ABSTRACT
Physical stresses such as high temperature or hyper- osmosis are known causes of intracellular ceramide (Cer) accumulation in mammalian epithelial cells; these stresses also result in the activation of the biosy- ntheses of glucosylceramide (GlcCer) or galactosyl- ceramide via ceramide glycosylation. We confirmed that intracellular Cer and GlcCer increased in mouse fibroblast Mop 8 cells under conditions of heat stress. When molecular species of Cer, GlcCer and sphingo- myelin (SM) were analyzed by matrix assisted laser desorption ionization time of flight mass spectrome- try (MALDI-TOF MS), the molecular ion peaks of Cer (d18:1 - C16:0, Na+) and Cer (d18:1 - C22:0, Na+) increased under heat stress compared with those of Cer (d18:1 - C24:1, Na+) and Cer (d18:1 - C24:0, Na+). GlcCer and SM demonstrated the wide spectra of fatty acyl chains compared with that of Cer. The ratio of GlcCer consisted of hydroxy fatty acid to that con- sisted of non-hydroxy fatty acid increased 2-5-fold in heat stressed cells. Cer metabolism-related genes, se- rine palmitoyltransferase (Spt), ceramide synthase-1, -2, -4, -5 and -6 (CerS1, -2, -4, -5 and -6), neutral sphingomyelinase-1 and -2 (nSMase1 and nS-Mase2), sphingomyelin synthase-1 (SgmS1), and ceramide glu- cosyltransferase (GlcT), were activated after 16 h un- der heat stress at 42?C. Activation of Sg-mS1 and GlcT genes played a role as Cer scavengers in the decrease of intracellular Cer levels. Activation of Cer- S5 and/or CerS6 gene may contribute to the accu- mulation of Cer species of (d18:1 - C16:0) under heat stress.