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 li nes suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concl uded 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 aggregatio n 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 inhibi tor of ER Ca2+-ATPase), cyclopiazonic acid (CPA, a reversible inhibito r of ER Ca2+-ATPase), or caffeine (an agonist of ER ryanodine receptor ). Changes in intracellular calcium activity were evaluated by fluores cence microscopy using fura-2-loaded cells. Protein synthesis was dete rmined by measuring the incorporation of [H-3]leucine into proteins. T he degree of aggregation of ribosomes was evaluated by electron micros copy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused a bout 70% inhibition of PS, and PS recovered completely 60 min after tr eatment. Caffeine produced an inhibition of PS to about 50% of control . Loading cells with the calcium chelator BAPTA-AM (33.3 mu M) alone s uppressed PS without reversing TG- or caffeine-induced inhibition of P S, 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-tr eatment. After caffeine treatment of cells, we observed a heterogeneou s pattern of ribosomal aggregation: in some neurons ribosomes were alm ost completely aggregated while in other cells a significant portion o f polyribosomes were disaggregated. The results indicate that a deplet ion of neuronal ER calcium stores disturbs protein synthesis in a simi lar way to the effects of transient forms of metabolic stress (ischemi a, hypoglycemia or status epilepticus), thus implying that a disturban ce in ER calcium homeostasis may contribute to the pathological proces s of stress-induced cell injury. (C) 1997 Elsevier Science B.V.