Iron/ascorbate-induced lipid peroxidation changes membrane fluidity and muscarinic cholinergic receptor binding in rat frontal cortex

Abstract

Lipid peroxidation (LP) is a complex process which involves the formation of lipid free radicals and leads to oxidative damage. LP has also been implicated in several neurodegenerative diseases as well as aging. In the present study, we evaluated the effects of the induction of LP in vitro on muscarinic cholinergic (Mch) receptor binding and membrane fluidity in rat brain. Membranes from the rat frontal cortex were peroxidized by adding ferrous sulphate (84 microM) and ascorbic acid (400 microM). Peroxidation was measured as the amount of thiobarbituric acid reactive products formed (nmol malondialdehyde/mg protein). Mch receptor binding was measured 10, 20 and 30 min after peroxidation. Membrane fluidity was evaluated by fluorescence polarization studies using two probes; 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4(trimethylamino)phenyl]-1,3,5-hexatriene (TMA-DPH). Significant alterations in Mch receptor binding (decreased Bmax and increased Kd) were found after peroxidation. Membrane fluidity was also significantly decreased after peroxidation as observed with both probes. The decrease in membrane fluidity was due to an increased cholesterol to phospholipid molar ratio after peroxidation. These data suggest that lipid peroxidation induces changes in membrane dynamics as detected by the fluorescent probes and such changes in membrane microviscosity may be the cause for alterations in Mch receptor kinetics.