[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 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.

[Ultrastructural characteristics of rat adrenal cortex cultured cells in the norm and after calcium ionophore A23187 and adrenocorticotropic hormone application]. / Osoblyvosti ul'trastruktury kul'tural'nykh klityn kory nadnyrkovykh zaloz shchura u normi ta pislia aplikatsiï kal'tsiievoho ionoforu A23187 ta adrenokortykotropnoho hormonu.

The ultrastructure of a fasciculata-reticularis zone cells of a rat adrenal cortex in norm, and after application of adrenocorticotropic hormone (ACTH) and calcium ionophore A23187 was investigated. In the control it has been revealed three types of cells which differed on the ultrastructure. ACTH or ionophore A23187 application resulted in disappearance of a difference in ultrastructure of cells of different types, also all cells got morphological attributes of accelerated steroidogenesis. The probable role of cells with different types of ultrastructure for fasciculata-reticularis zone function, and also prospective participation of calcium ions in ACTH influences on ultrastructure of a fasciculata-reticularis zone is discussed.

[Ultrastructural characteristics of lipid droplets in rat adrenocortical cells from zona fasciculata-reticularis]. / Osoblyvosti ul'trastruktury lipidnykh krapel' u klitynakh puchkovo-sitchastoï zony kory nadnyrkovykh zaloz shchura in vitro.

One day cultured adrenocortical cells from zona fasciculata-reticularis were used in morphological experiments. The electron-microscopic and imaging analysis methods were used for the investigation of intracellular ultrastructure of these cells. Experiments which conducted in control conditions, allowed us to allocate three types of cells which differed by ultrastructure of the mitochondria, lipid droplets, smooth endoplasmic reticulum and dense bodies. It was shown that lipid droplets with light and homogeneous matrix, met more often in cells with morphological attributes of high intensity of steroidogenesis. On the contrary, lipid droplets, with dark matrix and a dense edging, met more often in cells which having morphological attributes-of low intensity of steroidogenesis. Ionophore A23187 or adrenocorticotropic hormone application resulted in reduction of lipid droplets diameter and in simultaneous increase in density of their arrangement in cytoplasm. At the same time droplet matrix became light and homogeneous in all cells. Thus, the ultrastructure of lipid droplet matrix is sensitive to change of calcium ions concentration in cytoplasm. Processes which result in change of lipid droplet ultrastructure, probably, are connected to steroidogenesis, nevertheless, this question requires further investigation. The lipid droplets" ability to form morphological contacts with smooth endoplasmic reticulum, mitochondria, nuclear and cellular membranes is also discussed.

[Intracellular calcium homeostasis in sensory neurons during hypoxia]. / Vnutrishn'oklitynnyi kal'tsiievyi homeostaz sensornykh neironiv pry hipoksychnykh vplyvakh.

Hypoxia is the main reason leading to neuronal death during different forms of brain diseases. The main phenomenon observed at hypoxia is excessive growth of intraneuronal Ca2+ concentration leading to irreversible cell damage. Despite extensive studies of this process, the intracellular mechanisms responsible for disturbance in Ca2+ are still unclear. The aim of present investigations was to explore these mechanisms. Ca2+ was measured by spatial screening of isolated dorsal root ganglion (sensory) neurons loaded with fluorescent dye Fura-2AM after exposing them hypoxic solution. Hypoxia resulted in a reversible elevation of Ca2+, which could be partly prevented by several pharmacological agents. We concluded that in sensory neurons hypoxia-induced elevation of cytosolic Ca2+ is induced by primary changes in ionic channels and secondary in function of mitochondria.

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons.

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.

Electron microscopic evidence for multiple types of secretory vesicles in bovine chromaffin cells.

It has been previously shown that the neuron-like chromaffin cells from the bovine adrenal medulla are heterogeneous. Among other differences, the cells also differed in secretory vesicles represented in their cytoplasm. The present study investigates the types of secretory vesicles in bovine chromaffin cells by electron microscopy. Morphometric analysis revealed five types of electron-dense secretory vesicles in chromaffin cells. These were as follows: elementary large catecholamine-storing chromaffin granules of rounded shape, large dense core vesicles of ovoid and rod-like shapes, small dense core vesicles as well as ribosome-coated vesicles of intermediate density. Among the electron-lucent vesicles there were small synaptic-like microvesicles, endocytotic clathrin-coated vesicles, growth cone vesicles, and emptied large light core vesicles. The structural and functional backgrounds of different types of secretory vesicles are described, focusing on their formation and potential role.

Ultrastructural features of medullary chromaffin cell cultures.

