Depletion of Neuronal Endoplasmic Reticulum Calcium Stores by Thapsigargin: Effect on Protein Synthesis

Abstract

We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+‐ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 µM caused a permanent inhibition of protein synthesis in hippocampal slices from 3‐week‐old rats but no inhibition in slices prepared from 2‐month‐old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3‐week‐ and 2‐month‐old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24‐h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with ∼5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats

“…Finally, in addition to acting as a Ca 2ϩ buffering organelle, the ER can also release stored Ca 2ϩ , contributing to the overall NMDA-⌬Ca 2ϩ waveform (12). Prolonged blockade of the SERCa with TG, as performed in our study, also leads to the depletion of ER Ca 2ϩ store (45). Thus our results showing that TG resulted in an enhancement, rather than a reduction, in the NMDA-⌬Ca 2ϩ signal indicate that Ca 2ϩ release from the ER does not contribute to the ⌬Ca 2ϩ signals following NMDA receptor activation in MNCs.…”

Mutant Huntingtin Expression Induces Mitochondrial Calcium Handling Defects in Clonal Striatal Cells

“…Because ER contamination of the mitochondrial preparation was a possibility, we confirmed the mitochondrial nature of the Ca 2ϩ uptake in our mitochondrial preparations, as the addition of uncoupler (FCCP) caused release of Ca 2ϩ , and pretreatment of the cells with thapsigargin (which blocks the Ca 2ϩ uptake pump of the ER Ref. 31) did not produce any change in the Ca 2ϩ uptake capacity (Fig. 2D).…”

Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

“…Primary neuronal cell cultures were prepared from cortices of embryonic rat brains at days 15-17 of gestation, essentially as described (17). Cells were kept in MEM supplemented with 5% horse serum, 30 mM glucose, 2 mM glutamine, 10 units͞ml penicillin, and 10 ng͞ml streptomycin for 10 days before being used for experiments.…”