THE RESPONSE OF THE NEURONAL MEMBRANE TO ACETALDEHYDE TREATMENT
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1 CELLULAR & MOLECULAR BIOLOGY LETTERS Volume 6, (2001) pp 265 Ł 269 Received 13 May 2001 Accepted 28 May 2001 Short Communication THE RESPONSE OF THE NEURONAL MEMBRANE TO ACETALDEHYDE TREATMENT ANTON LIOPO 1, OLGA CHUMAKOVA 1, ILYA ZAVODNIK 1, AKSANA ANDREYEVA 1, MARIA BRYSZEWSKA 2 and SERGEY CHIZHIK 3 1 Institute of Biochemistry, Academy of Sciences of Belarus, BLK 50, Grodno Belarus, 2 Department of General Biophysics, University of ó odz, ó odz, Poland, 3 Metal-Polymer Research Institute, Academy of Sciences of Belarus, 32A Kirov St., Gomel , Belarus Abstract: To assess the effects of acetaldehyde (AA) on the native synaptosomal membranes, we used the atomic force microscopy (AFM) and fluorescent methods. A wide range of concentrations (from 2 to 4000 µm) of AA was studied. The visualization of synaptosomes by AFM showed structural changes in the synaptosomal surface at an AA concentration of 50 µm, which is comparable with the AA concentrations occurring during ethanol intoxication. In our study, we observed that AA at small concentrations (up to 50 µm) considerably decreased the microviscosity of the hydrophobic region of the bulk membrane lipids (the ratio of the monomeric and excimeric probe fluorescence decreased). These findings correlate with the AFM study on the effect of 50 µm AA on the washed synaptosome membranes. Key Words: Isolated Nerve Endings, Neuronal Membranes, Striatum INTODUCTION Acetaldehyde (AA) is formed from ethanol in, for example, the brain [1, 2]. The results of investigations on the role of AA in ethanol neurotoxicity are contradictory. The contradictions may be explained both by a high AA biological activity and the difficulties of the determination of AA level in situ [3]. Structural changes in the neuronal membrane may play an important role in acetaldehyde-induced brain toxicity [1, 4], and even low doses of AA may cause such changes. We used the atomic force microscopy (AFM) and
2 266 CELL. BIOL. MOL. LETT. Vol. 6. No. 2A fluorescent methods to investigate isolated nerve endings (synaptosomes) of rat striatum neuronal membrane surfaces, both untreated and after treatment with different AA concentrations. The aim of our study was to characterize neuronal membrane changes as a function of AA concentrations. MATERIALS AND METHODS Preparation of the synaptosomal fraction The synaptosomal fraction was isolated according to Weiler et al. (1981) [5] from the striatum of male albino rats of the Wistar line as described previously [6]. Biochemical control The isolated synaptosomal fraction was biochemically characterized by measuring the high-affinity choline uptake (HACU). HACU is a reliable presynaptic cholinergic marker. The procedure of HACU determination was carried out according to Simon and Kuhar [7] as described previously [6]. Incubation with acetaldehyde The samples containing a synaptosomal suspension were incubated for 20 min at 37 o C with AA at concentrations of 2, 20, 50, 200, 400, 2000, and 4000 µm in a 20 mm Na-phosphate buffer (ph 7.4) before the AFM- or fluorescent investigations. Atomic force microscopy Synaptosomal suspensions (4 µl) containing µg of protein in 0.1 ml of suspension were placed on a coverslips. AFM investigations were carried out with a NANOTOP-203 developed at the Metal-Polymer Research Institute of the National Academy of Sciences of Belarus [8]. The AFM measurements were conducted using the tapping mode. During scanning, the probe oscillation amplitude was maintained at a constant level. Fluorescent method The microviscosity of the hydrophobic region of the membrane lipid phase was measured [8]. The monomer (395 nm) to excimer (470 nm) fluorescence intensity ratio of pyrene (Sigma, Germany) incorporated into the lipid bilayer of the synaptosomal membrane was used. This ratio (I m /I ex ) was proportional to the probe environmental viscosity, and inversely proportional to its lateral mobility. Synaptosomal membranes (0.2 mg protein/ml) were suspended in sodiumphosphate buffer, ph 7.4 (22 o C) The pyrene concentration was 9.5 µm. The bulk lipid viscosity was measured by the direct excitation (319 nm) of the pyrene fluorescence using an Aminco-Bowman spectrofluorimeter (USA).
