Spectroscopic study of the effect of stress on chick synaptosomal membrane organization

TURINA A. V.; SCHREIER S.; PERILLO M.A.

San Sebastian-San Pablo

Workshop; 3rd International Workshop on Spectroscopy for Biology; 2010

Universidad de San Pablo- Universidad de Campinas-IUPAB

There is considerable evidence that stress induces changes in the number and/or the affinity ofbenzodiazepines receptors from synaptosomal membranes (SM). Results from our laboratorydemonstrated that GABAA-R from chick forebrain consistently exhibited an increase in receptordensity (Bmax) without changing the affinity, in animals submitted to several stressful situationssuch as a forced-swimming task (FST) [1]. The Bmax increase was associated to receptorrecruitment rather than synthesis or lower degradation rates (Martijena et al., 1992) whilethermodynamics of ligand binding and SM fatty acid composition suggested a decrease in membrane organization [2].In the present work, in SM from control chicks (Co) or from animals submitted to a FST (St) weinvestigated: a) [3H]-Flunitrazepam ([3H]-FNZ) binding to GABAA-R and b) the molecularorganization of SM by means of electron paramagnetic spectroscopy (EPR) using 0.15 mM doxylderivatives of stearic acid carrying the nitroxide moiety at C5 (5-SASL) and C12 (12-SASL), as well as by steady state fluorescence anisotropy analysis of 1,6-diphenilhexatriene (DPH) as a function of temperature.[3H]-FNZ binding showed an increase in Bmax values (Bmax,Co=1164±24 and Bmax,St=1789±72fmol/mg protein) with no statistically significant change in KD (KD,Co=2.18±0.15 nM andKD,St=2.6±0.2 nM).The FST induced a decrease in the order parameter (S) of 5-SASL spectra (SCo=0.757±0.003 and SSt=0.744 ± 0.007) while 12-SASL spectra showed a decrease of the empirical parameter h+1/h0 (h+1/h0,Co=0.422±0.004 and h+1/h0,St=0.412±0.001), indicating a more ordered/less mobile environment within the membrane core. Finally, the analysis of DPH anisotropy of fluorescence showed that the mean transition temperatue (Tm) of St membranes was higher (22ºC) than Tm,Co (19.5ºC).Taken together, our results indicate that the FST increased the stiffness in the deeper membraneregion as sensed by 12-SASL and DPH, which might be compatible with a topographicrearrangement or conformational change of membrane proteins, allowing for a recruitment ofreceptor sites. This might lead to an increased lipid packing, ordering the hydrocarbon chains, while an increase of membrane curvature could explain the order decrease at polar head region.[1]Martijena I.D. et al., Neural Transm Gen Sect. 87:97-104,1992.[2]García D. A. et al. Mol Membr Biol. 19:221-30, 2002.