[Lipoilcarnosine: synthesis, study of physico-chemical and antioxidant properties, biological activity]. / Lipoilkarnozin: sintez, izuchenie fiziko-khimicheskikh i antioksidantnykh svoistv, biologicheskaia aktivnost'.

Synthesis of lipoilcarnosine (LipC) - a conjugated molecule based on two natural antioxidants, carnosine and a-lipoic acid, is described. Its physico-chemical, antioxidant properties and biological activity are characterized. According to reversed-phase HPLC with a UV detector, purity of the final product was 89.3%. The individuality of the obtained sodium salt of LipC was confirmed by tandem HPLC-mass spectrometry. High resistance of LipC to hydrolysis with serum carnosinase was demonstrated. The antioxidant activity of LipC measured by reaction with the formation of thiobarbituric acid reacting substances and kinetic parameters of iron-induced chemiluminescence was higher than that of carnosine and lipoic acid. LipC did not affect viability of SH-SY5Y human neuroblastoma culture cells, differentiated towards the dopaminergic type, at concentrations not exceeding 5 mM. At the concentration range of 0.1-0.25 mM LipC protected neuronal cells against 1-methyl-4-phenylpyridinium (MPP + )-induced toxicity.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Dipeptide carnosine (ß-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.