The effect of amixin and agmatine on cytochrome C release from isolated mitochondria

Mitochondrial nicotinic acetylcholine receptors (nAChRs) control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in efficiency of therapeutic strategies against post-ischemic pathology.

Intracellular localization of nicotinic acetylcholine receptors in human cell lines.

AIMS: Previously we demonstrated that mouse liver mitochondria express functional α7 nicotinic acetylcholine receptors (nAChRs). The aim of this study was to investigate whether the nAChRs are found in mitochondria of non-neuronal human cell lines. MAIN METHODS: Three cell lines: U373 (astrocytes), U937 (monocytes) and Daudi (B lymphocytes) were examined by flow cytometry, Cell ELISA and fluorescent confocal microscopy using the antibodies against extracellular epitopes of α3, α4, α7, α9, ß2 and ß4 nAChR subunits. KEY FINDINGS: It is shown that the studied cells expressed different sets of nAChR subunits on the plasma membrane suggesting the presence of α7 nAChRs on all cells, of α3ß4 nAChRs on U373 cells and of α4ß2/α4ß4 nAChRs on U937 cells. In addition to nAChRs exposed on the surface, all cells contained a considerable intracellular pool of α3- and α7-containing nAChRs. The binding of α3-, α7- and ß4-specific antibodies partially overlapped with that of mitochondrial outer membrane translocase-specific antibody. Binding of α7-specific antibody also overlapped with that of MitoTracker Green, which binds to active mitochondria. SIGNIFICANCE: The data obtained suggest that a part of intracellular α3ß4 and α7 nAChRs in U373, U937 and Daudi cells is located on mitochondria. This finding complements our previous observation of α7 nAChRs in mouse liver mitochondria and reveals new intracellular targets for cholinergic regulation.

[Nicotine effects on mitochondria membrane potential: participation of nicotinic acetylcholine receptors].

The effect of nicotine on the mouse liver mitochondria was studied by fluorescent flow cytometry. Mice consumed nicotine during 65 days; alternatively, nicotine was added to isolated mitochondria. Mitochondria of nicotine-treated mice had significantly lower basic levels of membrane potential and granularity as compared to those of the control group. Pre-incubation of the isolated mitochondria with nicotine prevented from dissipation of their membrane potential stimulated with 0.8 microM CaCl2 depending on the dose, and this effect was strengthened by the antagonist of alpha7 nicotinic receptors (alpha7 nAChR) methyllicaconitine. Mitochondria of mice intravenously injected with the antibodies against alpha7 nAChR demonstrated lower levels of membrane potential. Introduction of nicotine, choline, acetylcholine or synthetic alpha7 nAChR agonist PNU 282987 into the incubation medium inhibited Ca2+ accumulation in mitochondria, although the doses of agonists were too low to activate the alpha7 nAChR ion channel. It is concluded that nicotine consumption worsens the functional state of mitochondria by affecting their membrane potential and granularity, and this effect, at least in part, is mediated by alpha7 nAChR desensitization.

[Analysis of signaling pathways activated by nicotinic acetylcholine receptors in B-lymphocyte-derived cells].

The expression of nicotinic acetylcholine receptors (nAChRs) in the chicken pre-B-lymphoma DT40 cell line was investigated. DT40 cells were shown to express at least alpha7-containing nAChRs; their amount increased upon incubation with 10 microM nicotine. Addition of 10 microM choline favoured the inclusion of 3-[4.5dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT); the effect of choline was inhibited by 2.5-25 nM methyllicaconitine (MLA) or 10-100 nM alpha-cobra-toxin indicating the alpha7 nAChR role in maintaining the proliferative potential of DT40 cells. Nicotine and choline potentiated the effect of 0.5 microM ionomycin, which suppresses cell viability via Ca2+ ions influx. Contrariwise, the suppressive effect of 1 microM hydrogen peroxide, mainly affecting cell mitochondria, was weakened by choline, but was increased by 2.5 nM MLA. MEK1/2 and PKC kinases activity was necessary for maintaining the proliferative potential of DT40 cells. MLA increased the effect of the MEK1/2 kinase inhibitor (U0126), while suppressive effect of MLA itself was decreased. The presence of CaMKII kinase inhibitor (KN-62) also decreased MLA effect. MLA favoured cell survival in the presence of PKC inhibitor (chelerythrine). These data indicate that MEK1/2 and CaMKII kinases are involved in alpha7-containing nAChR signaling in DT40 cells and that PKC plays a key role in this process.

