Enhancement of ganglioside GM3 synthesis in okadaic-acid-treated granulosa cells

Abstract

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphlrylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAc α2 → 3Gal β1 → 4Glc β1 → 1Cer (GM3) and Gal β1 → 3GalNAc β1 → 4[NeuAc α2 → 3]Gal β1 → 4Glc β1 → 1Cer(GM1), demonstrated by metabolic labeling of glycosphingolipids with [ 3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12- O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.