MODULATION BY SPHINGOLIPIDS OF CALCIUM SIGNALS EVOKED BY EPIDERMAL GROWTH-FACTOR

Receptor activated breakdown of complex sphingolipids has been propose d as a mechanism for generating sphingoid base-containing putative sec ond messenger molecules whose actions may modulate responses to extrac ellular signals, In human epidermoid carcinoma A431 cells, sphingosine (1-10 mu M) by itself had no effect on intracellular free calcium con centrations ([Ca2+](i)), yet within seconds, markedly enhanced the epi dermal growth factor (EGF)-evoked Ca2+ influx (by up to 2.fold), but f ailed to alter Ca2+ release from the intracellular stores, Ca2+ signal s evoked by serum were not affected by sphingosine. The response to sp hingosine was dose-dependent and saturable, exhibiting an EC(50) of 2. 3 mu M. In contrast, a ceramide, N-acetylsphingosine (10 mu M), sphing osine 1-phosphate (10 mu M) and sphingosylphosphorylcholine (10 mu M) inhibited EGF-evoked elevations in [Ca2+](i) The latter two compounds by themselves transiently increased [Ca2+](i). N-Octanoylsphingosine, N,N-dimethylsphingosine, sphingomyelin, and stearylamine were inactive . The potentiation of calcium signals by sphingosine occurred at all c oncentrations of EGF tested (0.15-15 nM) and did not alter the EGF rec eptor protein kinase activity as determined by antiphosphotyrosine imm unoblotting. Antiphosphoserine immunoblotting revealed that sphingosin e (10 mu M for 3 min) increased the phosphoserine content of two prote ins with approximate molecular masses of 40 and 70 kDa, Serine hyperph osphorylation of the 40-kDa protein was also observed in cells treated with EGF alone, whereas the intensity of the 70 kDa band was highest in cells treated with both sphingosine and EGF. The modulation of grow th factor receptor-regulated signaling, including changes in [Ca2+](i) may constitute a mechanism by which elevations in cellular levels of specific sphingolipids, which occur transiently upon activation of cer tain receptors and chronically in sphingolipid storage diseases, exert their physiological and pathophysiological effects.