Relation of neuronal endoplasmic reticulum calcium homeostasis to ribosomal aggregation and protein synthesis: implications for stress-induced suppression of protein synthesis.
Results from experiments performed with permanent non-neuronal cell lines suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concluded that disturbances in ER calcium homeostasis may contribute to the suppression of PS triggered by a severe metabolic stress (W. Paschen, Med. Hypoth., 47 (1996) 283-288). To elucidate how an emptying of ER calcium stores of these cells would effect PS and ribosomal aggregation of non-transformed fully differentiated cells, experiments were run on primary neuronal cell cultures. ER calcium stores were depleted by treating cells with thapsigargin (TG, a selective, irreversible inhibitor of ER Ca(2+)-ATPase), cyclopiazonic acid (CPA, a reversible inhibitor of ER Ca(2+)-ATPase), or caffeine (an agonist of ER ryanodine receptor). Changes in intracellular calcium activity were evaluated by fluorescence microscopy using fura-2-loaded cells. Protein synthesis was determined by measuring the incorporation of [3H]leucine into proteins. The degree of aggregation of ribosomes was evaluated by electron microscopy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused about 70% inhibition of PS, and PS recovered completely 60 min after treatment. Caffeine produced an inhibition of PS to about 50% of control. Loading cells with the calcium chelator BAPTA-AM (33.3 microM) alone suppressed PS without reversing TG- or caffeine-induced inhibition of PS, indicating that the suppression of PS was caused by a depletion of ER calcium stores and not by an increase in cytosolic calcium activity. TG-treatment of cells induced a complete disaggregation of polysomes which was not reversed within the 4 h recovery period following TG-treatment. After caffeine treatment of cells, we observed a heterogenous pattern of ribosomal aggregation: in some neurons ribosomes were almost completely aggregated while in other cells a significant portion of polyribosomes were disaggregated. The results indicate that a depletion of neuronal ER calcium stores disturbs protein synthesis in a similar way to the effects of transient forms of metabolic stress (ischemia, hypoglycemia or status epilepticus), thus implying that a disturbance in ER calcium homeostasis may contribute to the pathological process of stress-induced cell injury.
Doutheil
J
J
Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Cologne, Germany.
Gissel
C
C
Oschlies
U
U
Hossmann
K A
KA
Paschen
W
W
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology*; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009474:Neurons / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012270:Ribosomes / Q000187:drug effects / Q000502:physiology*; D013315:Stress, Psychological / Q000378:metabolism* / Q000503:physiopathology
D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium