Neurophysiology, Vol. 43, No. 1, July, 2011 (Original Vol. 43, No. 1, January-February, 2011)

Probable Mechanisms of the Effects of Cerebral on Morphological Characteristics of Rat Pheochromocytoma Cells N. Kh. Pogorelaya,1 D. A. Vasilenko,1 A. N. Makarenko,2 and S. I. Savos’ko2 Neirofiziologiya/Neurophysiology, Vol. 43, No. 1, pp. 18-28, January-February, 2011

Effects of a recently proposed organic preparation, Cerebral, used in the treatment of some vascular brain pathologies, were examined on cultured rat pheochromocytoma PC-12 cells. The normal culture media in six of the seven groups of cell cultures were replaced by similar media with the addition of 0.2 or 2.0 mg/ml Cerebral, 400 mg/ml nerve growth factor (NGF), 1.0 µM verapamil, a blocker of high-threshold calcium channels, or combinations of the above Cerebral concentrations with 1.0 µM verapamil. Within six days of the test period, we measured mean values of the density of cultured cells, proportions of the units possessing significant processes, and projective areas of the cell somata; according to the latter parameter, conventional diameters of the cells (diameters of the circles equivalent to the above area) were calculated. Culturing of PC-12 cells under control conditions (in the non-modified medium) was not accompanied by transformation of their overwhelming majority into neuron-like units. Cerebral in both tested concentrations significantly suppressed proliferation of PC-12 cells, intensified the formation of processes (some of which demonstrated a typical neurite-like structure), and led to an increase in the dimension of the cells. Under conditions of the action of NGF, similar effects were observed, but their intensity was greater. Manifestations of cell differentiation induced by Cerebral developed with a greater delay and were smoothed earlier, as compared with the respective NGF effects. The addition of 1.0 µM verapamil to the culture medium promoted the process of cell differentation; the effects observed were comparable with results of the action of Cerebral. Combinations of verapamil with Cerebral in both above-mentioned concentrations provided somewhat more intense modulatory effects on PC-12 cells than isolated applications of Cerebral, but the effects observed were not additive. The probable mechanisms of changes in the morphological characteristics of PC-12 cells under the influence of Cerebral and the dependence of such changes on the state of calcium signalization are discussed. The data obtained agree with a supposition that the active substances of Cerebral correspond to trophinotropins initiating and/or intensifying synthesis of NGF.

Keywords: pheochromocytoma PC-12, differentation, proliferation, Cerebral, nerve growth factor (NGF), verapamil.

INTRODUCTION

treatment of hemorrhagic insults is insufficient, and this disease is, as a rule, accompanied by subsequent invalidization. A search for therapeutic means that demonstrate neuroprotective capabilities, increase the survival rate after the above disease, and decrease the frequency and severity of invalidization within the post-insult period are exceptionally urgent. An organic preparation, Cerebral (Dniprofarm, Ukraine) was proposed as one of such means. This is a purified mixture of peptides and amino acids produced in the brain of animals subjected to induction of autohemorrhagic insult. Results of clinical tests demonstrated that Cerebral exerts a positive therapeutic effect in some pathologies of the cerebral vessels, including hemorrhagic insult. Peptides whose molecules are built from 3 to 12 amino acid residues

Disorders in the vascular system of the brain (in particular, insults of different geneses) are very widespread and dangerous diseases. Ischemic insults are more frequent (their relative number corresponds to 80-90%). Hemorrhagic insults occur much more rarely, but they are more dangerous; the mortality related to this type of disease is noticeably higher [1, 2]. The risk of cerebral insult exists in nearly all age categories of population. The efficacy of contemporary methods of Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 2 Taras Shevchenko Kyiv National University, Kyiv, Ukraine. Correspondence should be addressed to D. A. Vasilenko (e-mail: [email protected]) and A. N. Makarenko (e-mail: [email protected]. 1

14 0090-2977/11/4301-0014 © 2011 Springer Science+Business Media, Inc.

