EFFECT OF HEAT-SHOCK, [CA-2+]I, AND CAMP ON INOSITOL TRISPHOSPHATE IN HUMAN EPIDERMOID A-431 CELLS

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 mug/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. These data are the first to suggest that the heat-induced increase in InsP3 results from both an increase in intracellular Ca2+ concentration and activation of G proteins, while the adenosine 3',5'-cyclic monophosphate-induced increase is due to a rise in intracellular Ca2+ alone. This increase in InsP3 is not related to heat-induced intracellular acidification.