The role of alpha7 nicotinic acetylcholine receptors in B lymphocyte activation.

The involvement of nicotinic acetylcholine receptor (nAChR) a7 subtype in B lymphocyte activation has been investigated. B lymphocytes were magnetically separated from the spleens of C57Bl/6J mice. The purified lymphocytes were treated with fluorescently labeled IgM-, CD40-, CD16/32 or CD23-specific antibodies and unlabeled alpha7-specific antibody and examined by flow cytometry. The alpha7-specific antibody binding interfered with that of anti-CD40 but not of anti-IgM, anti-CD16/32 or anti-CD23 suggesting that alpha7 nAChRs are located close to CD40. B lymphocyte activation either in vitro with anti-CD40 or in vivo by immunization with cytochrome c resulted in increased alpha7 nAChR expression. Anti-CD40-induced B lymphocyte proliferation studied by [3H]thymidine incorporation was increased upon alpha7 nAChR inhibition with methyllicaconitine, choline or antibiotic gentamicin, as well as in the presence of the inhibitor of acetylcholine synthesis hemicholine-3. Mice injected with both cytochrome c and methyllicaconitine responded with IgM anti-cytochrome c antibodies faster than those injected with cytochrome c alone, while the secondary IgG responses were similar. It is concluded that alpha7 nAChRs negatively control CD40-mediated B lymphocyte proliferation but did not affect the IgM-IgG class switch or memory B cell activation. Endogenous acetylcholine may be regarded as an auto/paracrine regulator of B lymphocyte activation.

The role of nicotinic receptors in B-lymphocyte development and activation.

We studied the binding of [(3)H]-epibatidine and [(125)I-]alpha-bungarotoxin, as well as subunit-specific antibodies with purified B lymphocytes of C57Bl/6J mice and found that these cells contained 12,200+/-3200 of alpha4(alpha5)beta2 and 3130+/-750 of alpha7(alpha5beta4) nicotinic acetylcholine receptors per cell. According to flow cytometry data, the highest expression of alpha4(alpha5)beta2 receptors was observed in immature newly generated B lymphocytes of the bone marrow, while the number of alpha7(alpha5beta4) receptors grew up along with the B cell maturation in the spleen. By using alpha4, beta2 or alpha7 knockout and chimera mice, it was shown that both receptor subtypes supported the survival of B cell precursors and increased the size of B-lymphocyte population in the bone marrow. In contrast, propagation of mature B lymphocytes in the spleen was controlled by alpha7-containing subtype only. Moreover, mature B lymphocytes became sensitive to nicotine only in the absence of beta2-containing receptors. Knockout mice had less serum IgG, IgG-producing cells and natural IgG antibodies than their wild-type counterparts, while the absence of beta2-containing receptors resulted in increased B-lymphocyte activation and antibody immune response. The data obtained indicate that nicotinic receptors are involved in regulating B-lymphocyte development and activation, possibly, by affecting expression and/or signaling of CD40, the two subtypes playing different roles.

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits.

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.

[The role of nicotine in regulation of lymphocyte proliferation].

The effect of nicotine on both the expression of nicotinic acetylcholine receptors (nAChRs) and proliferation of hybridoma cells and normal mouse lymphocytes has been investigated. By means of immunoenzyme assay, nicotine was shown to regulate the number of nAChRs in both hybridoma cells and normal rat splenocytes. According to the data of triazolyl blue inclusion and ELISpot assay, nicotine stimulated proliferation of both hybridoma cells and normal plasma cells generated in the course of immune response in vivo. The cell sensitivity to nicotine depended on the number of nAChRs expressed on the membrane, as well as on their functional activity affected, in particular, by adhesive contacts. The use of the open channel blocker benzohexonium revealed that proliferative signal through nAChR in hybridoma cells was mediated by ion channel opening. The data obtained demonstrate the proproliferative role of nicotine for B lymphocytes, and may account for the development of lymphoproliferative disorders in tobacco smokers.

[The role of fibrin clot degradation products in the regulation of vital functions of PC-12 cells].