Effects of Cerebral on Rat Pheochromocytoma Cells probably represent the most active components of the preparation providing the above effects. As is supposed, these peptides function as regulators of production of nerve growth factor, NGF. The latter factor, as is known, is continuously present in the brain of animals and humans, especially within the early stages of ontogenetic development. In the case of damage to the brain tissues, production of NGF increases considerably. As was shown, introduction of Cerebral under conditions of experimental hemorrhagic insult results in increase in the intensity and secretion of NGF. At the same time, Cerebral practically did not affect synthesis and secretion of this growth factor in intact animals; in other words, active substances of Cerebral probably are rather specific trophinotropins [3]. It is known that differentiation of cultured immature neural and neuron-like cells is induced and/or intensified under the influence of NGF and some other factors. This is manifested in the retardation or termination of the process of proliferation of such cells and intensification of growth of their processes (neurites). There are reasons to believe that such processes are Ca-dependent [4-9]. Considering the above-mentioned data, we examined the effect of Cerebral on cultured cells of rat pheochromocytoma PC-12. Cells of this strain descend from cells of the cortical layer of the adrenals; they possess a few properties similar to those of the neurons and are capable, under certain conditions, of transforming into neuron-like cell units. Cells of PC-12 are extensively used in experiments directed toward elucidation of the mechanisms underlying cell growth and differentiation. We compared the effects of Cerebral added to the culture medium in two concentrations (differing from each other by an order of magnitude) with the effects of the agent, which is probably a target for the regulatory influence of this preparation, i.e., NGF per se. We also analyzed the effects exerted on cultured PC-12 cells by verapamil, a blocker of L-type calcium channels. In addition, the effects of combined action of Cerebral and verapamil were studied. We believe that the respective data will help to elucidate the mechanism of action of Cerebral on the processes of proliferation and differentiation of neuron-like cell elements. This, in turn, can be useful for interpretation of the mechanisms of therapeutic effects of Cerebral under conditions of insult-evoked impairment of the brain.

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METHODS Experiments were carried out on cells of rat pheochromocytoma, strain PC-12. Culturing of this strain has for a long time been maintained in the Bogomolets Institute of Physiology of the NAS of Ukraine. Under conditions of our experiment, the cells were cultured for 7 days. Samples of the culture (suspension in the normal culture medium, 85% RPMI-1640, 10% thermoinactivated horse serum, and 5% embryonic calf serum, with 400 µg/ml gentamycin added; all components from Sigma, USA) were passed in plastic Carrel flasks and incubated at 37°C in an atmosphere of moistened air enriched with CO 2 to 5%. Six to nine hours after passage, the initial morphometric characteristics of the cells settled on the substrate were measured. For this purpose, photomicrographs of different zones with cultured cells (prepared using an inverted phase-contrast microscope) were subjected to the respective measurements. At the end of the lag period, 24 h after passage, the culture medium in six examined groups of cultures (except the control group 1) was replaced by modified media containing the tested agents. The initial composition of the medium in group 1 was not subjected to modification. In groups 2 and 3, 0.2 and 2.0 mg/ml Cerebral (Dniprofarm, Ukraine) were added, correspondingly, to the normal medium. Cells of group 4 were cultured in the medium containing 400 ng/ml NGF (Sigma, USA); those of group 5 were cultured in the presence of 1.0 µM verapamil (Darnitsa, Ukraine). Finally, cells of groups 6 and 7 were subjected to combined actions of 0.2 and 2.0 mg/ml Cerebral + 1.0 µM verapamil, respectively. After replacing the media, i.e., on the 2nd day of culturing, the examined cultures were photographed again, and this procedure was repeated every 24 h, up to the end of the culturing test period. On the photomicrographs obtained, we measured the densities of cultured cells (their numbers per 1 mm 2 of the substrate), the relative numbers of cells possessing well-expressed processes, and the projective areas of the cell somata; the latter parameter was measured using a semiautomatic image analyzer.

RESULTS The proliferative activity of the cells under study was characterized according to changes in their density on the surface of settling within different

