[Morphological bases of dopamine effect on HEP-2 tumor cells viability].

The purpose of the present investigation was to study the morpho-functional organization of a classical object of cytological research - cultured HEp-2 tumor cells, using dopamine as a penetrating agent, inducing the polymerization of cytosolic actin. It was demonstrated that dopamine introduced into the incubation medium reduced viability and caused morphological disturbances of cultured HEp-2 cells; these effects were proportional to dopamine concentrations (1.0 x 10(-4) M to 1.0 x 10(-3) M) and exposure duration (2 to 3 days). These cells, according to ultrastructural data, underwent fusion and lysis because of the appearance of actin filaments network in the loci of globular actin prevalence in control cells. Dopamine receptors had no effect on cytotoxic effect of dopamine. This was indicated by fluorescent microscopical evidence of dopamine penetration into experimental cells in the presence of haloperidol, as well as destruction of HEp-2 cells under the action of pyrimidinethione, similar to dopamine by characteristics, but lacking its own receptors. It is suggested that cytoplasmic target for dopamine is globular actin and that induced polymerization of this cytoskeletal protein caused injury to tumour cells.

[Cytochemical and ultrastructural characteristic of BHK-21 cells exposed to dopamine].

BHK-21 cells were incubated in a medium containing dopamine (DA) and then their catecholamine content evaluated by using the Falck cytochemical method. The significant intensification of cell fluorescence as compared to that one in control preparations was detected; this effect was proportional to DA concentration and exposure duration and was more pronounced in cells in suspension than in those attached to the substrate. Simultaneous ultrastructural investigation has shown that an increased intensity of the cytoplasm fluorescence correlated with the appearance of the dense network of fibrils that were morphologically identified as F-actin microfilaments. Prior blockade of dopaminergic receptors by haloperidol did not change the following DA effect both on the fluorescence intensity and cell ultrastructure. The data obtained suggest that DA chronically acting on the living cells was able to penetrate into the cytoplasm, causing actin polymerization and incorporating into the newly formed actin cytoskeleton. Structurally, this may be manifested by cytoskeleton and its derivative hypertrophy, that could have a substantial effect on general morphology of the cell.

[The influence of dopamine on the ultrastructure of BHK-21 cells].

The influence of dopamine on the haloperidol of BHK-21 cells being in suspension or attached to substrate was investigated. It was shown that the ultrastructural changes affected mainly the cellular loci enriched by the cytoskeleton actin such as intercellular desmosome-like contacts, microvilli and cortical layer or mesh just beneath the plasmatic membrane. The desmosome-like contacts were hypertrophied, their electron density was increased and fibrilar bridges appeared in specialized contacts. Many microvilli fused with each other and with plasma membrane of the neighboring cells, or, on the contrary, split up. Frequently, the membrane surface between microvilli and particularly their apical parts was seen to be pierced by thin thread, morphologically similar to actin filaments. The cytoplasmatic matrix onto ultrathin sections had blotched appearance and at the ultrastructural level was represented by numerous randomly oriented actin filaments. The effect of dopamine was more pronounced in the BHK-21 cells when being in suspension than in attached to the substrate ones, which presumably occurred due to known lesser differentiation of the cytoskeleton in the formers. Finally, it was established that the preliminary blockade of cellular D2 receptors with haloperidol neither affected the ultrastructure of BHK-21 cells nor prevented the following effect of dopamine. The data obtained suggest the direct interactions of dopamine with the actin cytoskeleton.

[The cultivation of fragments of the parotid venom-secreting gland in the common adder (Vipera berus)]. / Kul'tivirovanie fragmentov okoloushnoi iadosekretiruiushchei zhelezy gadiuki obyknovennoi.

The primary culture of the Vipera berus poison-secretory parotid gland has been obtained. The morphology of the intact secretory epithelium and epithelial cells cultured in different conditions has been examined by light and electron microscopy. The secretory epithelium cells were able to survive in the cultural medium and to adapt to in vitro conditions maintaining their nearly normal ultrastructure corresponding to the stage of active poison secretion commonly observed in epithelial cells of the native gland.

[Changes in the rates of glucose consumption and lactate release by cells in perfused and nonperfused cultures]. / Izmenenie skorostei potrebleniia gliukozy i vydeleniia laktata kletkami v perfuziruemoi i neperfuziruemoi kul'turakh.