The products of the fibrin clot hydrolysis performed by PC-12 cells modulated dose-dependently the rate of cell proliferation and favored their survival when seeded in suboptimal density. Co-incubation of PC-12 cells with fibrin degradation products enhanced cell adhesion to tissue culture plastic, as well as the number of nicotinic acetylcholine receptor alpha3 and alpha5 subunits expressed. It was demonstrated that, in fact, such a heterogeneous and comprehensive influence was a sum of effects exerted by different fibrin fragments. Low molecular weight fraction (below 30 kDa), but not a purified alphaC-domain, stimulated PC-12 cell proliferation, diminished their adhesion to plastic, increased nicotinic receptor expression and caused processes outgrowth. On the contrary, high molecular weight products, in particular D, DD and E fragments, enhanced PC-12 adhesion to plastic and, as a result, slowed cell division. Both high and low molecular weight fragments favored the survival of PC-12 cells. These results showed that fibrin degradation products support the vital functions of neuron-like cells, favor their contacts with extracellular surrounding and act as neurotrophic factors.

[Alfa-subunit composition of neuronal nicotinic acetylcholine receptors of the submucosal plexus in the guinea-pig small intestine]. / Alfa-subodynychnyi sklad nikotynovykh kholinoretseptoriv neironiv pidslyzovoho spletennia tonkoho kyshechnyka mors'koï svynky.

The alpha-subunit composition of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using the whole-cell patch-clamp technique and affinity-purified antibodies (Abs) against alpha3-, alpha4, alpha5 and alpha7-subunits of nAChRs. The independent addition of anti-alpha3 and anti-alpha5 Abs to extracellular bath solution induced depression of the acetylcholine (ACh)-evoked currents by 60% +/- 1.56% and 65% +/- 1.62% correspondingly. Successive application of anti-alpha5 Abs in the presence of anti-alpha3 Abs did not have any additional blocking effect on ACh-evoked currents. Anti-alpha7 Abs evoked depression of Ach-induced currents by 24% +/- 1.51% in 80% of investigated neurons and by 67% +/- 1.5% in 20% of neurons. The addition of anti-alpha4 Abs to extracellular bath solution did not have effect on membrane currents of the investigated neurons. Our data provide evidence of participation of alpha3-, alpha5 and alpha7-containing receptors in the response to ACh. Alpha3- and alpha5-subunits are associated in the same functional nAChRs that provide greater part of response to ACh in the most of submucosal neurons. However, in some neurones alpha7-containing nAChRs provide greater part of response to agonist.

Functional nicotinic acetylcholine receptors in the neurons of rat intracardiac ganglia.

The alpha subunit composition of nicotinic acetylcholine receptors (nAChRs) expressed in the neurons of intracardiac ganglia of the rat was investigated using monoclonal and polyclonal antibodies against alpha3, alpha4, alpha5 and alpha7 nAChR subunits. The acetylcholine-induced membrane potentials in the neurons of the subepicardial plexus isolated from the left atrium of the heart were studied by intracellular recording performed in the presence of subunit-specific nAChR-blocking antibodies applied either separately or in various combinations. It was found that intracardiac ganglia neurons express alpha3alpha5; alpha7; alpha7(alpha5) and alpha4-containing nAChRs. The neurons were heterogeneous as to the nAChR subtypes expressed that possibly indicated their functional differences.

[Neuronal nicotinic receptors: structure and function in different types of cells]. / Nikotynovu retseptory neironnoho typu: budova i finktsii u klitynakh riznoho pokhodzhennia.

The review deals with the up-to-date literature data on the subunit composition and functions of nicotinic acetylcholine receptors in both excitable (brain and autonomic ganglia) and non-excitable (respiratory epithelium, vascular endothelium, skin, blood) tissues. Special attention is paid to the expression and role of nicotinic receptors in the immune cells.

[PC-12 cells hydrolyze the fibrin clot by producing both plasminogen and its tissue activator]. / Klityny PC-12 hidrolizuiut' fibrynovyi zhustok shliakhom produktsiï plazminohenu ta ioho tkanynnoho aktyvatora.

It has been shown that rat pheochromocytoma PC-12 cells degraded fibrin clots in vitro. SDS-PAGE performed in the gels of different density and Western blotting using monoclonal antibodies against DD- and D-fibrin fragments demonstrated that both high and low molecular weight degradation products similar to those of fibrin hydrolysis by plasmin had been formed. Enzyme electrophoresis, chromogenic assay and enzyme-linked immunosorbent assay using tPA-specific antibodies demonstrated that PC-12 cells constitutively secreted both plasminogen and tPA. The results obtained allow using PC-12 cells as a model to examine the interaction of nerve cells with the fibrin clot.

[Regulation of fibroblast adhesion by polyreactive antibodies]. / Rehuliatsiia adheziï fibroblastiv polireaktyvnymi antytilamy.