N. Kh. Pogorelaya et al.

16 periods of culturing. The variability of this parameter within different regions of these surfaces was quite noticeable; this is why we calculated mean values of the density in each group. For this purpose, we measured the number of cells within a few (up to 15) randomly selected square test regions 400 × 400 µm 2, and the values obtained were re-calculated for a 1.0 mm 2 area. The initial averaged values of the density measured 6 to 9 h after passage varied in different groups from 73 ± 6 to 105 ± 7 mm – 2. To provide more objective characterization of the process of proliferation in these groups, we used normalized values of the above parameter; the mentioned initial values in each group were taken in each case as 100%. Twenty-four hours after passage, i.e., at the beginning of the test period, the mean normalized density of the cells in different groups varied from 130 to 94% with respect to the initial values. The differences between the initial densities and the above day-2 values in each group in most cases did not reach the significance level. The intergroup comparison of the densities measured on day 2 of culturing, i.e., on the 1st day of the test period, also demonstrated the absence of significant differences (Fig. 2A-C). Within the 1st to 3rd days of the test period (days 2 to 4 of culturing), the density of PC-12 cells in group 1 (control) increased approximately four and half times. On the 4th day, this parameter stayed at a value rather close to the mentioned above. At the end of the culturing period, the cell density in the control group decreased noticeably but was still more than three times higher than the initial one (A-C). Culturing of PC-12 cells in the medium containing 0.2 mg/ml Cerebral (group 2) resulted in considerable (within days 4 to 6, highly significant) suppression of proliferative activity. Within days 2 to 4 of the test period, the density of the cells remained nearly constant (varying, on average, from 179 to 190% of the initial value). Later on, this parameter began to increase slowly; on the 6th (last) day of the test period, the density reached 255% of the initial one (Fig. 2A). When a greater amount of Cerebral was added to the medium (2.0 mg/ml, group 3), suppression of proliferative activity of PC-12 cell was more intense. On the 3rd day of the test period, the increment in the cell density, as compared with the initial value, did not exceed 40%, and nearly the same level was maintained on the 4th day. Only on the 6th day, the mean value of the density reached 240% of the initial one (Fig. 2B).

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C Fig. 1. Examples of photomicrographs of pheochromocytoma cells at different terms of culturing under different conditions. A-C) Cells on day 1 of culturing (7 h after passage) in the normal medium (A), on day 3 of the test period in the presence of 2.0 mg/ml Cerebral (group 3; B), and on day 3 of the test period in the presence of 400 ng/ml NGF (group 4; C).

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Fig. 2. Dynamics of proliferative activity of PC-12 cells under different culturing conditions. A-C) Graphs of changes in the mean normalized values of the density of cells on the substrate (ordinate, %) during the test period (abscissa, days); s.e.m. values are also shown. Values of the cell density, mm‒2, in different groups before replacing of the normal medium by the media containing test agents are, in all cases, taken as 100%. Group 1, control; 2-7, culturing in the presence of 0.2 and 2.0 mg/ml Cerebral (2 and 3, respectively), 400 ng/ml NGF (4), 1.0 µM verapamil (5), and combinations of 0.2 and 2.0 mg/ml Cerebral + 1.0 µM verapamil (6 and 7, respectively).

The medium containing 400 ng/ml NGF practically entirely suppressed proliferative activity of cultured PC-12 cells in group 4. On the 2nd day after the4 beginning of action of this factor, the density of the cells did not increase but even demonstrated a more than twofold drop (on average, to 49% of the initial value). During the subsequent 3 days of culturing, this parameter was maintained at levels lower than the initial one (58-76%). Only on the 6th day, the average density of PC-12 cells surpassed the initial value, but only by 13% (Fig. 2C). The addition of 1.0 µM verapamil to the medium (group 5) resulted, in general, in some decrease in proliferative activity as compared with the control, but the effect was not as powerful as in the case of action of NGF. On the 2nd day of the test period, the density of PC-12 cells showed nearly no increase with respect to the initial value; then, this parameter began to increase, but noticeably more slowly than in the control group 1. On the 6th day, the cell density in group 5 exceeded the initial value, but less than three times (reaching about 280%). In this group, a decrease in the density during days 5 and 6 of the test period typical of group 1 (control) was not observed (Fig. 2C). The combined influence of Cerebral and verapamil on cultured PC-12 cells resulted, in general, in somewhat more intense suppression of proliferation than isolated influence of Cerebral in both tested doses. The graphs reflecting the dynamics of the effects of 0.2 and 2.0 mg/ml Cerebral + 1.0 µM verapamil (groups 6 and 7) were mostly located below the curves corresponding to the isolated actions of Cerebral in both

