Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells

Abstract

The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC [high pressure liquid chromatography] methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells contained GM3, GM2, GM1, GD3 and GD1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GalNAc(.beta.1 .fwdarw. 4) Gal(.beta.1 .fwdarw. 4)Glc(.beta.1 .fwdarw. 1)Cer (GgOse3Cer) and GalNAc(.beta.1 .fwdarw. 3)Gal(.alpha.1 .fwdarw. 4) Gal(.beta.1 .fwdarw. 4) Glc(.beta.1 .fwdarw. 1)Cer (GbOse4Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mM-EGTA [ethyleneglycol bis(.beta.-aminoethyl ether)N,N,N'',N'',N.sbd.tetraacetic acid] showed a 2- to 3-fold increase in GM3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mM-EGTA plus 0.4 mM-EDTA a similar increase in GM3 was observed, but this change was accompanied by decreases in GM2, GM1, GgOse3Cer, and GbOse4Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca2+ sufficient to bind all chelator. Mn2+ replacement reversed the chelator effects differentially; GM2 and GM1 levels were the most sensitive to increases in Mn2+ concentration; GgOse3Cer and GbOse4Cer were also sensitive; GM3 was the least affected. Ca apparently serves an important regulatory role on GM3 levels, and Mn concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.