The effect of polyreactive antibodies on fibroblast adhesion was studied. Dose-dependent decrease of adhesion was found at the stage of adhesive complex formation. A possible mechanism of this effect was determined as a steric influence on integrin agregation and clastering.

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations.

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.

Nicotinic acetylcholine receptor subtypes in rat superior cervical ganglion neurons as studied by sequential application of two alpha-subunit-specific antibodies.

All cultured neurons of rat superior cervical ganglion (SCG) were stained with nicotinic acetylcholine receptor (nAChR) alpha5- or alpha7-subunit-specific oligoclonal antibodies (Abs) and could additionally bind alpha3-subunit-specific monoclonal antibody (mAb). About 60% of the neurons were stained with alpha4-specific Ab and could not bind alpha3-specific mAb. The acetylcholine-induced membrane currents recorded with the whole-cell patch clamp method and partially blocked with alpha3-specific mAbs, could be additionally blocked with alpha5- and alpha7- specific Abs, and vice versa. The results suggest that: (1) each neuron of rat SCG expresses several nAChR subtypes with different alpha-subunits; (2) the alpha3-, alpha5- and alpha7-subunit-containing nAChRs are probably located far enough from each other thus enabling joint binding to the cell of the corresponding alpha-subunit specific Abs, in contrast to the alpha4-subunit-containing nAChRs which are probably located too close to the alpha3-containing ones to allow their joint binding.

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies.

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different alpha subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the alpha(181-192) region of alpha3, alpha4, alpha5, and alpha7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among alpha3, alpha4, alpha5, and alpha7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the alpha3, alpha5, and alpha7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly alpha4-positive. In contrast, only about one-third of the intracardiac neurons were alpha3-positive, about 50% were alpha4-positive, one-seventh were alpha5-positive, and one-fifth were alpha7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that alpha3, alpha5, and alpha7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas alpha4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

The immune response to cytochrome c in BALB/c mice is delayed due to inability of their non-specific antigen-presenting cells to provide its immunodominant epitope.

The antibody response to horse cytochrome c (cyt.c) in BALB/c mice developed slowly and a substantial production of IgG antibodies was observed only 26-30 days after immunization. Lymph node cells (LNC) of unimmunized mice proliferated weakly in response to both native cyt.c and its synthetic peptides. On day 8 after immunization, LNC could not be stimulated with native cyt.c and peptide 92-104. However, they did proliferate in response to cyt.c peptides 1-6, 1-13, 2-13, 14-22, 46-56, 57-77, 61-77 and 61-69 which are closely related in horse and mouse cyt.c. On day 26, both native cyt.c and the peptides, including 92-104, were equally active in stimulating LNC proliferation. Both plastic-adherent and cyt.c-specific cells panned from day 8 cells enhanced the response of unprimed cells to native cyt.c. Elimination of B cells demonstrated that primary recognition of cyt.c was mediated, at least partly, by non-specific antigen-presenting cells (APC) while later B cells of additional specificities were involved. It is concluded that immunization with horse cyt.c initiated an autoimmune response resulting in T-dependent anergy. Peptide determinants processed by non-specific APC stimulated corresponding autoreactive T cells. Specific B cells which appeared as a result of the response maturation processed successfully the immunodominant epitope and finally mediated proliferative and antibody responses.

[Dynamics of antibody formation, isotypes and specificity of antibodies in the immune response of Balb/c mice to cytochrome c]. / Dinamika antiteloobrazovaniia, izotipy i spetsifichnost' antitel v immunom otvete myshei Balb/c na tsitokhrom c.

The production of IgG antibody (Ab) to cytochrome c in BALB/c mice reached its maximum four weeks after primary immunization which is significantly later than the response to the same dose of keyhole limpet hemocyanin (KLH). The relative amounts of IgG subclasses varied during the immune response to KLH: IgG2a Abs dominating on day 11 were replaced by IgG1 on day 30 after immunization. This was not the case with anti-cytochrome c Abs: their IgG subclasses varied significantly among individual mice within the strain. The heme elimination altered the cytochrome c antigenic structure, although some antigenic determinants remained intact. The heme with its neighbouring residues represented by heme peptide 14-22 was not recognized by anti-cytochrome c Abs. Serum IgG Abs interacted with peptides 1-65 and 1-80 obtained by BrCN hydrolysis of cytochrome c; however, they did not bind to peptide 66-104. The Abs affinity purified on cytochrome c bound to peptides 1-65 and 1-80, while the Abs purified on peptide 1-65 or apo-cytochrome could recognize native cytochrome c as well.