Meta-Cresol Affects Lipid Raft Organization in Membrane-Model Systems and Increases Membrane Leakage in Neural Cells

Abstract

m-cresol is an excipient stabilizer used in numerous pharmaceutical formulations, including injectable insulin and vaccines. Therefore, we studied the effects of m-cresol in a range of concentrations from 10nM to 3mM on membrane model systems mimicking lipid-rafts and living neural-cells. First, the intrinsic fluorescence of m-cresol was studied. Both its fluorescence lifetime and anisotropy increased in the presence of liposomes, indicating a decreased mobility of the molecule. This interaction was dependent on membrane lipid composition. To elucidate this process, liposomes were labeled with several membrane probes spanning a range of in-depth locations and with preference for distinct lipid domains. For the probes located in the bilayer core (DPH and trans-parinaric acid), no effect was detected even for an m-cresol concentration of 300M, whereas for the more superficial NBD-DOPE and NBD-DPPE, >30M m-cresol induced a significant fluorescence lifetime decrease. Atomic force microscopy experiments were performed on ternary supported lipid bilayers containing raft-like liquid ordered domains (Lo). Indeed, it was observed that upon addition of m-cresol in the M range, a reduction of the Lo occurs without changing their thickness. For higher m-cresol concentrations, raft-like domains are not detected at all. Whole-cell voltage-clamp recordings from pyramidal-neurons isolated from the CA1 region of rat hippocampus (p21-p29) and from N1E-115 neuroblastoma cells were also performed. m-Cresol was applied during constant superfusion and the following parameters were monitored: series-resistance, whole-cell capacitance, holding-current (Vm=-70 mV), and another read-out for the leak-current. Results show that only the leak current was altered by m-cresol (>100 M). As a whole, we show that m-cresol interacts with the membrane, affecting lipid raft organization, with functional implications on neural-cell integrity. We thank F.C.T. Portugal for financial support (Ciência2007, SFRH/BD/64442/2009, PEst-OE/QUI/UI0612/2011).