PATHWAYS OF GLYCOSPHINGOLIPID SYNTHESIS DURING MDCK CELL DIFFERENTIATION

PESCIO LUCILA; ROMERO DANIELA; STERIN SPEZIALE NORMA

Congreso; 56th International Conference on the Bioscience of Lipids (ICBL); 2015

Previously we reported that hypertonicity induces MDCK cell differentiation by a glycosphingolipid (GSL) ? dependent mechanism. GSL can be synthesized de novo or from hydrolysis of sphingolipids (salvage pathway), or by recycling from the endosomal pathway through the Golgi. In this study we evaluate the three pathways in the hypertonicity - induced MDCK cell differentiation. Confluent MDCK cells were subjected to hypertonicity for 24h or 48h or kept under isotonicity as control cultures. GSL metabolism was determined by using [14C]Galactose ([14C]Gal) and [14C]Palmitic Acid ([14C]PA) as precursors, in the presence or absence of sphingolipid metabolic inhibitors: L-cycloserine (CS), Fumonisin B1 (FB1) and Amitriptyline, as inhibitors of serine palmitoyltransferase, ceramide synthase and acid sphingomyelinase, respectively. Treatment with Amitriptyline showed an increase in the incorporation of [14C]Gal and [14C]PA to GlcCer either in control cells as in cells subjected to hypertonicity. Interestingly, the levels of [14C]Gal complex GSL decreased. Treatment with Amitriptyline + CS induced a decrease in labeled GSL. The susceptibility to CS was less evident in cells subjected to hypertonicity for 24h. Treatment with Amitriptyline + FB1 showed similar results than Amitriptyline + CS. We studied the MDCK cell differentiation by immunofluorescence and found that cells subjected to hypertonicity in the presence of Amitriptyline did not develop a mature apical membrane. These results could indicate that in the presence of Amitriptyline de novo synthesis of sphingolipids increased, but the pool generated is not the required to reverse the effect of the absence of GSLs synthetized by recycling, thus suggesting that GSLs necessary for the differentiation of the MDCK cells derive from endolysosomal degradation of SM by acid sphingomyelinase.