lactosyl-lysosphingolipid and sphingosyl-beta-glucoside

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

Topics: Animals; Cations; Chromatography; Chromatography, Ion Exchange; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Gaucher Disease; Leukodystrophy, Globoid Cell; Lipid Metabolism; Lipids; Mice; Neutral Glycosphingolipids; Psychosine; Sphingosine

Recently, we demonstrated that the GSL (glycosphingolipid), GlcCer (glucosylceramide), modulates Ca2+ release from intracellular stores and from microsomes by sensitizing the RyaR (ryanodine receptor), a major Ca2+-release channel of the endoplasmic reticulum, whereas the lyso derivative of GlcCer, namely GlcSph (glucosylsphingosine), induced Ca2+ release via a mechanism independent of the RyaR [Lloyd-Evans, Pelled, Riebeling, Bodennec, de-Morgan, Waller, Schiffmann and Futerman (2003) J. Biol. Chem. 278, 23594-23599]. We now systematically examine the mechanism by which GlcSph and other lyso-GSLs modulate Ca2+ mobilization from rat brain cortical and cerebellar microsomes. GlcSph, lactosylsphingosine and galactosylsphingosine all mobilized Ca2+, but at significantly higher concentrations than those required for GlcCer-mediated sensitization of the RyaR. GlcSph-induced Ca2+ mobilization was partially blocked by heparin, an inhibitor of the Ins(1,4,5) P3 receptor, and also partially blocked by thapsigargin or ADP, inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), but completely blocked when both acted together. In contrast, neither lactosylsphingosine nor galactosylsphingosine had any effect on Ca2+ release via either the Ins(1,4,5) P3 receptor or SERCA, but acted as agonists of the RyaR. Finally, and surprisingly, all three lyso-GSLs reversed inhibition of SERCA by thapsigargin. We conclude that different lyso-GSLs modulate Ca2+ mobilization via different mechanisms, and discuss the relevance of these findings to the GSL storage diseases in which lyso-GSLs accumulate.

Topics: Animals; Brain; Calcium; Calcium-Transporting ATPases; Glycosphingolipids; Inositol Phosphates; Microsomes; Models, Biological; Psychosine; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sphingosine; Thapsigargin