inositol-1-4-5-trisphosphate and Shock

The effect of heat shock on agonist-stimulated intracellular Ca2+ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma x glioma hybrid cells (NG 108-15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5 degrees C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca2+]i rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca2+]i rise was abolished and the [Ca2+]i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca(2+)-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP3) production was not altered by heat shock at 12 h after heat shock, whereas IP3 receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP3 receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IP3-mediated intracellular Ca2+ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca2+]i rise.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channels; Glioma; Heat-Shock Proteins; Hot Temperature; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Mice; Neuroblastoma; Oligonucleotide Probes; Quercetin; Rats; Receptors, Cytoplasmic and Nuclear; Shock; Tumor Cells, Cultured

The basal levels of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3) in A-431 cells incubated in Na(+)-Hanks' solution were, respectively, 1.23 +/- 0.18, 0.17 +/- 0.03, and 0.69 +/- 0.07% of the total radioactivity in the cell. When cells were heated, InsP3 increased in a temperature-dependent manner related to the duration of heating. The active form of InsP3, inositol 1,4,5-trisphosphate, increased 237 +/- 17% after heating (45 degrees C, 20 min) then returned to baseline within 15 min after the return to 37 degrees C. The heat-induced increase in InsP3 was not observed in the absence of extracellular Ca2+ or with amiloride treatment. Treatment with the nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate stimulated that component of the InsP3 increase due to G proteins. U-73122, an inhibitor of phospholipase C-mediated processes, blocked the increase in InsP3 resulting from heat exposure. Both pertussis toxin (30 ng/ml, 24 h), an inhibitor of G inhibitory protein, and cholera toxin (1 microgram/ml, 1 h), a stimulator of G stimulatory protein, increased InsP3 in unheated cells, and heating failed to induce a further increase, suggesting that heat activates G proteins. Likewise, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 3-isobutyl-1-methylxanthine, Ro 20-1724, or forskolin increased InsP3 in unheated cells, and heat did not cause an additional increase. The InsP3 increase induced by 8-BrcAMP was inhibited by removal of extracellular Ca2+ or treatment with verapamil, suggesting that an influx of extracellular Ca2+ stimulates InsP3 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acids; Calcium; Carcinoma, Squamous Cell; Cyclic AMP; GTP-Binding Proteins; Hot Temperature; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Osmolar Concentration; Shock; Tumor Cells, Cultured; Type C Phospholipases