M.N. Karpenko - Ph.D. (Biol.), Institute of Experimental Medicine (St.-Petersburg); Associate Professor, Department of «Biophysics», Peter the Great St.-Petersburg Polytechnic University (St.-Petersburg). E-mail: mnkarpenko@mail.ru N.S. Pestereva - Student, Department of «Biophysics», Peter the Great St.-Petersburg Polytechnic University (St.-Petersburg). E-mail: Nelvina@yandex.ru I.S. Pestereva - Student, Department of «Biophysics», Peter the Great St.-Petersburg Polytechnic University (St.-Petersburg). E-mail: Irixanchik@mail.ru V.A. Schukina - Post-graduate Student, Institute of Experimental Medicine (St.-Petersburg). E-mail: sch_viktoriya@mail.ru

Calpains - calcium-dependent intracellular proteases, which modulate a number of physiological functions including synaptic transmission. Current study analyses relation between the level of calpain activation and the level of dopamine secretion. It have been shown that incubation of extracted nerve endings - synaptosomes with 1mM EDTA, 50 uM calpain inhibitor I (N-Ac-Leu-Leu-norleucinal) or 50 uM calpain inhibitor II (N-Ac-Leu-Leu-methioninal) leads to a decrease in the activity of synaptosomal m-calpain, while application of 1mM СаСl2 leaded to the activation of m-calpain. Furthermore, introduction of both these inhibitors in combination with 1mM СаСl2 also resulted in the decrease of calpain activity. The level of dopamine in the current samples outside the synptosomes, analysed with a means of high-performance liquid chromatography with electrochemical detector, also differs. Addition of 1mM EDTA leaded to a decrease (р=0,03), and 1 mM СаСl2 - increase (р=0,02) of the dopamine level in the extrasynaptosomal domain in comparison with the sample without additives. Samples preincubated with synthetic calpain inhibitors showed increase in the level of extrasynaptosomal dopamine in comparisom with control without additives (р=0,03 and р=0,04 for calpain inhibitors I and II correspondingly), but did not reach the level of positive control (р=0,04 for both inhibitors in comparison with addition of 1mM СаСl2). The effect of inhibitors was similar in presence of calcium ions: simultaneous addition 1mM СаСl2 and 50uM of corresponding inhibitor resulted in an increase of dopamine level in non-synaptosomal liquid in comparison with a sample having only 1mM CaCl added (р=0,02 и р=0,03 for calpain inhibitors I and II correspondingly). Most probable reason for the level of extrasynaptosomal dopamine to be increased in the condition of calpain suppression can be connected to its ability to cleave intracellular domain of DAT (dopamine transporter). As a result of such proteolysis transporter looses its activity, which leads to the decrease of the extracellular dopamine. The work was supported by the RFFI grant № 14-04-00587.

 

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