3 CELLULAR & MOLECULAR BIOLOGY LETTERS 267 RESULTS AND DISCUSSION The kinetic parameters of the HACU (K m, µm, and V max, nmol /mg protein min) were calculated by linearization of the dependence of the initial rate of the process on the concentration of the substrate (choline) in Lineweaver-Burk coordinates: K m was 0.53±0.03 µm; V max was 528.1±53.04 nmol choline/mg protein min, for the Na + - dependent process. These values snow a high cholinergic activity for the synaptosomal fraction. AA at different concentrations induced changes in the synaptosomal surface, detected by means of the AFM technique. The dose-dependent changes in the synaptosomal surface were observed in the presence of AA, beginning at 50 µm, when enlarged separate synaptosomes stuck together (Fig.1b). With increasing AA concentration up to 4000 µm, damage manifested itself as a substantial degradation of synaptosomes. In our study, we observed that AA at low concentrations (up to 50 µm) considerably decreased the microviscosity of the hydrophobic region of the bulk membrane lipids (the ratio of the monomeric and excimeric probe fluorescence decreased) (Fig. 2). At higher AA concentrations, no further alteration of membrane microviscosity was observed. These findings correlated with the AFM study on the effect of 50 µm AA on washed synaptosome membranes. The height of the cross-sectional profile of an untreated membrane film was approximately 2225 nm. 50 µm AA caused a clear flattening of the membrane film down to 911 nm. a b Fig. 1. AFM- imaging of striatal synaptosomes: a Ł untreated, b - after 20 min incubation at an acetaldehyde concentration of 50 µm.
4 268 CELL. BIOL. MOL. LETT. Vol. 6. No. 2A I m /I ex 2,60 2,55 2,50 2,45 2,40 2,35 2,30 2,25 2, Acetaldehyde [um] Fig. 2. The change in the microviscosity of the bulk lipid of the synaptosomal membrane in the presence of acetaldehyde (λ = 319 nm, 22 o C). I m /I ex - the ratio of monomer to excimer fluorescence intensity for pyrene. From our results we can conclude that the dose-dependent structural changes in the surface of both the native synaptosomes and washed synaptosomal membranes observed in the presence of AA began at 50 µm. Acknowledgment. Supported by Grant No B from the Belarus FFI. REFERENCES 1. Eysseric, H., Gonthier, B., Soubeyran, A., Bessard G, Saxod R, Barret L. Characterization of the production of acetaldehyde by astrocytes in culture after ethanol exposure. Alcohol. Clin. Exp. Res. 21 (1997) Zimatkin, S.M., Liopo, A.V. and Deitrich, R.A. Distribution and kinetics of ethanol metabolism in rat brain. Alcohol. Clin. Exp. Res. 22 (1998) Fukunaga, T., Sillanaukee, P. and Eriksson, C.J.P. Problems involved in the determination of endogenous acetaldehyde in human blood. Alcohol Alcohol. 28 (1993) Pratt, O.E., Rooprai, H.K., Shaw, G.K. and Thomson, A.D. The genesis of alcoholic brain tissue injury. Alcohol Alcohol. 25 (1990) Weiler, M.H., Gundersen, C.B. and Jenden, D.J. Choline uptake and acetylcholine synthesis in synaptosomes: investigations using two different labeled variants of choline. J. Neurochem. 32 (1981)
5 CELLULAR & MOLECULAR BIOLOGY LETTERS Chumakova, O.V. and Liopo, A.V. Acetylcholinesterase and choline uptake in striatum from rats with varying sleeping times. Alcohol Alcohol. 31 (1996) Simon, J.R. and Kuhar, M.J. High affinity choline uptake: ionic and energy requirements. J. Neurochem. 27 (1976) Chizhik, S.A., Gorbunov, V.V. and Dubravin, A.M. Experimental and computing complex based on STM/AFM NANOTOP -2. Proc. of Int. Conf. Computer Methods and Inverse Problems in Nondestructive Testing and Diagnostics. Minsk (1995) Gala, H.-J. and Sackmann, E. Lateral diffusion in the hydrophobic region of membranes: use of pyrene excimer as optical probes. Biochim. Biophys. Acta 339 (1974)
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