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 µ M 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 m M 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 n M 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.