above-indicated doses (groups 2 and 3, Fig. 2A, B), while differences between the corresponding values of one day of observation or another were usually insignificant. Therefore, we can, in general, conclude that 1.0 µM verapamil potentiates, to some extent, the antiproliferative effects of Cerebral. Dynamics of the Number of Cells Possessing Processes. Before beginning of culturing, PC-12 cells in suspension were spherical or ellipsoidal. After settling on the substrate (6 to 9 h after passage), such cells mostly preserved the above shape (Fig. 1A). In 24 h, however, these cells, after their fixation to the substrate, rather frequently demonstrated primary signs of morphological differentiation. Such units were spread out across the substrate, their somata acquired a polygonal form, and the cytoplasm of many cells formed protrusions comparable in their length with the dimension of the cell soma or even exceeding this value. Such formations having no typical neuritelike structure are usually qualified as pseudoprocesses [7-9]. In the control group, the proportion of cells possessing such processes then progressively decreased, while the number of cells of spheroidal or ellipsoidal shapes increased. On the 1st day of the test period, units of the control group 1 possessing clearly expressed processes constituted more than one-third of the population. At the end of the above period, the percentage of such cells only somewhat exceeded 7% (Fig. 3A-C). Test culturing of PC-12 cells in the media containing factors promoting the process of cell differentiation resulted in significant intensification of growth of the processes. In many cells, such processes were rather

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Fig. 3. Dynamics of relative numbers, %, of PC-12 cells possessing clearly manifested processes within the test period, days. Designations of the experimental groups are similar to those in Fig. 2.

long and acquired a typical neurite-like form. In a part of them, ramifications of the 2nd and even 3rd order were present. The latter type of processes was observed in a small part of the units usually not exceeding 1 or 0.5% (Fig. 1B, C). In other units, characteristics of the cytoplasmic protrusions (even those having a rather considerable length) corresponded to the structure of pseudoprocesses (B). At the same time, many cells had no long processes and preserved a smoothed polygonal or rounded form. Such heterogeneity of the observed pictures was the reason for the mode of our calculations. When examining the corresponding cell groups, we took into account all units with processes longer than the dimension of the cell soma, independently of the structure of these processes (neurite-like or other). Normalized numbers of the cells possessing processes, when measured on the 1st day of the test period, i.e., after replacement of the normal medium by the test media, varied in different experimental groups from 32.2 to 27.8%. In other words, these values were quite comparable with the corresponding values in group 1. In group 2 influenced by 0.2 mg/ml Cerebral, the proportion of cells with processes was maintained approximately stable up to the 3rd day of the test period. On the 4th day, this index increased to 36.1% and then noticeably dropped (Fig. 3A). The dynamics of the number of cells with processes in group 3, where 2.0 mg/ml Cerebral was added to the medium, demonstrated a certain similarity with the above described one, but in this group a rather sharp rise in the mentioned proportion (to 37.2%) occurred earlier, on the 3rd day of the test period (B). A dramatic increase in the number of cells having processes was observed in the case of action of NGF (group 4). This shift developed as early as on the

next day from the beginning of action of this factor. In group 4, the normalized number of such units exceeded 50% during the 2nd and 3rd days of the test period. Later on, this index decreased noticeably, but even at the end of the culturing period more than one-third of the observed cells possessed rather long processes (Fig. 3C). The action of 1.0 µM verapamil (group 5) also resulted in significant increases in the number of cultured cells with processes. This effect, however, was not as intense as in the case of the NGF action, and its maximum was reached only on the 4th day after replacing the media. At the end of the test period, the respective index noticeably decreased but remained considerably higher than that in the control group 1. Under conditions of culturing in the medium containing a combination of 0.2 mg/ml Cerebral and verapamil (group 6), the number of cells possessing processes was somewhat greater within the first 2 days of the test period than the analogous values under conditions of isolated action of this Cerebral concentration. Later on, such a difference was practically not observed (Fig. 3A). If verapamil was added to 2.0 mg/ml Cerebral (group 7), there was no noticeable difference between the numbers of cells with processes and the respective values in group 3 (isolated action of Cerebral in the mentioned concentration) (B). Dynamics of the Dimensions of PC-12 Cells. As was mentioned in Methods, the projective area of the cell soma was the directly measured parameter characterizing the dimensions of cultured cells. When this index was measured, processes thinner than 0.2 to 0.15 of the soma diameter were ruled out from consideration. The technique of measurement of the projective area of a cell unit, which is extensively

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Fig. 4. Dynamics of mean normalized values, %, of the conventional diameter, Dcon, of the somata of PC-12 cells within the test period, days. Values of the mean Dcon, in different groups before modifications of the media (i.e., beginning of action of the test agents) are taken in all cases as 100%. Other designations are similar to those in Fig. 2.

