Determination Of The Lipid Membrane Composition Of J774 Macrophages Cells Surexpressing Mrp Protein (resistant To Ciprofloxacin)

Abstract

Ciprofloxacin (CIP) is a fluoroquinolone antibiotic with an activity towards both extracellular and intracellular bacteria (Seral et al., 2005). Diffusion and efflux processes modulate accumulation of this drug within eukaryotic cells. When J774 macrophages were grown in presence of ciprofloxacin, the antibiotic is subject to constitutive efflux through the activity of an MRP-related transporter (Michot et al., 2004).In view of the critical role of lipids for both drug uptake and activity of MRP proteins (Hinrichs et al, 2004), together with the ability of fluoroquinolones to interact with lipids (Bensikaddour et al., 2008 (a,b)), we investigated the composition of lipids in resistant and sensitive J 774 macrophages to ciprofloxacin.Firstly, we characterized by thin layer chromatography the phospholipids composition of J774 macrophages cells sensitive (WT) and resistant to ciprofloxacin (CIP). Results showed that sphingomyelin (SM) decreased 2 times whereas phosphatidylinositol increased 1.5 fold in resistant cells. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and cholesterol didn't show any significant change. Secondly, we studied membrane fluidity of liposomes mimicking WT and CIP resistant J774 cells, by fluorescence polarization spectroscopy. In this respect, we observed a decrease in the melting temperature in vesicles mimicking the membrane composition of CIP resistant cells, indicating that changes in the fluidity of these membranes may be due to the decrease of SM. Studies are currently performed to investigate potential changes in other components of rafts like glucosylceramides. These data might have important relevance to relate the interaction of fluoroquinolones with lipids and change in the processes involved in cellular accumulation of fluoroquinolones.