recovery of neuronal protein synthesis after irreversible inhibition of the endoplasmic reticulum...

Research

Recovery of neuronal proteinsynthesis after irreversible inhibitionof the endoplasmic reticulumcalcium pump

J. Doutheil, 1 M. Treiman, 2 U. Oschlies, 1 W. Paschen 1

1Department of Experimental Neurology, Max-Planck-Institute for Neurological Research, Cologne, Germany2Department of Medical Physiology, Biotechnology Center for Cell Communication, The Panum Institute, Copenhagen, Denmark

Summary In the physiological state, protein synthesis is controlled by calcium homeostasis in the endoplasmicreticulum (ER). Recently, evidence has been presented that dividing cells can adapt to an irreversible inhibition of theER 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 completesuppression of protein synthesis and disaggregation of polyribosomes indicating inhibition of the initiation step ofprotein synthesis. Protein synthesis and ribosomal aggregation recovered to 50–70% of control when cells werecultured in medium supplemented with serum for 24 h, but recovery was significantly suppressed in a serum-freemedium. Culturing cells in serum-free medium for 24 h already caused an almost 50% suppression of SERCA activityand protein synthesis. SERCA activity did not recover after Tg treatment, and a second exposure of cells to Tg, 24 hafter the first, had no effect on protein synthesis. Acute exposure of neurons to Tg induced a depletion of ER calciumstores as indicated by an increase in cytoplasmic calcium activity, but this response was not elicited by the sametreatment 24 h later. However, treatments known to deplete ER calcium stores (exposure to the ryanodine receptoragonists caffeine or 2-hydroxycarbazole, or incubating cells in calcium-free medium supplemented with EGTA) causeda second suppression of protein synthesis when applied 24 h after Tg treatment. The results suggest that after Tgexposure, restoration of protein synthesis was induced by recovery of the regulatory link between ER calciumhomeostasis and protein synthesis, and not by renewed synthesis of SERCA protein or development of a newregulatory system for the control of protein synthesis. The effect of serum withdrawal on SERCA activity and proteinsynthesis points to a role of growth factors in maintaining ER calcium homeostasis, and suggests that the ER acts as amediator of cell damage after interruption of growth factor supplies.

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INTRODUCTION

Results of experiments performed on permanent celllines indicate that, in the physiological state, protein syn-thesis is controlled by calcium homeostasis within theendoplasmic reticulum (ER) [1], and a similar relationshiphas been found in terminally differentiated cells such as

Received 22 February 1999Revised 21 May 1999Accepted 21 May 1999

Correspondence to: Dr Wulf Paschen, Department of ExperimentalNeurology, Max-Planck-Institute for Neurological Research. Gleuelerstrasse50, 50931 Cologne, Germany. Tel: +49 221 47260; fax: +49 221 4726 298;e-mail: [email protected]

primary neurons [2]. A depletion of ER calcium storescauses a suppression of the initiation of translation, asindicated by an increased phosphorylation of the eukary-otic initiation factor 2α [3,4], and a disaggregation ofpolyribosomes [5,6]. The double-stranded RNA-activatedprotein kinase (PKR) is involved in the signal transduc-tion pathway between ER calcium homeostasis and pro-tein synthesis: depletion of ER calcium stores induces anactivation of PKR which then phosphorylates eIF-2α [7,8].A second protein kinase (PERK), fulfilling the same func-tion, has recently been identified [9].

Suppression of global protein synthesis is a commonresponse to stress (e.g. to the metabolic form of stressproduced by transient ischaemia), and the similarities in

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the manner of suppression of protein synthesis aftertransient ischaemia and depletion of ER calcium storeshave led to the conclusion that transient ischaemia maycause disturbances in ER calcium homeostasis [10,11].Indeed, depletion of ER Ca2+ stores have been observedrecently in neurons of the hippocampal CA1-subfieldafter transient cerebral ischaemia in gerbils [12].Following ischaemia, global protein synthesis is com-pletely suppressed [13,14]; it recovers in non-vulnerableareas but not in vulnerable ones [13,14], implying thatthe ability to recover from an inhibition of synthesisdetermines the final outcome. Adaptation of proteinsynthesis is also thought to be involved in the develop-ment of tolerance, since the recovery of protein synthesisfrom a state of post-ischaemic suppression is facilitatedin animals with induced tolerance [15].