The energetic state of Chinese hamster fibroblasts was investigated under stationary cultural conditions and under condition of culture medium perfusion immediately above the cells. Specific rates of glucose utilization and lactate formation under the former conditions (1.88 +/- 0.2) x 10(-13) and (4.3 +/- 0.56) x 10(-13) Mole/cell/h at the logarithmic growth phase, and (0.21 +/- 0.08) x 10(-13) and (0.58 +/- 0.06) x 10(-13) Mole/cell/h at the stationary phase, respectively. In the perfused culture, specific rates of glucose utilization and formation of lactate are (4.86 +/- 0.56) x 10(-13) and (11.0 +/- 1.8) x 10(-13) Mole/cell/h at the logarithmic growth phase, and (1.57 +/- 0.14) x 10(-13) and (4.11 +/- 0.5) x 10(-13) Mole/cell/h at the stationary phase, respectively. It has been proposed that under conditions of stationary culture the fall of the rates, as the culture reaches the survival phase, is due to diffusion-dependent limitations of mass transfer between the medium and the culture. Under perfusion conditions, the fall of the rates can be explained by some deficiency of necessary components and by excessive amounts of metabolic products in the multilayer structure.

[The relationship of the saturation density of multilayer cell cultures to their mass exchange with the medium]. / Zavisimost' plotnosti nasyshcheniia mul'tisloinykh kul'tur kletok ot ikh massoobmena so sredoi.

Chinese hamster fibroblasts (CHF) and NIH 3T3 cells were cultured on a glass substrate at different distances from the porous membrane separating the cells from the perfusing medium. It is shown that with perfusion of medium above the membrane there is no movement of the medium near the cells. In both the types of culture, the cells grow in multilayers, however the multilayer character of growth in CHF is more pronounced than in NIH 3T3 cells. The saturation density of the cultures depends on the cell-membrane separation, and at separations of no more than 0.2 mm exceeds the saturation density in the monolayer by 8-10 fold. The dependences of the saturation density on separation are different for CHE and NIH 3T3 cells, indicating qualitative differences in the inhibition of cell growth in monolayers between these cultures. The results obtained indicate that the inhibition of cell growth in monolayer is due to mass exchange limitations, rather than to intercellular contact interactions.

[The proliferative characteristics of cells in culture during perfusion of the medium]. / Osobennosti proliferatsii kletok v kul'ture pri perfuzii sredy.

The proliferation of Chinese hamster fibroblasts (CHF) and NIH 3T3 cells was studied at different flow rates immediately above the cells. It is shown that there is a limiting density of saturation of the perfused culture that accounts for 1.7 x 10(6) - 2.0 x 10(6) cells/cm2 for NIH 3T3 cells, and for 6 x 10(6) - 7 x 10(6) cells/cm2 for CHF. The growth curves and the distribution of cells with respect to the phases of the cell cycle during cultivation with and without perfusion are presented. Based on the results obtained it is assumed that the limit of saturation density of perfused CHF culture is due to the mass transfer of the growth-inhibiting metabolites inside the multilayer structures. The assumption seems to hold true for NIH 3T3 cells, too, even though the multilayer character of growth of this culture is less pronounced than in CHF.

[The multilayer growth of epithelium-like ESK cells in perfused cultures]. / Mul'tisloinyi rost épiteliopodobnykh kletok SPEV v perfuziruemykh kul'turakh.

The multilayer growth of an epithelium-like ESK [correction of SPEV] cell culture was achieved under condition of culture medium perfusion. The growth of the ESK [correction of SPEV] cells is described under conditions excluding medium movement above the cells in the chamber with a semipermeable membrane separating the cells from the perfused medium. Under these conditions the multilayer (30-40 layers) growth of culture was observed. The data observed are in favour of our suggestion that it is the diffusional limitations in mass exchange, rather than intracellular interaction contacts, that may be responsible for cell growth in culture. A dependence of culture growth on the initial inoculation density, under condition of medium perfusion immediately above the cells, was determined. The dependence of the multilayer saturation density on the perfusion rate and on the complete replacement of the perfused medium is discussed. The existence of a limiting saturation density of perfused culture was shown.

[A flow diffusion chamber for research on cells in culture]. / Protochno-diffuzionnaia kamera dlia issledovaniia kletok v kul'ture.

A flow diffusion chamber designed for studying cells and tissues in culture is described. The chamber contains a plate with a great number of isolated holes, which enables one to perform the cultivation of cells at different distances from the porous membrane separating the cells from the perfused medium. An individual porous membrane can be placed above each hole. Evidence for the selective permeability of domestic membranes under the conditions of cell culture in chamber is presented. The chamber makes possible a simultaneous cultivation of a great number of various cultures with different conditions of mass exchange with common perfused medium, which contributes to intensification of studies.