used in morhpometric studies and was applied earlier in our laboratory [7-9], allows one to objectively characterize the dimensions of 2D objects having a complex geometric form. Nonetheless, a disadvantage of this approach is the following. The values obtained are proportional not to the linear dimensions of the object but to these values squared. Attempts to directly measure the above linear dimensions (e.g., diameters of an approximating ellipse or perimeter of the cell profile), as a rule, cannot provide sufficient accuracy. This is why we used in our study an earlier proposed approach, namely calculation of a conventional diameter, D con, of the measured unit. This is the diameter of a circle equivalent in its area to the measured projective area. Such a calculation makes it possible to reduce the characteristics of linear dimensions of the complex objects to a single parameter, although a conventional one. This measure significantly simplifies statistical processing. Such a technique is rather elementary; nonetheless, it demonstrated sufficiently high validity [10]. Values of the D con calculated according to the values of projective areas varied in different groups within the 1st day of culturing (6 to 9 h after passage) from 20.8 ± 0.4 to 22.0 ± 0.3 µm. These values were taken in all cases as 100%, and the dynamics of dimensions of cultured cells were, respectively, characterized by changes in the normalized mean values in the D con. The latter values for the PC-12 cell somata varied on the 1st day of the test period from 106.7 to 113.9% of the initial values. Let us recall that within this day the cells settled on the substrate and were spread out. They acquired a flat form, and many of them formed pseudoprocesses; these protrusions were not taken into account in the area measurements. In the control

group 1, the mean D con first insignificantly decreased but within the subsequent 2 days increased to 114.4% (P > 0.05). At the end of the culturing period, this index practically returned to the initial value (Fig. 4). In group 2 subjected to the action of 0.2 mg/ml Cerebral, the D con significantly increased on the 3rd day of the test period (to 128.2%, as compared with the initial value, P < 0.05); then this parameter dropped (Fig. 4A). A higher concentration of Cerebral (2.0 mg/ml) induced a rather significant increase in the dimension of the cells examined as early as on the 2nd day of the test period (D con reached 138.0%; P < 0.01). Practically the same value was preserved on the 3rd day, but then this index, similarly to what was observed in group 2, decreased (Fig. 4B). Application of NGF resulted in rapid and rather significant increases in the D con of cultured PC-12 cells. This was observed within the first 3 days of the test period. The average value of this parameter on the 3rd day reached 166.4% of the initial index (this shift reflected an increase in the mean projective area to nearly 280%). Then, such index was followed by a rather rapid drop. At the end of the test period, the mean D con in group 4, however, still exceeded the initial value by about 20%. The action of 1.0 µM verapamil (group 5) was manifested in a relatively moderate, by about 25%, increase in the D con of PC-12 cells on the 3rd day of the test period; later on, this index decreased (Fig. 4B). If 1.0 µM verapamil was added to 0.2 mg/ml Cerebral (group 6), the former agent noticeably augmented the effect of the organic preparation tested; increases in the dimensions of the cells were noticeably greater. On days 2 and 4 of the test period, the differences between the effects of isolated Cerebral application

20 and combined action were significant (Fig. 4A). At the same time, the addition of verapamil to 2.0 mg/ml. Cerebral practically did not modify the effect of the latter on the dimensions of cultured cells. The respective graphs were very close to each other, and values of the D con within all days of the test period did not noticeably differ from the analogous values under conditions of isolated actions of Cerebral in the mentioned high dose (Fig. 4B).

DISCUSSION The data obtained in our study can be summarized in the following manner. Pheochromocytoma cells belonging to the PC-12 strain, when cultured under control conditions (using one of the modifications of the standard technique [11]), demonstrate rather intense proliferation up to about day 5 of the test period; then this process is noticeably suppressed. A considerable part (more than one-third) of the control cells, after completion of the lag period and fixation to the substrate, had clearly expressed processes. The structure of the latter, however, corresponded to that of the so-called pseudoprocesses (even though some part of them had branches of the 2nd order). In the course of culturing under control conditions, typical neurite-like processes were not formed by PC-12 cells (with extremely rare exceptions). The normalized number of units possessing processes demonstrated an approximately fivefold decrease at the end of the culturing period. It should be supposed that this decrease, at least partly, was determined by retraction of the processes. In the course of culturing of the control group, the dimensions of the cell somata of the examined units changed comparatively moderately. Therefore, culturing of PC-12 cells in the normal (control) medium was not accompanied by differentiation of the overwhelming majority of these cells into neuron-like units, while some part of the cells preserved signs of primary differentiation even at the end of the test period. Introduction of 400 ng/ml NGF in the culture medium completely suppressed proliferation of PC-12 cells and rather rapidly evoked intense formation of the processes by such cells. Under conditions of NGF action, the relative number of cells having processes of a considerable length exceeded 50% on days 2 and 3 of the test period. In many such cells, their processes rather strictly corresponded to neurites in their morphological characteristics. Their length exceeded the dimensions of the somata many times,