Two different adaptive responses have been describedin cells exposed to agents disturbing ER functions.Results from experiments performed with different per-manent cell-culture systems suggest that cells that areexposed to thapsigargin (Tg), a specific irreversibleinhibitor of the ER calcium pump (SERCA), or to otherperturbants of ER function adapt to this severe form ofstress within several hours, as indicated by recovery ofprotein synthesis to 40–70% of control [16]. Althoughthe mechanisms underlying this adaptive response havenot yet been fully elucidated, it has been suggested thatactivation of grp78 synthesis plays a role in the recoveryprocess [17]. In a second series of experiments, Tg-resis-tant cells were selected by exposing permanent cell linesto low Tg concentrations over a period of 10 months [18].This adaptive response was mediated, at least in part, bya mutation of the SERCA gene resulting in a substitutionat amino acid 256 of SERCA [19]. Whether non-dividingcells such as primary neuronal cells have the capacity toadapt to severe ER dysfunction has not yet been investi-gated. This is a topic of significant interest becausesuppression of protein synthesis is the most commonresponse to a severe form of stress and recent studieshave indicated that ER dysfunction plays a central role inthe pathological process culminating in cell injury invarious pathological states of the brain [11].

We decided to investigate the adaptive processactivated by blocking ER Ca2+-ATPase (SERCA) throughexposure of neurons to Tg. This enzyme is particularlysensitive to oxygen radicals [20,21], i.e. reactive oxygenspecies which have been implicated in the pathologicalprocess of ischaemic cell injury[22]. Results presentedhere indicate that exposing neurons to Tg, which blocksprotein synthesis almost completely immediately afterexposure, stimulates cells to adapt in such a way thatprotein synthesis recovers to a considerable extent evenin the absence of SERCA activity. This recovery of proteinsynthesis is paralleled by a restoration of ribosomal

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aggregation. The observation that, after recovery from Tgexposure, protein synthesis cannot again be blocked byTg but still responds to caffeine, 2-hydoxycarbazole orcalcium chelator, implies that the regulatory linkbetween ER calcium homeostasis and protein synthesisrecovers at least partially even after an irreversibleinhibition of SERCA activity.

MATERIALS AND METHODS

Culture of primary cortical neurons

Primary neuronal cell cultures were prepared from thecerebral cortex of embryonic rat brains at days 16–18 ofgestation [2]. After dissociation with papain and DNAse I,filtration through a nylon mesh and centrifugation, cellswere suspended in minimal essential medium (MEM)supplemented with 30 mM glucose and 2 mM glutamine,10 IU/ml penicillin and 10 ng/ml streptomycin, and 5%horse serum. Cells were plated on polyethylenimine-coated dishes at a density of 106 cells/ml, and placed in ahumidified atmosphere of 95% air and 5% CO2 at 37°C.Cells were used for experiments after 9–10 days in vitro.

Depletion of endoplasmic reticulum calcium stores

Endoplasmic reticulum (ER) calcium stores weredepleted by exposing cells to thapsigargin (Tg, an irre-versible inhibitor of ER Ca2+-ATPase). Stock solutions (1mM) were prepared freshly in dried DMSO. Cells weretransferred to serum-free MEM medium to which 1 µMTg had been added. After 30 min exposure, the Tg-con-taining medium was washed off, and the mediumchanged to MEM supplemented with 5% serum. Controlcultures were exposed to DMSO (0.1%) for 30 min. ERcalcium stores were also depleted by drawing calciumions out of the ER by extracellular (EGTA) chelation, orby an activation of the ER ryanodine receptor (caffeine or2-hydroxycarbazole exposure). Cell were exposed for 30min to calcium-free medium supplemented with 1 mMEGTA, to 10, 20 or 40 mM caffeine or to 100–500 µM 2-hydroxycarbazole followed by the addition of L-[4,5-3H]leucine for evaluation of protein synthesis in thepresence of drugs (see below).

