D-Erythro-Sphingosine Hydrochloride

Ceramides (Cers) have recently been identified as key signaling molecules that mediate biological functions such as cell growth, differentiation, senescence, apoptosis, and autophagy. However, the functions of Cer accumulation in necrotic cell death remain unknown. The aim of this study was to clarify the relationship between Cer accumulation with inhibition of the conversion pathway of Cer and concomitant necrotic cell death. In order to minimize the effect of apoptosis against necrotic cell death, A549 cells having the inhibiting effect of caspase 9 by survivin were used in this study. Consequently, Cer accumulation in A549 cells would likely be associated with a pathway other than the mitochondrial caspase-dependent pathway of apoptosis. Here, we showed that the dual addition of a glucosyl-Cer synthase inhibitor and a ceramidase inhibitor to A549 cell culture induced palmitoyl-Cer accumulation with Cer synthase 5 expression and necrotic cell death with lysosomal rupture together with leakage of cathepsin B/alkalization after 2-3 h, although it is unknown in this study whether the necrotic cell death was caused by the lysosomal rupture. This Cer accumulation was followed by a steep increase in sphinganine base levels via the activation of serine palmitoyltransferase activity brought about by the increase in palmitoyl-coenzyme A concentration as a substrate after 5-6 h. The increase in palmitoyl-coenzyme A concentration was achieved by activation of the fatty acid synthetic pathway from acetyl coenzyme A.

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca(2+) activated Cl(-) channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720-P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10-20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.