N. Kh. Pogorelaya et al. and a part of such processes had ramifications of the 2nd and even of the 3rd order. At the same time, morphological peculiarities of the processes in another part of the cells of the NGF group 4 corresponded to those of pseudoprocesses. They were relatively thick and had no clear border with respect to the cell soma. In other cells, cytoplasmic pseudoprocesses were relatively small, and a noticeable proportion of the cell preserved, under conditions of action of NGF, spherical or ellipsoidal shapes and had no processes at all. Such heterogeneity of the observed morphological characteristics determined the mode of classification of the cells in our experiments. Within experimental groups 2-7, we did not differentiate cells possessing either neurites of pseudoprocesses but calculated total numbers of the units with clearly manifested processes (longer than the dimension of the soma). Besides this, in our study we did not analyze in detail the dynamics of the lengths of the processes understanding that the measurements of such a parameter are subjective to a considerable extent; the estimates obtained inevitably would be rather inaccurate. An insufficient level of synchronization before passage and testing of the effects of the agents used is the most probable reason for the observed heterogeneity of morphological characteristics of PC-12 cells subjected to induced differentiation. A considerable (on average, more than by 50%) increase in the linear dimensions of the examined units, a phenomenon clearly observed throughout the nearly entire test period, represents another aspect of the influence of NGF on cultured PC-12 cells. Thus, our data completely agree with the earlier obtained numerous proofs that NGF is a powerful inductor of the process of cell differentiation [4, 5]. Cerebral in both test doses used considerably suppressed proliferation of PC-12 cells and significantly modified the growth curves. After a short initial stage of increase, the cell density in the corresponding groups was maintained within certain segments of the test period at more or less constant levels (significantly lower than in the control). Under the influence of Cerebral, formation of the processes was noticeably intensified (though not to the same extent as under direct NGF influence). A certain part of the cells subjected to the influence of Cerebral in both concentrations used formed processes demonstrating a typical neurite-like structure (Fig. 1B). Cerebral also influenced dimensions of the cells in the respective groups (their D con), and differences from the control were significant within considerable segments of the test period. Certain dose dependence

Effects of Cerebral on Rat Pheochromocytoma Cells was observed in the effects of Cerebral. Suppression of proliferation, stimulation of the formation of processes, and increase in the cell dimensions of PC-12 cells were noticeably more intense in the case of a higher concentration of the preparation in the culture medium (2.0 mg/ml). The Cerebral-induced effects had a smaller intensity, as compared with those of NGF, and they were also more limited in time. These effects were manifested later with respect to the moment of replacement of the media and became smoothed, in general, somewhat earlier than manifestations of the NGF effect. Thus, results of our study agree well with a concept that the effects of Cerebral, the preparation probably including a few trophotropic factors of a peptide nature, are based, to a considerable extent, on augmentation of NGF synthesis. As was found, Cerebral is capable of facilitating the NGF production. The corresponding phenomena are manifested not only in vivo, but also under conditions of culturing of the cell strains, which are able to differentiate and to be transformed into neuron-like elements (in particular the PC-12 cell strain). Among the generally recognized criteria of morphological differentiation, a triad of the respective signs has been usually mentioned. These are: (i) suppression of mitotic activity, (ii) changes in cell dimensions, and (iii) formation of neurites [4, 8]. Our experiments demonstrated that processes of induced differentiation in the examined culture did not develop in a synchronized and single-valued manner. The observed morphological pattern was characterized by significant heterogeneity. Transformation of a considerable part of PC-12 cells into neuron-like cell units developed, within certain time intervals, in fact in a parallel mode with prolonged intense proliferation, and the dynamics of changes in the dimensions of examined cells were not strictly similar to the dynamics of the number of cells with significant processes. Among the latter, only a part could be considered corresponding to neurites in all their characteristics. Such heterogeneity of the morphological pattern and divergence of the processes of cell growth and differentiation were, in fact, sufficiently clearly manifested in other preceding studies carried out on rat pheochromocytoma cells [8, 9, 12, 13] and on another object, cells of murine neuroblastoma N1E115 [7], but this aspect was not specially emphasized in the respective publications. Our experiments demonstrated a certain dependence of the observed effects of Cerebral on the action of an agent influencing calcium signalization, verapamil

