Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

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Abstract
The monovalent cationic ionophore monensin reduces the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induces a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. Further studies on the effects on monensin and comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts are presented here. Results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10-7 M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10-8 M. These effects of monensis were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides. The effects of the K+ ionophore valinomycin and the lysosome-disrupting cation NH4+ were different from those of ouabain and monensin and showed a general enhancement of incorporation of labeled galactose into all glycosphingolipids and no accumulation. These findings suggest that manipulation of ionic flux may variably regulate anabolism and, consequently, the composition of plasma membrane glycosphingolipids.