The ultrastructural organization on the fourth day of culture of chromaffin cells isolated from the bovine adrenal medulla was characterized based on electron microscopic and morphological analysis. We established that medullary chromaffin cells could be divided into four morphologically different subtypes. Most cells (49.1% of those examined) had a dense cytoplasm and fine dense granules. Cells with dense cytoplasm and large granules represented a second type of chromaffin cell (21.1%). Cells of the third type had a light cytoplasm, granules with a light halo and a well-developed Golgi apparatus (26.3%). The fourth type of chromaffin cell was characterized by moderately dense cytoplasm with well-expressed varicose rough endoplasmic reticulum (about 3.5%). Among concomitant cell types, cortical adrenal cells from the zona fasciculata and zona glomerulosa, epithelial cells, fibroblasts, lymphocytes, brown lipoblasts and glial Schwann cells were present. Morphological analysis implies that cells with dense cytoplasm and fine granules and those with light cytoplasm and haloed granules (75.4% in total) are adrenaline-containing cells, whereas the cells with dense cytoplasm and large granules (26.3%) contain noradrenaline. Cells with moderately dense cytoplasm and varicose reticulum share common morphological properties with classical glandular cells and, by their properties, were closer to noradrenaline-containing cells. It is concluded that chromaffin cells, which are the main cell type among cultured cells from adult bovine adrenal medulla, are morphologically quite heterogeneous. Other cell types of different nature may also be present in the culture and can locally influence the properties of the investigated medullary chromaffin cells used in electrophysiological experiments.

Electron cytochemical study of carbohydrate components in different types of cultured glial cells of snail Helix pomatia.

Using a variety of colloidal gold-labelled lectins with different sugar specificities, the structure and topography of carbohydrate determinants of the surface membrane of in vitro cultured glial and nerve cells of the snail Helix pomatia have been electron cytochemically studied. Heterogeneity of carbohydrate pools among different types of glial cells and between glial and nerve cells was established. It was found that satellite glial cells having the ultrastructural signs of cells with high metabolic level (type II cells) selectively bind GNA which is specific to terminal alpha-D-mannose residues and do not bind other mannose-specific lectins, Con A and LCA. GNA determinants are absent in satellite type I glial cells, fibrous glial cells, microglia and neurons. It has been found that glial cells (satellite type I and II glial cells, filamentous glial cells and microglial cells) do not bind PVA and LABA. LTA did not bind to any glial cells and binds weakly to neurons. Con A and WGA determinants which are abundant on the neurons are completely absent on satellite type II glial cells but present on satellite type I glial cells and filamentous glial cells. Microglial cells contain Con A and LCA determinants and the density of PNA determinants on these cells is the highest compared to other types of glial cells or neurons. It is concluded that some lectin determinants (for RCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a definite type of glial cells and can serve as a marker of these cells. The role of specific carbohydrate determinants in the functioning of a neuron-glial complex is discussed.

Newly identified nerve cells of the snail, Helix pomatia, associated with the generation of pacemaker activity.

Data on new, previously unidentified nerve cells of the snail Helix pomatia are presented in this paper. The identified neurons described may serve as a convenient model for the investigation of the cellular mechanisms of pacemaker activity, the role of neuropeptides in the generation and regulation of pacemaker activity, peptidergic transmission, and the functional role of the inward calcium current.

[Electron-cytochemical study of carbohydrate components in the surface membrane of cultured Helix pomatia neurons]. / Elektronno-tsitokhimicheskoe issledovanie karbogidratnykh komponentov poverkhnostnoi membrany kul'tiviruemykh neironov vinogradnoi ulitki.

The structure and topography of carbohydrates on the surface of nerve cells of snail Helix pomatia cultured in vitro have been characterized with a series of colloidal gold-labelled lectins of different sugar specificity. The analysis of the lectin binding has shown substantial differences in the carbohydrate pattern between the soma of monoaminergic and peptidergic neurons. It has been found that the surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine (WGA+) and N-acetyllactosamine (RCA-1-) determinants and does not contain neuraminic acid (LPA-) and complex branched N-glycosyl chains (PVA-). At the same time N-acetylgalactosamine (HPA+) was detected on the peptidergic neuron membrane only. It has been concluded that terminal residues of sialic acid are absent on the most of snail nerve cells. Differences in lectin binding between monoaminergic and peptidergic neurons can serve as a basis for formation of specific connections of cells by different types in the developing brain.

