Vol 10, No 4 (2016)

P-glycoprotein/ABCB1 (Pgp) is a well known protein of cell defense system. It is localized in cell membrane and pumps different drugs out of various cells using ATP energy. Its overexpression is associated with the development of multidrug resistance (MDR) in cancer cells. The data showing that Pgp also has other functions appeared recently, and this review surveys these data. In particular, (1) Pgp can protect cells from apoptosis; it suppresses the expression of endogenous protein TRAIL and decreases the activity of caspases 8 and 3; (2) Pgp is able to act as an outwardly directed flippase; (3) Pgp participates in a proper development of the innate immune response to intracellular pathogens and in the development of inflammation; (4) functionally active Pgp can be transferred from drug-resistant to drug-sensitive cells by microvesicles (MV). This is a new way of the Pgp-mediated MDR emergence in populations of tumor cells. Thus, Pgp functions as a regulator of some cellular processes. Molecular mechanisms of the Pgp influence on tumor cell viability are related not only with the drug efflux but also with some other functions.

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K_d = 1.85 ± 0.16 nM, B_max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K_d = 1.57 ± 0.27 nM, B_max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K_d = 1.36 ± 0.03 nM) and 84% (B_max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K_d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B_max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K_d1 = 1.13 ± 0.09, K_d2 = 6.07 ± 1.06 nM, B_m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K_d1 = 0.61 ± 0.02, K_d2 = 3.41 ± 0.13 nM, B_m1 = 1.88 ± 0.028, B_m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B_max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.

The use of luteinizing hormone (LH) and its structural and functional homolog human chorionic gonadotropin (hCG) leads to hyperstimulation of LH-dependent signalling pathways and to desensitization of LH receptors. Therefore, the development of low molecular weight agonists of the LH receptor without these disadvantages has been carried out in recent years. These agonists, unlike gonadotropins, are active when administered orally. The greatest prospects are associated with the development of the thienopyrimidine derivatives structurally similar to compound Org 43553. The purpose of this work was the synthesis of new thienopyrimidine derivatives TP03 and TP04, the study of the regulation of LH-sensitive adenylyl cyclase signalling system in rat testes, and the evaluation of the ability of TP03 and TP04 to stimulate testosterone synthesis in male rats at different routes of administration. In the concentration range 10^–7–10^–3 M, TP03 and TP04 induced an increase of the basal adenylyl cyclase (AC) activity in rat testicular membranes with the EC₅₀ values of 390 and 759 nM, respectively. At a concentration of 10^–4 M, AC-stimulating effects of TP03 and TP04 were 213 and 122%, respectively, which indicates a higher activity of TP03 under in vitro conditions. At the same time, the AC-stimulating effect of TP03 was approximately 2.5-fold weaker than that of hCG (10^–8 M). Upon simultaneous administration of hCG and thienopyrimidine derivatives, their stimulating effects on the AC activity were additive due to the different localization of their binding sites in the LH receptor. Compound TP03 (25 mg/kg), when administered intraperitoneally to male rats, was more efficient than TP04: the maximal increase in the testosterone level (3 h after administration) was by 60% higher than that induced by TP04. TP03 increased the testosterone level after oral administration (50 mg/kg) as well, but the effect was weaker than in the case of the intraperitoneal injection. Orally administered TP04 exhibited a low activity. The results suggest that 5-amino-N-tert-butyl-2-(methylsulfonyl)-4-(3-(nicotinamide)phenyl) thieno[2,3-d]pyrimidine-6-carboxamide (TP03) can be used for the development of the drugs that stimulate steroidogenesis in Leydig cells.

In order to study the structural and functional specificity of different fragments of extracellular domains of the membrane receptors, their immunological blockade and analysis of changes in brain function in health and disease were carried out. Effects of the immunization against unstructured fragments of the neurotrophin receptor (p75 NTR) containing binding sites for a nerve growth factor, amyloid-beta, and proneurotrophin were studied. It was shown that immunization of NMRI mice with synthetic peptides with amino-acid residue sequences corresponding to the p75 NTR fragments 86–93 and 146–153 exerted an adverse effect both on the morpho-functional state of the cortical and hippocampal neurons and on the spatial memory of animals. In contrast, the immunization by the p75 NTR synthetic fragment 167–176 was accompanied by an improvement of these characteristics. The results of the structural-functional mapping of the extracellular domain in neuronal membrane receptors in vivo were in line with the published data of the X-ray analysis. The immunological approach developed in this work provides insight into the peculiarities of the receptor functioning in the course of the Alzheimer’s type neurodegeneration process and favors the development of effective immunological ways for selective therapy of the Alzheimer’s disease.

The distribution of initiation factor eIF3 in the cytoplasm of peripheral blood neutrophils in Alzheimer disease (AD) was investigated. Using a specially developed image analysis protocol, we found a significant increase in the heterogeneity of the eIF3 distribution in AD neutrophils as compared with control cells. We also obtained an evidence that the increase in the eIF3 heterogeneity in AD neutrophils is associated with the formation of stress granules–non-membrane structures that emerge in the cytoplasm of eukaryotic cells in response to the environmental stress.

Here we show that in primary culture of rat cortical neurons the number of episodes of epileptiform curents (EC) provoked by extracellular magnesium removal increases over time. We demonstrate that NMDA receptor agonists in low concentrations induce an elevation of frequency of miniature postsynaptic currents followed by their synchronization resulting in EC. Ifenprodil did not block EC but strongly inhibited NMDA-evoked whole-cell currents, which say for a key role of the ifenprodil-resistant synaptic GluN2A-containing NMDA receptors in the generation of EC. We suppose that in cultured neurons the onset of EC and a gradual increase of the EC amplitude over the time of culture period is directed by an increase of synaptic connections density and displacement of the GluN2B subunit by GluN2A in synapses.