Measurement of cytoplasmic calcium activity

The effect of Tg exposure on ER calcium pool depletionwas evaluated indirectly by measuring the increase incytoplasmic calcium activity. Cells were washed withbuffered salt solution (BSS) and loaded for 60 min withfura-2-AM (5 µM, stock solution: 1 mM in DMSO). Afterloading, cells were washed again with BSS and allowed torecover for at least 30 min before starting measurements.Cytoplasmic calcium activity was evaluated using an

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image system connected to an inverted fluorescencemicroscope. Fluorescence signals were converted to cyto-plasmic calcium activity as described by Grynkiewicz etal. [23]. A calibration curve was constructed from mea-surements of fura-2 and solutions with different calciumactivities. Before fluorescence images were taken andprocessed for quantification of cytoplasmic calciumactivity, cells in the area of interest were characterized bymorphological criteria using conventional microscopy.Only cells with the characteristic morphological featureof neurons were used for further analysis.

Measurement of Ca 2+-ATPase phosphorylatedintermediate (E~P) in microsomes from culturedcortical neurons

Harvested neurons were homogenized in batches ofapproximately 6–13 × 106 cells/2 ml of ice-cold buffercontaining sucrose 0.3 M, N-tris(hydroxymethyl)methyl-2-aminomethanesulphonic acid (TES) 20 mM(pH7), phenylmethylsulphonyl fluoride (0.5 mM) andleupeptin (5 µg/ml). Homogenates were spun at 10 000 gfor 10 min, and the resulting supernatants centrifuged at100 000 g for 60 min to obtain microsomal pellets.Autophosphorylation of microsomal Ca2+-ATPase wascarried out as described earlier [24], using the ‘reversedassay’ procedure to ensure full inhibition of the SERCAE~P formation by Tg. Briefly, the final assay mixture(100 µl volume) contained (concentrations in mM): TES40 (pH 7), KCl 120, CaCl2 0.6, EGTA 0.5. When present, Tgwas used at a final concentration of 100 nM. Fifty micro-grammes of microsomal protein was incubated on icefor 20 s with [γ-32P]ATP 50 nM (approximately 500Ci/mmole). The reaction was stopped by adding 100 µl ofice-cold 12% trichloroacetic acid/20 mM H3PO4. SDS-polyacrylamide gel electrophoresis was carried out underacid conditions as described previously [25]. After autora-diography of the dried gels, the phosphorylated bandswere cut out and the radioactivity determined by scintil-lation counting.

Protein synthesis

At the end of the experiments, protein synthesis wasevaluated by measuring the incorporation of L-[4,5-3H]leucine into proteins; 10 µCi/ml L-[4,5-3H]leucine wasadded to the last medium, and cells were incubated foran additional 30 min in the presence of the labelledleucine. At the end of the incorporation period, theL-[4,5-3H]leucine was washed off and proteins precipi-tated by adding cold TCA solution. After extensive wash-ing, precipitated proteins were dissolved in 1 mM NaOH.Aliquots were taken for determination of protein content


[26] and for measuring the incorporation of radioactivityinto proteins by liquid scintillation counting.

Electron microscopy

The state of ribosomal aggregation was evaluated byelectron microscopy [5]. Primary neuronal cells isolatedfrom the cortex of embryonic rat brains at days 16–18 ofgestation (see above) were cultured on polyethylenimine-coated polyester films, which were sterilized and incu-bated for 18 h in serum-containing MEM before use.After 9 days in vitro, cells were used for the experiments.At the end of the experiments, cells were fixed with warmglutaraldehyde fixative (2.5% glutaraldehyde in 0.09 Mcacodylate buffer, pH 7.2, containing 3 mM calciumchloride), and incubated in 1% OsO4 solution for 2 h.Specimens were dehydrated (50–100% ethanol solution,15 min each), and 0.5% uranyl acetate and 1% phospho-tungstic acid was added to the 70% ethanol solution toenhance contrast. After polymerization and slicing,polyribosomal aggregation was studied using a ZEISS EM109 electron microscope. The state of ribosomal aggrega-tion was quantified by calculating an index of ribosomalaggregation (IRA). Cells were classified as follows:1 = normal aggregation; 0.5 = partial disaggregation;0 = complete disaggregation.

Presentation of data

Data are presented as means±SD (n = 4–8/group).Statistically significant differences between groups wereevaluated by analysis of variance (ANOVA), followed byFisher’s PLSD test to correct for multiple comparisons. Aprobability of 95% was considered to indicate a signifi-cant difference.

