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.

NMDA receptors in immune competent cells.

This review presents analysis of literature data indicating the presence of NMDA-type glutamate receptors in several types of immune competent cells such as thymocytes, lymphocytes, and neutrophils. The possible role of these receptors in the function of these cells is discussed. The interaction of the receptors with certain ligands circulating in the bloodstream and their role in modulation of immune function is described. It is suggested that homocysteine ​​acts as modulator of these receptors, and its toxicity is largely explained by hyperactivation of the NMDA-type glutamate receptors.

Effect of homocysteine on properties of neutrophils activated in vivo.

We have found that neutrophils begin to express NMDA receptors on their membranes after in vivo activation. These receptors are the target for action of homocysteine (HC). After incubation of activated neutrophils with HC, the degranulation process is stimulated and generation of reactive oxygen species is increased. We conclude that expression of NMDA receptors on neutrophil membrane makes neutrophils sensitive to HC. Thus, hyperhomocysteinemia may induce additional stimulation of immune competent cells.

Effect of homocysteine and homocysteic acid on glutamate receptors on rat lymphocytes.

Homocysteine and homocysteic acid increased the stationary level of reactive oxygen species in rat lymphocytes, homocysteic acid being more potent in this respect. The effect of this compound was realized via ionotropic NMDA receptors and group III metabotropic glutamate receptors. Incubation of lymphocytes with homocysteic acid increased intracellular Ca(2+)concentration, activated of protein kinase C, and induced accumulation of reactive oxygen species, which reflected the involvement of homocysteic acid into cell signaling mechanisms.