Effect of intracellular administration of L-tyrosine and L-phenylalanine upon voltage-dependent calcium current in PC 12 pheochromocytoma cells

Using the voltage clamp technique under conditions of intracellular perfusion, we investigated changes in high-threshold voltage-dependent calcium current (I_Ca) in the surface membrane of PC 12 cells caused by intracellular administration of the aromatic amino acids L-tyrosine and L-phenylalanine. Administration of L-tyrosine (20 mmole/1) prevented decrease in I_Ca caused by perfusion of the cell with an artificial saline solution and had a transient restorative effect upon the cell. Administration of L-phenylalanine (20 mM) quickened the decrease in amplitude of I_Ca observed in the control. These effect of aromatic amino acids are maintained when ATP (2 mM) in the intracellular solution is replaced by an equivalent quantity of ADP. The tyrosine hydroxylase blocker -methyl D,L-tyrosine (20 MM) had an effect upon I_Ca analogous to that of L-tyrosine.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 105–111, January–February, 1991.     

Effect of an epoxy derivative of 2′5′-trioligoadenylate on human neuroblastoma cells

We studied the effect of an epoxy derivative of dephosphorylated 2′,5′-trioligoadenylate (5′,5′ApApAepoxy) resistive to the action of cellular phosphodiesterase on cells of human neuroblastoma IMR 32 cultured in vitro. Twenty-two hours after the addition of 5·10⁻⁶ M 2′,5′ApApAepoxy to the culture medium, the number of cells decreased by 20% (P < 0.05), while the content of protein in these cells increased, on average, by 52% (P < 0.01), as compared with the control. The activities of Na⁺,K⁺-and Ca²⁺, Mg²⁺-ATPases in a microsomal fraction obtained from cells cultured in the presence of 2′, 5′ ApApAepoxy decreased by 50% (P < 0.001) as compared with those in the control cells. Our data indicate that 2′,5′ApApAepoxy possess antiproliferative activity. According to our findings, the antiproliferative effect of 2′,5′ ApApAepoxy can, to a great extent, be explained by the fact that this oligoadenylate derivative significantly modulates the activities of Na⁺,K⁺-and Ca²⁺,Mg²⁺-ATPases. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 97–102, March–April, 2006.     

Effect of dephosphorylated 2′,5′-trioligoadenylate on the entry of sodium ions into human neuroblastoma cells

Using a radioisotope technique, we studied the effect of dephosphorylated 2′,5′-trioligoadenylate (2′,5′ ApApA) on the entry of sodium ions into cultured human neuroblastoma cells (IMR 32 strain). Short-term (nearly 1 h) action of 2′,5′ ApApA did not influence the entry of sodium ions through voltage-operated sodium channels in the absence of neurotoxins modulating the characteristics of these channels (toxin of a scorpion, Leiurus quinquestriatus, + veratrine). At the same time, 2′,5′ ApApA weakened in a dose-dependent manner the entry of sodium ions through sodium channels opened upon the action of the above neurotoxins. In cells cultured for 22 h in a medium containing 5 · 10⁻⁶ M 2′,5′ ApApA, the entry of sodium ions in the absence of neurotoxins was 25% greater, while in the presence of neurotoxins it was 24% smaller than that in the control cells. Tetrodotoxin (TTX, 4 · 10⁻⁷ M) blocked completely sodium entry through sodium channels in cells cultured in the absence of 2′,5′ ApApA, while in cells cultured in the presence of this adenylate TTX decreased the entry by 64%. It is hypothesized that long-lasting action of 2′,5′ ApApA results in the appearance of voltage-operated TTX-insensitive sodium channels in the plasma membrane of IMR 32 cells. Our data show that 2′,5′ ApApA is capable of modulating to a considerable extent the functioning of mechanisms controlling transport of sodium ions in cells of human neuroblastoma cells of the IMR 32 strain. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 3–8, January–February, 2008.     

Effect of long-term depolarization on the morphological characteristics of rat pheochromocytoma cells

Changes in the morphological characteristics of rat PC12 pheochromocytoma cells caused by incubation in the medium with a high KCl content were studied. On the third day of culturing in the modified medium containing 20 or 30 mM KCl, the proliferating activity of the cells was enhanced, and the total length of the dendrites and their number increased. Culturing of the cells in the medium containing 40 mM KCl resulted in significant suppression of the cell vital functions: retraction of the processes and blocking of proliferation were observed. Thus, chronic depolarization caused by a high potassium content in the extracellular medium activates proliferation of PC12 cells, which is probably related to the change in the level of free cytosolic calcium.     