RESULTS

Exposing primary neuronal cells to Tg (an irreversibleinhibitor of endoplasmic reticulum (ER) Ca2+-ATPase)induced an almost complete suppression of proteinsynthesis immediately after addition of Tg (Fig. 1). Proteinsynthesis recovered substantially, to about 70 and 58% ofcontrol values, by 6 h and 24 h after Tg-treatment, whencells were cultured in a medium supplemented with 5%horse serum (Fig. 1). In the absence of serum, however,protein synthesis rates recovered to only about 42% (6 h)and 26% (24 h) of levels in control cells incubated inserum-containing medium (Fig. 1), indicating that therecovery process was significantly suppressed by omit-ting serum from the incubation medium. Interestingly,a 24 h incubation of control neurons in serum-freemedium also caused a significant suppression of proteinsynthesis to 57% of the respective serum-containing

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Fig. 1 Effect of transient Tg exposure on recovery of neuronalprotein synthesis. Experiments were performed with primaryneuronal cell cultures prepared from the cortex of embryonic ratbrains at days 16–18 of gestation, and held in tissue culture inserum-containing medium for 9 days before being used. Cells wereexposed to Tg (1 µM) or the Tg solvent DMSO (0.1%) for 30 min.Recovery of 6 h (A) or 24 h (B,C) was induced by washing off theTg and transferring cells to a recovery medium with or withoutsupplementary 5% horse serum. Additional experiments wereperformed to study the effect on protein synthesis of a secondexposure of cells to Tg (1 µM), 24 h after the first treatment (C).Data are presented as means ± SD (n=4–8 cultures/group).Statistically significant differences between experimental groupsand controls are indicated by: *** p<0.001; between Tg-exposedcells incubated in the presence or absence of serum; by c; p<0.001between Tg-exposed cells with or without 24 h of recovery; by f;p<0.001 (analysis of variance, followed by Fisher’s PLSD test tocorrect for multiple comparisons).

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control (Fig. 1); a significant reduction to about 70% ofcontrol was already observed 12 h after serum with-drawal (data not shown).

The recovery of protein synthesis after Tg exposure ofneurons could be brought about by three differentmechanisms: (1) renewed synthesis of pump protein (ERCa2+-ATPase); (2) activation of an alternative, novel mech-anism for the uptake of calcium ions into the ER and (3)uncoupling of protein synthesis from ER calcium home-ostasis. To establish which of these mechanisms is actu-ally involved in the recovery process, we first studiedwhether a second Tg exposure of neurons (24 h after thefirst exposure) was able to suppress protein synthesis asecond time. Incubating cells in a medium supplementedwith 1 µM Tg 24 h after the first exposure of cells to Tgdid not suppress protein synthesis a second time (Fig. 1),implying that ER Ca2+-ATPase was not re-synthesized.Similar results were obtained when cyclopiazonic acid, areversible inhibitor of ER Ca2+-ATPase, was used 24 hafter Tg exposure of cells (data not shown).

In the second set of experiments, we measured ERCa2+-ATPase (formation of the phosphorylated intermedi-ate) activity in control neurons and cells subjectedto Tg (1 µM for 30 min) and exposed again after 24 h of

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recovery (Fig. 2). In addition, experiments were per-formed with cells cultured both in the presence andabsence of 5% horse serum. Culturing neurons in serum-free medium was sufficient to reduce ER Ca2+-ATPase to56% of control (Fig. 2), indicating a close relationshipbetween ER Ca2+-ATPase activity and protein synthesis(Fig. 1). Exposing cells to Tg caused a complete and irre-versible inhibition of ER Ca2+-ATPase, irrespective ofwhether or not serum was present during the 24 h recov-ery period (Fig. 2). These results indicate clearly thatrecovery of protein synthesis after Tg exposure of cellswas not paralleled by a recovery of ER Ca2+-ATPase activ-ity.

In the third set of experiments, we evaluated the effectof Tg on cytoplasmic calcium activity ([Ca2+]i). In the con-trol state, [Ca2+]i rises on exposure of neurons to Tg, sincecalcium ions released spontaneously from the ER cannotbe taken up again. In control cells studied 24 h after a 30min exposure to the Tg solvent DMSO (0.1%), cytoplas-mic calcium activity rose markedly on depletion of ERcalcium pools by Tg (Fig. 3A). In contrast, when cells hadalready been exposed to 1 µM Tg 24 h earlier, the secondexposure to Tg did not induce a rise in [Ca2+]i (Fig. 3B),



