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 C. 20 min then returned to baseline within 15 min in InsP3 was not observed in the absence of extracellular Ca2 or with amiloride treatment. Treatment with the nonhydrolyzable GTP analog GppNHp stimulated that component of the InsP3 increase due to G proteins. U-73122, an inhibitor of PL-C-mediated processes blocked the increase in InsP3 resulting from heat exposure. Both pertussis toxin 30 ngmL, 24 h, an inhibitor of G inhibitory protein, and cholera toxin I microgrammL, 1 h, a stimulator of G-stimulatory protein, increased InsP3 in unheated cells, and heating failed to induce a further increase, suggesting heat activates G proteins. Likewise, 8-Br-cAMP, IBMX, Ro 20-1724, or forskolin increased InsP3 in unheated cells, and heat did not cause an additional increase. The InsP3 increase induced by 8-Br-cAMP 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 cAMP- induced increase is due to a rise in intracellular Ca2 alone.