Nonuniformity of calcium efflux from pancreatic acinar cells and its analysis by mathematical model of calcium diffusion and buffering in extracellular solution

We studied spatial and temporal patterns of Ca²⁺ extrusion from pancreatic acinar cells evoked by acetylcholine(ACh)-induced activation of plasma membrane calcium pumps. Using a modification of an earlier developed model, we estimated the time course of extracellular calcium concentration changes near the basal pole of a cell in the case, when calcium ions are released from the same site on the cell surface, and in the case when they are extruded from the apical pole and diffuse to the basal one. It is concluded that at the first stage of ACh-induced Ca²⁺ extrusion the appearance of Ca²⁺ elevation near the basal pole of the cells cannot be explained as a result of diffusion, but is mainly determined by Ca²⁺ efflux from this pole. The results also show that there are plasma membrane calcium pumps in both apical and basal parts of pancreatic acinar cells, but the activity of the pumps is substantially higher in the apical region.     

Structural features of corticospinal connections in the cat

Structural features of connections between corticospinal fibers and neurons of the cervical and lumbar segments of the cat spinal cord were studied by the experimental degeneration method. It was shown by the Fink-Heimer method that the preterminals of these fibers are mainly located in the lateral part of Rexed's laminae V and VI (the lateral basilar region — LBR) and also, to some extent, in the medial basilar region (MBR). The diameters of the myelinated part of these fibers in LBR vary from 0.8 to 11µ. They form chiefly terminals of the F-type (with flattened synaptic vesicles), which undergo degeneration of the light type (lysis of the internal structures) or, less frequently, the dark type (increase in electron density), followed by phagocytosis by glial cells. No degenerating terminals are found in the glomerulus-like synaptic complexes, in axo-axonal synapses, or on dendrites with a dark matrix. Only a few degenerating axon terminals still remained 20 days or more after extirpation of the cortex. The relative number of terminals of different types was counted at this period. The number of axon terminals of F-type on the dendrites was reduced by 1.5 times, while the number on the soma remained relatively unchanged. The results confirm the earlier hypothesis that corticospinal fibers terminate on dendrites and their appendages in LBR as endings of the F-type. These neurons also receive many terminals from other intracerebral systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol.4, No.5, pp. 480–487, September–October, 1972.     

Experimental morphological study of primary afferent terminals at the base of the dorsal horn of the cat spinal cord

The distribution and ultrastructure of primary afferent terminals in the gray matter of the cervical and lumbar regions of the cat spinal cord were studied by the experimental degeneration method of Fink and Heimer. Most preterminals of primary afferents were shown to be concentrated in the region of the intermediate nucleus of Cajal (central part of Rexed's laminae VI–VII), in the substantial gelatinosa (laminae II–III), and in the nucleus proprius of the dorsal horn (central and medial parts of lamina IV). Fewer are found in the region of the motor nuclei. The number of degenerating axon terminals in the lateral parts of laminae IV and V differed: 31.5 and 0.4% respectively of all axon terminals. Many terminals of primary afferents in lamina IV contribute to the formation of glomerular structures in which they exist as terminals of S-type forming axo-axonal connections with other terminals. These results are in agreement with electrophysiological data to show that interneurons in different parts of the base of the dorsal horn differ significantly in the relative numbers of synaptic inputs formed by peripheral afferents and descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 406–414, July–August, 1973.     

Probable Mechanisms of the Effects of Cerebral on Morphological Characteristics of Rat Pheochromocytoma Cells

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.     

Effect of Recombinant Interferon-α2b (Laferon) on Transport of Sodium Ions in Human Neuroblastoma Cells

To elucidate molecular mechanisms of neurotropic action of a recombinant interferon, IFN-2b (laferon), its effect on transport of ²²Na⁺ through the membrane of cultured human neuroblastoma cells (line IMR 32) was investigated. Within the first minutes after treatment with IFN-2b, the influx of ²²Na⁺ ions was reduced by 20%, as compared with the control. Depolarization of the plasma membrane by a mixture of veratrine and scorpion (Leiurus quinquestriatus) toxin (200 and 10 g/ml, respectively) increased this flux by 50% in the control and by 70% in the IFN-2b-treated cells. A blocker of voltage-operated sodium channels, tetrodotoxin (TTX, 4 · 10^-7 M), suppressed the inward flux of ²²Na⁺ ions (completely in the control cells and by 75% in the IFN-2b-treated cells). The influx of ²²Na⁺ ions into neuroblastoma cells depended on the concentration of IFN-2b in the incubation medium, reaching a maximum at concentrations of 600-1000 IU/ml. This allows us to suggest that entry of Na⁺ ions into neuroblastoma cells caused by IFN-2b is basically performed through voltage-operated TTX-sensitive sodium channels.