Fig. 2 Inhibition of Ca2+-ATPase phosphorylated intermediate(E~P) formation following Tg treatment of cortical neurons inculture. The activity of SERCA pumps was evaluated by measuringthe formation of Tg-sensitive Ca2+-ATPase phosphorylatedintermediates. Microsomes were prepared from harvested cellsand the phosphorylation assay was carried out as described in theMaterials and Methods section. Following autoradiography, Ca+-ATPase phosphorylated intermediate was observed as a 100 kDaband in each lane. Cell treatment prior to harvesting and assay areindicated for each lane. The four lanes designated ‘-serum’represent those cells for which serum-free medium was substitutedfor the 24 h of the recovery period following the first treatment.Cells represented by four lanes designated ‘+serum’ weremaintained in the medium containing 5% horse serum during thecorresponding recovery period. The following treatments wereused: DMSO 0.1% for 30 min + recovery (‘control’ and ‘control +Tg’), 1 µM Tg for 30 min + recovery (‘Tg 24 h’), 1 µM Tg for 30 min+ recovery + 1 µM Tg for 30 min (‘Tg 24 h Tg’). Lanes marked‘control+Tg’ show the E ~ P formation observed when 100 nM Tgwas included in the phosphorylation assay.

Fig. 3 Tg-induced changes in cytoplasmic calcium activity. Toverify whether SERCA activity was restored 24 h after exposure ofcells to Tg the acute effect of Tg treatment on cytoplasmic calciumactivity ([Ca2+]i) was evaluated as an indicator of a release ofcalcium from the ER. Primary cortical neurons were exposed to1 µM Tg for 30 min followed by 24 h recovery in mediumsupplemented with 5% horse serum. Control cells were treatedwith the Tg solvent DMSO (0.1%) for 30 min followed by the samerecovery conditions. [Ca2+]i was evaluated by measuring thefluorescence of cells after loading with the fluorescent calciumindicator fura-2. In control cells (A), Tg induced a marked transientincrease in [Ca2+]i. On the other hand, in cells subjected to 30 minTg exposure and 24 h recovery in serum-containing medium, asecond exposure to Tg did not cause an increase in [Ca2+]i(B). Traces shown illustrate changes in calcium activity of individualcells.

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implying that the ER Ca2+-ATPase activity had indeedbeen completely blocked by the first Tg treatment.

The suppression of protein synthesis induced by deple-tion of ER calcium stores is characterized by a block oftranslation at the initiation process, as indicated by anincrease in the phosphorylation of the eukaryotic initia-tion factor 2α, and a disaggregation of polyribosomes[5,6]. We therefore studied whether neuronal ribosomesreaggregate within 24 h of exposure of cells to Tg (Fig. 4),i.e. at a time when protein synthesis has largely beenrestored. In control cells, ribosomes aggregated to formpolyribosomes (Fig. 4A) and the index of ribosomalaggregation (IRA, for definition see above) amounted to0.98. Immediately after exposure of cells to Tg, polyribo-somes were completely disaggregated (Fig. 4B) and IRAwas reduced to 0. Ribosomal aggregation recovered sub-stantially within 24 h of exposure of cells to Tg, whencells were cultured in serum-containing medium (Fig.4C), and IRA recovered to 0.48. Recovery of ribosomalaggregation was, however, clearly impaired when cellswere left to recover from Tg exposure in serum-freemedium (Fig. 4D), and IRA amounted to 0.03.

In the last series of experiments, we studied the effectsof treatments, known to deplete ER calcium stores, onneuronal protein synthesis 24 h after the first Tg treat-ment. Neurons were incubated in MEM supplementedwith Tg for 30 min, and then left to recover for 24 h in


medium supplemented with 5% horse serum. Cells werethen exposed either to the ryanodine receptor agonistscaffeine or 2-hydroxycarbazole or to calcium-free buffersupplemented with the extracellular calcium chelatorEGTA. The effect of the 2-hydroxycarbazole on ER cal-cium levels was evaluated in control cells by measuringthe increase in cytoplasmic calcium activity caused byTg-induced depletion of ER calcium stores with or with-out pretreating cells with 2-hydroxycarbazole (Fig. 5). Inuntreated cells, Tg exposure caused a marked rise in

