HEAT-TREATMENT INDUCES AN INCREASE IN INTRACELLULAR CYCLIC-AMP CONTENT IN HUMAN EPIDERMOID-A-431 CELLS

The basal level of intracellular cyclic AMP (cAMP(i)) in A-431 cells incubated at 37-degrees-C in Na+-containing Hanks solution is 2086 +/- 139 fmol/10(6) cells. When cells are exposed to 45-degrees-C for 10 min, cAMP(i) increases by 40 +/- 4%, and then returns to basal levels within 30 min. Incubating cells in Ca2+-free or Mg2+-free Hanks solution has no effect on the heat-induced increase in cAMP(i), but the increase is inhibited by acid-loading cells to intracellular pH 7.0 or 6.8. In unheated cells, cAMP(i) increases by 16 +/- 8%, 53 +/- 7%, or 39 +/- 8% when incubated with 3-isobutyl-1-methylxanthine (1 mM), Ro 20-1724 (0.5 mM), or theophylline (1 mM) respectively. However, heat treatment further elevates cAMP(i) in cells treated with phosphodiesterase inhibitors, indicating that heat treatment and phosphodiesterase inhibitors elevate cAMP(i) by a different pathway(s). Heat treatment increases adenylate cyclase activity 2.5-fold. When forskolin (150-mu-M), an adenylate cyclase stimulator, is applied to cells, the basal cAMP(i) increases 28 +/- 6-fold compared with controls. Subsequent heating of these cells lowers cAMP(i) levels to 7.0 +/- 0.5 times that in control cells. This decrease is prevented by pretreatment with pertussis toxin (30 ng/ml, 24 h), suggesting that G-proteins are involved in the process of heat-induced cAMP(i) increase. 2-Deoxy-D-glucose (10 mM), NaN3 (10 mM) and 2,4-dinitrophenol (1 mM) also increase cAMP(i) in A-431 cells. However, application of these metabolic inhibitors to cells before heat treatment does not result in cAMP(i) levels greater than that observed in cells with heat alone. Similar observations are obtained in heat-treated cells previously exposed to adenosine, but not to AMP or ADP. These data are the first to suggest that thermally induced increase in cAMP(i) is due to a combination of activation of adenylate cyclase and G-proteins, and an increase in adenosine owing to ATP breakdown caused by hyperthermia.