[Quantitative characteristics and reconstruction of the surface topography of histochemical markers on the nerve cell plasmalemma]. / Chislennye kharakteristiki i rekonstruktsiia poverkhnostnoi topografii gistokhimicheskikh markerov na plazmalemme nervnykh kletok.

An electron microscopic study of topography of colloidal gold-bound lectins conjugating to glycocalix carbohydrate residues on the somatic membrane surface of the cultivated spinal neurons has been carried out. The quantitative procedures are suggested for analyzing the surface pattern of the markers: two stochastic functions are considered to correspond properly to the particle distribution observed in the electron micrograms. The analysis of these functions permits obtaining required numerical characteristics. The Monte Carlo reconstructing model is described, and results of its work (on the basis of the above experimental data) are demonstrated in the form of "averaged" surface topography of the studied markers within the membrane fragments bordered. Possible connection of the obtained data with cooperative properties of the membrane is discussed.

[Immunocytochemical study of the distribution of cytoskeletal proteins in cultured neurons of the spinal cord of mouse embryos]. / Immunotsitokhimicheskoe issledovanie raspredeleniia tsitoskeletnykh belkov v kul'tiviruemykh neironakh spinnogo mozga myshinykh émbrionov.

The distribution of tubulin, actin and neurofilamentous protein (molecular weight 160 kD) in the spinal cord neurons of mice embryos cultivated in monolayer was studied by means of monoclonal FITC- and rhodamine labeled antibodies. It was found that nerve cell development induced migration of tubulin from the neuronal soma to its processes. The tubulin filling in different processes was uneven at certain stages of cell differentiation. The actin content in the neuron was negligible. Its point concentration was observed along the neuronal processes in the vicinity of focal contacts with the lining cells. Main content of neurofilamentous protein is concentrated in the neuronal body and proximal parts of neurites. The results obtained present the pattern of distribution of cytoskeletal proteins in the spinal cord neurons.

[Special forms of synaptic junctions in cultures of dissociated mammalian spinal cord and spinal ganglia]. / Osobye formy sinapticheskikh soedineii v kul'ture dissotsiirovannogo spinnogo mozga i spinal'nykh gangliev mlekopitaiushchikh.

The ultrastructural study of cells in the monolayer culture of dissociated spinal cord and spinal ganglia of mouse embryos has been performed. The results obtained show that in the course of differentiation of completely isolated cells of the spinal cord special forms of synaptic contacts may appear. They are typical of the spinal cord of phylogenetically inferior animal species. These "mixed" synaptic contacts having properties both of electrical and of chemical synapses seem to represent phylogenetically determined processes of synaptogenesis.

The axosomatic contacts on the bursting neuron of the snail Helix pomatia. II. Ultrastructural localization of adenylate cyclase.

Fluoride and peptide-stimulated adenylate cyclase activity was investigated by electron histochemistry on serial sections of the RPAI neuron of the snail Helix pomatia. Fluoride-stimulated adenylate cyclase was detected in the surface membrane of the RPAI neuron, the postsynaptic membrane of axosomatic contacts, and the surface of glial cells forming a multilayer capsule around the neuron. Peptide-stimulated adenylate cyclase was located in the membrane of glial cells surrounding the neuron, their processes (trophospongia) invaginating deeply in the neuronal soma, and the membrane of somatic protrusions forming the system of lacoons in the region of the axosomatic contact. No peptide-stimulated adenylate cyclase was revealed in the remaining part of the surface of the somatic membrane. The localization of adenylate cyclase activity in the postsynaptic membrane in the region of the axosomatic contact is in accordance with the hypothesis based on electrophysiological experiments that the cyclase system participates in the genesis and regulation of the bursting activity of the RPAI neuron.

The axosomatic contacts on the bursting neuron of the snail Helix pomatia. I. Ultrastructural features of the axosomatic contacts.

The analysis of serial ultrathin sections of the RPAI bursting neuron of the snail Helix pomatia reveals the presence of axosomatic contacts on its surface membrane. These contacts have a number of specific features: the presynaptic axon contains synaptic vesicles and electron-dense granules, typical of peptidergic terminals; the terminal part of the axon forms many finger-like processes which invaginate the neuronal soma; the width of the cleft (80 nm) in the area of the contact is larger than that in usual synaptic contacts; and there is a system of lacoons in the region of the axosomatic contact; this system is formed by protrusions of the soma and it accompanies the contact along its extent. It is suggested that the system of lacoons which communicates with the space between the terminal and the soma may serve as a ramified synaptic cleft into which the secretion from the terminal is released. This system may contribute to a considerable prolongation of the time of action of the secretory product on the membrane of the RPAI neuron.