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Fig. 4 Electron microscopic evaluation of Tg-induced changes in ribosomal aggregation. Primary cortical neurons isolated from embryonicrat brain at days 16–18 of gestation were cultured on polyethylenimine-coated polyester films. Eight days after isolation, cells were taken forexperiments. Cells were exposed to Tg (1 µM) for 30 min, followed by 24 h recovery in serum-free or serum-containing medium. At the endof the experiments, cells were fixed, and, after embedding, polymerization, and slicing of specimens, the state of polyribosomal aggregationwas evaluated using a ZEISS EM 109 electron microscope. In control cells (A) ribosomes aggregated to form polyribosomes. Immediatelyafter Tg exposure of cells (B), polyribosomes became completely disaggregated. After 24 h recovery from Tg treatment, ribosomalaggregation was largely restored when cells were cultured in serum-containing medium after Tg exposure (C), while reaggregation wasclearly suppressed in cells cultured in serum-free medium (D); magnification: × 48 000.

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cytoplasmic calcium activity (Fig. 5A). This response wasseverely suppressed after pre-exposing cells to 2-hydrox-ycarbazole (Fig. 5B), indicating that activation of theryanodine receptor by 2-hydroxycarbazole caused amarked depletion of ER calcium stores.

Treating cells with 2-hydroxycarbazole (100–500 µM)24 h after Tg exposure caused a concentration-depen-dent suppression of protein synthesis (Fig. 6A) and a sim-ilar relationship was obtained by treating cells withcaffeine (10–40 mM; Fig. 6B). Finally, cells were incu-bated in calcium-free medium supplemented with EGTA(1 mM). This treatment inhibited the protein synthesismeasured 24 h after severe exposure of cells to Tg(Fig. 6C). The blocking effect of EGTA on protein synthe-sis was partially reversed when, after preincubation withEGTA for 30 min, cells were transferred to calcium-con-taining medium during L-[4,5-3H]leucine incorporation(Fig. 6C). Taken together, these observations strongly

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suggest that the regulatory link between ER calciumhomeostasis and protein synthesis recovered substan-tially after exposure of cells to Tg, although the ERcalcium pump was permanently blocked.

DISCUSSION

In the physiological state, protein synthesis is controlledby the level of endoplasmic reticulum calcium stores [1].In the present study, neuronal ER calcium stores weredepleted by exposing cells to Tg, a procedure whichdepletes ER calcium stores completely by suppressingthe uptake mechanism. If protein synthesis is, indeed,controlled by ER calcium homeostasis, studying the rateof protein synthesis should enable us to draw conclu-sions about the levels of ER calcium stores under acuteexperimental conditions through which ER calciumhomeostasis can be manipulated. To date, it has still not



Fig. 5 Verification of the effect of 2-hydroxycarbazole on ER calcium homeostasis. The effect of an activation of the ryanodine receptor by2-hydroxycarbazole on ER calcium levels was evaluated by measuring changes in cytoplasmic calcium activity caused by Tg-induceddepletion of ER calcium stores. Cells were pre-loaded with fura-2 and Tg (1 µM)-induced changes in cytoplasmic calcium activity wereevaluated with or without previous exposure of cells to 2-hydroxycarbazole. In control cells, Tg exposure caused a marked rise incytoplasmic calcium activity (A). When cells were pre-treated with 2-hydroxycarbazole (500 µM) before being exposed to Tg, Tg-inducedrise in cytoplasmic calcium activity was suppressed indicating a marked 2-hydroxycarbazole-induced reduction of ER calcium levels (B).Traces shown illustrate changes in calcium activity of individual cells.

Fig. 6 Effects of agents known to deplete ER calcium stores, onprotein synthesis 24 h after Tg exposure of cells. Cells wereincubated in medium supplemented with 1 µM Tg or the Tg solventDMSO (0.1%), followed by 24 h recovery in serum-containingmedium. Cells then underwent one of three treatments: (A)exposure to 2-hydoxycarbazole (2HC); (B) exposure to caffeine(Ca), another agonist of the ER ryanodine receptor; (C) incubationin calcium-free medium supplemented with 1 mM EGTA.Treatments were performed for 30 min, followed by the addition of10 µCi/ml L-[4,5-3H]leucine to the medium in the presence of drugs,to evaluate their effect on protein synthesis. In addition, in theexperiments where a calcium-free medium supplemented withEGTA was used to draw calcium ions out of the ER compartment(C), L-[4,5-3H]leucine incorporation was also measured afterchanging the medium. Calcium ions were added and EGTAomitted, to study the effect of emptying and refilling ER calciumstores on the recovery of protein synthesis 24 h after Tg exposureof cells. Data are presented as means±SD (n=4–8 cultures/group).Statistically significant differences between these measurementsand those obtained under other conditions are indicated by: **:p<0.01 or ***: p>0.001 for the deviation from control values; (a):p<0.05 or (c): p<0.001 in relation to cells exposed to Tg 24 h afterthe first exposure; and (e): p<0.01 or (f): p<0.001 in relation to cellsexposed to calcium-free buffer 24 h after Tg exposure followed byincubation in calcium-containing buffer (analysis of variance,followed by Fisher’s PLSD test to correct for multiple comparisons)

