CC BY
359th multidisciplinary international
Conference of Biological Psychiatry
«Stress and Behavior»
Proceedings of the 9th International Multidisciplinary Conference «Stress and behavior» Saint-Petersburg, Russia, 16-19 May 2005 Editor: Allan V. Kalueff, PhD
CONFERENCE ABSTRACTS 5. PSYCHONEUROIMMUNOLOGY
SIMULATION OF STRESS RESPONSE IN ANIMALS BY THE DELIVERY OF HEAT SHOCK PROTEIN 70 kDa
L.I. Andreeva, I.V. Guzhova, B.A. Margulis, D.V. Nikiforova, A.A. Lebedev, E.E. Voevodin, P.D. Shabanov Military Medical Academy, Institute of Cytology RAS, Saint-Petersburg, Russia Heat shock proteins (Hsps) are thought to exert two major functions: proper folding of cellular protein (chaperonic activity) and cytoprotection. Unless known as almost exclusively intracellular protein Hsp70 was shown to persist in extracellular locales and biological fluids of an organism, for example, in blood (Febbraio M.A. et al., 2002) and in saliva (Fabian T.K. et al., 2003). It was shown that the appearance of Hsp70 in circulation in most cases is due to its export from living cells, rather than to the release from dying cells (Hunter-Lavin C. et al., 2004). Extracellular Hsp70 was found also to enter macrophages or endothelial cells and into other cells, for example brain cells. The quantity of Hsp70 in blood increases after an action of different stressors such as heating, exercises. The main possible site of Hsp70 internalization and externalization is cellular membrane lipid rafts, which are sphingolipid-cholesterol-rich structures (Broquet A.H. et al., 2003). Besides the latter Hsp70 can associate with some cellular membrane receptors such as toll-like receptors, CD14 (Asea A. et al., 2000), CD40 (Becker T. et al., 2002) and CD91 (Noessner E. et al., 2002). Basing on the data obtained in vitro models we suggest that the main function of exogenous internalized Hsp70 is to protect cells from damaging factors (Tytell M. et al, 1986, Guzhova I.V. et al., 2001). It is also worth-mentioning that Hsp70 after its entry to a cell can modulate the activity of certain cellular proteins (Guzhova I.V. et al., 1997), receptors, and to influence the expression of some genes whose products take part in stress reaction and determine inflammatory status of an organism. The goal of this investigation was to simulate stress response of the whole organism by the delivery of Hsp70 preparation in a brain and to explore how this delivery can influence on key functions of control or subjected to stressful conditions animals.
Methods. The preparation of Hsp70 was obtained from the bovine muscle and was consisted of two isoforms: Hsp70 and Hsc70. Hsp70 preparation was injected into normal and stressed males of unbred and Wistar rats in cisterna magna (up to 10 ug), central nucleus of amygdala (up to 1 ug), and in both nostrils (10 ug). To reveal the possible sites of Hsp70 localization the protein was labeled with NHS-ester-Alexa Fluor (Molecular Probes, USA). Control and stressed rats were behaviourally tested. Biological samples were analyzed using biochemical and morphological methods.
Results and discussion. First experiments revealed that Hsp70 preparation exerted dose-dependent suppressive effect on exploratory and motor activity of rats, which resembles some stress situation when depression of behavior activity takes place (Andreeva L.I. et al., 2004). Simultaneously, emotionality of animals was found to elevate in rats accepted Hsp70. After the protein (0.2 and 0.5 ug) delivery into cisterna magna the behavior activity in the open field test was not altered even there was a tendency to some activation. When doses of the protein were elevated up to 1 — 10 ug, dose-dependent inhibitory effect was observed. Hsp70 in a maximal doses caused increased emotionality of rats. High doses of Hsp70 increased in some extent the content of corticosteron in serum 4 h after injections (514 ± 125 nM saline; 656 ± 104 and 614 ± 93 nM, 3 and 10 ug of Hsp70 respectively). Repeated intranasal HSP70 administration (once a day, 3 days) also inhibited motor and exploratory activity compared with controls. Corticosteron content in sera of 43% rats received Hsp70 was significantly lower than in control group (saline) indicating that anti-stress effect of Hsp70 was profound in a half of animals. This effect was also found under double Hsp70
Psychopharmacol. Biol. Narcol. 2005. Vol. 5, N 2. P. 908-909
Psyhopharmacology & biological narcology
ISSN 1606-8181
intranasal administration following both psycho-emotional and physical stressors representing «terrorist-hostage» model and characterized by hyper-excitability of animals. The behavior testing showed that the Hsp70 administration led to restriction of excitation after the end of stress treatment. Biochemical analysis demonstrated the decreasing of calcium concentration, urea, creatinine, some intracellular enzymes in serum after applying of exogenous Hsp70. The tissue content of inducible isoform of Hsp70 (stress marker) in brain and in liver of stressed rats was also lower as compared with control stressed animals (saline). The results of experiments with injection of Hsp70 into central nucleus of amigdala («center of emotions») showed that Hsp70 (0.5 and 1 ug) was able to activate self-stimulation in rats. On the contrary, when the protein was applied to rats which were subjected to the social isolation and alcogolization the protein was found to suppress self-stimulation. Preliminary data on localization of exogenously delivered Hsp70 showed that fluorescent label localized to sites proximal to ependimal cells, vessel endothelial cells and to Purkinje cells of cerebellum.
Conclusion. Simulation of stress response in a whole organism by Hsp70 administration (a transition from norm to stress) revealed a dose-dependent effect of the protein from slight activation to growing depression of motor and exploratory behavior. Increased emotionality after Hsp70 administration may be explained by interactions of Hsp70 with limbic structures. Hsp70 plays an important role in development of stress reaction. Alterations of motor activity maybe associated with internalization of Hsp70 by motor neurons (Purkinje cells). The study was supported by the Russian Foundation for Basic Research (project № 03-03-33024).
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Psychopharmacol. Biol. Narcol. 2005. Vol. 5, N 2. P. 908-909
Psyhopharmacology & biological narcology
ISSN 1606-8181
