the Tg-induced rise in gene expression. This small inhibitory effect may result from the severe suppression of protein synthesis caused by BAPTA-AM. The results suggest that the increase in stress gene expression induced by exposure of neurons to Tg is triggered by a decrease in ER calcium activity and not by the corresponding increase in cytoplasmic calcium activity.

Erp72 expression activated by transient cerebral ischemia or disturbance of neuronal endoplasmic reticulum calcium stores.

Stress-induced activation of the expression of the endoplasmic reticulum (ER)-resident chaperon and member of the protein disulfide isomerase family erp72 was studied after transient cerebral ischemia in vivo using the four-vessel occlusion method and experimental depletion of ER calcium stores in primary neuronal cell cultures. After 8 days in vitro, neurons were exposed to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase, or the Tg solvent DMSO. In separate experiments neurons were pre-loaded with the cell-permeant calcium chelator BAPTA-AM before Tg exposure. Stress-induced changes in erp72 expression were analysed by quantitative PCR. Transient cerebral ischemia produced a significant increase in erp72 mRNA levels which rose to about 200% of control (hippocampus) or 300% of control (cortex). After depletion of ER calcium stores neuronal erp72 mRNA levels rose markedly, peaking at 12 h of recovery. Counteracting the Tg-induced rise in cytoplasmic calcium activity by preloading cells with the chelator BAPTA-AM did not influence erp72 expression significantly, suggesting that the activation of erp72 expression resulted from the depletion of ER calcium stores and not from the corresponding increase in cytoplasmic calcium activity. An activation of erp72 expression is indicative of a disturbance of ER function. The results of the present study therefore provide evidence to support the notion that transient cerebral ischemia induces disturbances of neuronal ER function, probably through a depletion of ER calcium stores.

Temporal analysis of changes in neuronal c-fos mRNA levels induced by depletion of endoplasmic reticulum calcium stores: effect of clamping cytoplasmic calcium activity at resting levels.

Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Tg-induced rise in [Ca2+]i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca2+]i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c-fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c-fos mRNA levels (approximately 10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c-fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c-fos. Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.

Calcium ionophore A23187 as a secretagogue for rat mast cells: Does it bypass inhibition by calcium flux blockers?

It is widely accepted that an increase in cytoplasmic calcium activity is a stimulus-secretion coupling event required to initiate secretion in mast cells and basophils. This study is concerned with the notion that inhibition of secretion by certain agents thought to be calcium influx inhibitors, including dibutyryl cyclic AMP (db-cAMP), quercetin, verapamil and ruthenium red can be ‘bypassed’ with the calcium-carrying ionophore A23187. The importance of the bypass concept lies in its implication that, in the absence of A23187, these agents inhibit secretion solely by impairing physiologic calcium gating.