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been clearly elucidated whether protein synthesis is con-trolled by whole ER calcium stores or by calcium storesof ER subcompartments such as those sensitive to anactivation of the IP3- or the ryanodine receptor [27,28].The observation that, in control neurons, protein syn-thesis was severely suppressed immediately after Tgexposure, indicates that ER Ca2+-ATPase (SERCA) wascompletely inhibited and that no other mechanisms werepresent to pump back calcium ions from the cytosol intothe ER. Although a depletion of neuronal Ca2+ stores byTg elevates the cytosolic-free Ca2+ (Fig. 3A), we have


shown recently [5] that it is the depletion, per se, ratherthan an increase in [Ca2+]i, that is responsible for the-inhibition of neuronal protein synthesis, as confirmed inthe present study by use of Ca2+ chelation (Fig. 6).

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After exposing cells to Tg, protein synthesis recoveredconsiderably within 24 h. Recovery of protein synthesisafter an irreversible inhibition of SERCA could bebrought about by three different mechanisms: (1) by arenewed synthesis of SERCA protein [29]; (2) by anuncoupling of the regulatory link between ER calciumhomeostasis and protein synthesis; and (3) by a recoveryof the regulatory link between ER calcium homeostasisand protein synthesis, even in the absence of SERCAactivity. After 24 h recovery from Tg exposure of cells norenewed synthesis of active SERCA protein was observedin the present study and protein synthesis responded toagents known to deplete ER calcium stores. The mostplausible explanation for the results of this study is,therefore, a restoration of the regulatory link betweenER calcium homeostasis and protein synthesis. Thisrecovery process may have been induced by the develop-ment of a new system for the uptake of calcium ionsinto that ER subcompartment which controls proteinsynthesis.

This is to our knowledge the first report investigatingthe mechanisms underlying the adaptive response to ERdysfunction activated in primary neurons (i.e. non-dividing cells). A different response has been observed inpermanent cell lines where Tg-resistant cells wereselected by exposure to increasing Tg concentrationsover a period of 10 months [18,30]. Tg resistance of thesecells was induced through several adaptive changes[19,31] including a mutation of the SERCA gene resultingin a substitution at amino acid 256 of SERCA [19]. Noneof these molecular processes can apply to the systemused in the present study, i.e. to the restoration of theregulatory link between ER calcium homeostasis andprotein synthesis after short-term Tg exposure of primaryneurons. In another series of experiments, an acute adap-tive response was observed in permanent cell linesexposed to Tg or other perturbants of ER function [16].However, these studies did not establish whether therecovery of protein synthesis (e.g. after Tg exposure ofcells) was caused by new synthesis of SERCA protein, byrestoration of the regulatory link between ER calciumhomeostatsis and protein synthesis, or by the develop-ment of a new system for the control of protein synthesis.

Under physiological conditions, calcium ions aresequestered within the ER by the action of SERCApumps, a family of Ca2+-ATPases highly sensitive toblockade by Tg [32,33,34]. Exposure of cells to Tg resultsin an irreversible inhibition of calcium accumulationwithin the ER [32,35,36]. The ER calcium activity isseveral orders of magnitude above that of the cytoplasm[37,38], and an active transport system is, therefore,required to pump back calcium ions against a steep con-centration gradient. The nature of the uptake systemresponsible for restoration of the ER Ca2+ pool after the