[Effect of nutrient medium perfusion on the secretory activity of rat hepatocytes and pancreatic islet cells]. / Vliianie perfuzii pitatel'noi sredoi na sekretornuiu aktivnost' gepatotsitov i ostrovkovykh kletok podzheludochnoi zhelezy krys.

The data are reported on albumin secretion by rat hepatocytes and insulin secretion by pancreatic beta-cells of newborn rats during cell cultivation on flat synthetic membrane in conditions of continuous medium perfusion. Albumin and insulin secretion by the appropriate cultures was higher in continuous medium perfusion than in the control. Enhanced sensitivity of pancreatic beta-cells to glucose, as compared to the control was revealed. It is concluded that continuous medium perfusion of hepatocytes and pancreatic beta-cells in the primary culture had a favourable effect on albumin and insulin secretion by the appropriate cultures.

[Growth of a cell culture on porous capillaries]. / Rost kul'tury kletok na poristykh kapilliarakh.

The growth of BHK-21 culture cells on the outer surface and in the clear space of capillaries made of phenylone or polyacrylnitrile was studied. The cultivation of cells in the clear space of the capillary extends their survival time and outlines possible ways of continuous cultivation of tissue cells which are cultivated in the attached state.

[Function of hormone-secreting cells cultured under continuous perfusion with nutrient medium]. / Funktsiia gormonsekretiruiushchikh kletok, kul'tiviruemykh v usloviiakh nepreryvnoi perfuzii pitatel'noi sredoi.

The data are presented as to prolactin and ACTH secretion by rat adenohypophysis cells and insulin secretion by pancreatic beta-cells of the newborn rats during cell cultivation on flat synthetic membranes with pores differing in diameter under continuous medium perfusion. The cultured cells were discovered to be viable and to possess secretory activity. Secretion of prolactin and ACTH and that of insulin in the appropriate cultures increases in the course of long-term incubation under continuous medium perfusion. It is concluded that application of the porous membranes enables a selective study of the release of one or another hormone into the medium. It is believed that cultivation of hormone-secreting and hormone-susceptible cells under closed- and unclosed-type nutrient medium perfusion is fairly promising.

[Controlled cell cultivation. III. The flow-diffusion chamber]. / Upravliaemoe kul'tirovanie kletok. III. Protochno-diffuzionnaia kamera.

A perfuse chamber is described intended for a continuous culturing of cells and tissues, for in vitro observations and for the screening of biologically active substances. The chamber is dismountable. A ring-shaped partition of the chamber, separating the pericellular zone from the external flowing one, contains to diametrically opposite diffuse filters in the form of cylindrical stoppers made of porous glass. Depending on the angle of rotation of the diffuse filter axis with respect to the axis of pipe connections of the chamber, the wanted rate of material transfer between pericellular and flowing zones is achieved. Biological tests have shown that the survival time of isolated molluscan neurones of Limnaea stagnalis in this chamber considerably exceeds that in conventional perfusion chambers without partition. The chamber can be used in studies of embryogenesis, for incubating fertilized oocytes under conditions of controlled culturing.

[Controlled cell cultivation. 2. Cultivation of mammalian cells and tissues in the perfusion chamber]. / Upravliaemoe kul'tivirovanie kletok. 2. Kul'tivirovanie kletok i tkanei mlekopitaiushchikh v perfuzionnoi kamere.

The results of biological testing of a perfusion chamber are presented. The chamber can be used for a prolonged cultivation of cells and tissues of different types on controlling the composition of nutrient medium and gas phase. The possibility is shown to apply the chamber for a prolonged cultivation and the observation of the behavior of live cells under different influences.

[Controlled cell culture. I. A modified perfusion chamber]. / Upravliaemoe kul'tivirovanie kletok. I. Modifitsirovannaia perfuzionnaia kamera.

The construction of a modified perfusion chamber is presented, which can be used for a prolonged cultivation of mammalian cells and tissues, for observation of the behavior of living cells as well as for the study of different effects on these cells. The chamber is made as non-demountable of optical glass, with a diffusive barrier separating the pericellular zone from that with a perfusion medium. The scheme of the equipment for cultivation of cells and tissues in this diffusion chamber on controlling the composition of nutrient medium and gas phase is given.