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(blocker of high-threshold calcium channels). Both cell growth and differentiation are highly dependent on the level of free calcium in the cytoplasm ([Ca2+]i), and this dependence is rather complex [4, 6, 9, 14]. Comparison of the results of different studies led to the conclusion that suppression of proliferation and differentiation of the cells occurs only within rather narrow ranges of [Ca 2+] i [15]. This is why blockers of calcium channels in experiments on some objects stimulated the growth of neurites, while in other objects this process under such conditions either was suppressed or underwent no changes (see [8, 9]). As is supposed, shifts of the [Ca 2+] i can either inhibit or activate cell differentiation depending on the initial value of the above index in one sort of cells or another. In addition, the effects of the above blockers, naturally, depend on their concentrations, and the sensitivity of the cells under study to these agents significantly changes in the course of culturing. As was shown in earlier studies carried out in our laboratory, calcium channel blockers, nifedipine and verapamil, can exert rather diverse effects on the cells of rat pheochomocytoma and murine neuroblastoma. In particular, it was found that 1.0 µM verapanil significantly augmented manifestations of morphological differentiation of neuroblastoma N1E115 cells, while a 100 µM concentration significantly suppressed the general viability of the mentioned cells [7]. Results of our above-described experiments on another object, pheochromocytoma PC-12 cells, agree well with the above data. Results of earlier experiments on cultured pheochromocytoma cells using another calcium channel blocker, nifedipine [8], are also quite comparable. In our tests, 1.0 µM verapamil somewhat suppressed proliferation of PC-12 cells within the observation period, maintained the proportion of the cells possessing processes at a relatively constant level (higher than that in the control), and provided some increase in the dimensions of the studied cells within certain segments of the test period. Therefore, it can be concluded that verapamil in the above dose promoted, in general, the process of differentiation of PC-12 cells, though not as intensely as NGF did. Effects of verapamil were, on the whole, comparable in their intensity with the effects of 0.2 mg/ml Cerebral but somewhat weaker than the effects of the latter in the higher concentration. As is supposed, blockers of calcium channels applied in certain concentrations are able to limit excessive calcium entry into the cell and stabilize definite [Ca 2+] i values that are optimum for the process

22 of differentiation [7, 8]. It should be taken into account that the latter process per se increases expression of calcium channels (including low-threshold, LVA, ones); this effect should increase [Ca 2+] i and can lead this index out from the optimum range [6-8, 15]. The action of blockers of high-threshold calcium channels results in noticeable modulation of not only highthreshold but also of low-threshold components of the calcium current; changes in these components within early stages of culturing can exert a crucial effect on the process of differentiation [7, 8]. Thus, the mechanisms of actions of blockers of calcium currents on cell growth and differentiation are rather complicated. Moreover, the roles of high- and lowthreshold calcium channels and interrelations between these channel structures, the level of intracellular Ca, and the state of the cytoskeleton (i.e., between the key factors influencing the above processes) can be significantly dissimilar within different stages of development of neuron-like cells [4, 6, 12, 16]. When analyzing our findings, we should probably emphasize not only the observed dependence of the effects of Cerebral on modulation of Ca entry in PC-12 cells provided by the influence of the blocker of calcium channels, but also the limited pattern of such a dependence. It should be noted that the effects of Cerebral in both concentrations used and those of 1.0 µM verapamil were not additive. As was mentioned, the combined action of these agents augmented, to a certain extent (usually insignificantly), the antiproliferative activity, as compared with the isolated effects of Cerebral. At the same time, such combination led to some increase in the number of cells having processes and also some increase in the dimensions of PC-12 cells. This effect was, however, manifested only in the case where a smaller Cerebral concentration (0.2 mg/ml) was used. At a higher dose of Cerebral, verapamil did not intensify the above aspects of the effects of this preparation.

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