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exposure of cells to Tg has not yet been elucidated.Primary candidates for this role would have to includeTg-insensitive Ca2+-ATPases of non-mitochondrial Ca2+

stores. Occurrence of such enzymes has been noted inseveral systems (e.g. [18,32,39,40]), including the brain[41]. Indeed, the small residual amount of the phos-phorylated intermediate found in the presence of a max-imally inhibitory concentration of Tg (Fig 2) is consistentwith the presence of such novel Ca2+-ATPase in culturedcortical neurons. It should be noted that to explain thepresent data, one key requirement for any Ca2+ uptakesystem must be its ability to acquire an association denovo with ER Ca2+ stores: a Tg-insensitive Ca2+ uptakemechanism was evidently not contributing to store fillingbefore the first exposure to Tg, since this first exposurewas as effective in causing store depletion and inhibitionof protein synthesis as was the release of calcium ionsfrom the ER by activation of the ryanodine receptor withcaffeine (Fig. 6). It has been suggested that distinct Ca2+

store subcompartments may exist, characterized bydifferent types of Ca2+ pumps and ion channels[32,39,42]. It could be speculated that a high degree ofCa2+ depletion from ER, reported to cause majorrearrangements of ER structure [43,44], might lead to theestablishing of new luminal connections, thereby allow-ing a Tg-insensitive Ca2+-uptake system, normally limitedto a separate compartment, to assume a new role in load-ing of Ca2+ pools which were previously associated withSERCA pumps.

One interesting finding of the present study is theobserved effect of serum on SERCA activity. Culturingneurons in serum-free medium caused a marked sup-pression of both SERCA activity and protein synthesis.This implies that the decrease in SERCA activityobserved in cells cultured in serum-free medium hadalready caused disturbances of ER calcium homeostasiswhich then induced the suppression of protein synthesis.It is worth noting here that disturbances of ER calciumhomeostasis have been shown to induce apoptosis [45].This observation is difficult to understand at first,because disturbances of ER calcium homeostasis evoke asuppression of global protein synthesis (see above),whereas the pathological process of apoptosis is knownto be blocked by cycloheximide, a well-establishedinhibitor of protein synthesis. However, for the interpre-tation of the effect of cycloheximide on the developmentof apoptosis in different experimental models, it shouldbe considered that blocking of the apoptosic process bycycloheximide is not necessarily caused by a suppressionof protein synthesis. Indeed, cycloheximide, at concen-trations that only partially block protein synthesis, acti-vates the transcription and translation of stress genessuch as Bcl-2 [46], implying that the protective effect ofcycloheximide is brought about by activation of stress



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gene expression and not by suppression of protein syn-thesis.

Notably, in the absence of serum, recovery of rib-osomal aggregation and protein synthesis was signifi-cantly hindered after irreversible blocking of SERCAactivity by exposure of cells to Tg. The effect of serumwithdrawal on SERCA activity and protein synthesispoints to a role of growth factors in maintaining ER cal-cium homeostasis, and suggests that the ER acts as amediator of cell damage after interruption of growth-factor supplies.

The observation that both SERCA activity and proteinsynthesis are suppressed when primary neuronal cellsare placed in serum-free medium may significantly alterthe interpretation of results from cell-culture studies ofglutamate excitotoxicity. A frequently used experimentalmodel for these studies is transient exposure of primaryneuronal cell cultures to glutamate or glutamate ago-nists, followed by washing-off of the glutamate andrecovery in serum-free medium [47]. Serum is usuallyomitted because cell damage is quantified by measuringlactate dehydrogenase released from damaged cells intothe medium, an assay which is obscured by enzymeactivity present in the serum. Results of the present seriesand other experiments imply that in these in-vitromodels, designed to study glutamate toxicity, ‘controlcells’ are also brought into a pathological state by serumwithdrawal. Indeed, evidence has been presented indicat-ing that glutamate toxicity can be prevented completelyby addition of horse serum to the medium [48]. Ourinterpretation of the effect of serum on neuronal calciumhomeostasis and protein synthesis is corroborated by thefact that the experimental condition (serum withdrawal)used to study glutamate excitotoxicity, has recently beenused as a model for studying mechanisms of inducedapoptosis [49,50]. It must now be concluded that 24 hafter serum withdrawal neurons are pre-apoptotic, a statein which the functioning of the ER and protein synthesismachinery is already markedly impaired.

ACKNOWLEDGEMENTS

The excellent technical assistance of Änne Pribliczki andCordula Strecker is gratefully acknowledged. M.T. wassupported by the Danish Government BiotechnologyProgram.

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