Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations

Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase.     

Modulation of voltage-gated sodium channels by recombinant interferon-alpha2b in the human neuroblastoma cell line IMR-32

Clonal human neuroblastoma cells imr-32 are a suitable model system for studies of neuronal excitability modulation. The ability interferon-alpha 2b "laferon" to modulate the mechanisms of electrical activity was studied in whole-cell patch-clamped undifferentiated human neuroblastoma cells IMR-32. It was shown that 1 h incubation of IMR-32 cells at 37 degrees C in medium with laferon (600 U/ml) exerted changes in voltage-dependent properties of Na(+)-channels. The results of the present study demonstrate that laferon decreased of Na(+)-channels sensitivity to changes of membrane potential leading of IMR-32 cells electrical excitability decrease.     

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 interferon-α 2b on cells from human embryonic nerve tissue in the early stages of neurogenesis

We studied the effects of interferon (IFN)- 2b on cells obtained from the brain of human embryos (4 to 12 weeks of gestation). It was demonstrated that IFN exerts modulatory effects on biochemical and physico-chemical properties of cells of embryonic nerve tissue in the early stages of embryonic development (from 4 weeks of gestation). IFN decreased the content of protein, inhibited the activity of Na⁺,K⁺-ATPase, and induced changes in the superficial charge of the plasma membrane. Based on the obtained experimental data, we suppose that IFN- 2b is involved in regulation of neurogenesis.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 363–369, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

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.     

The Ca2+-activated photoprotein obelin as a calcium transport inducer in proteoliposomes in the membrane of the T-system of skeletal muscles]

The calcium-binding photoprotein obelin extracted and purified from the luminescent hydroid Obelia longissima was used to record the processes of Ca2+ release from proteoliposomes. It has been shown that lecithin proteoliposomes with incorporated rabbit skeletal muscle T-system membranes possess a BAY K-8644-activated permeability which is inhibited by nitrendipine. The Ca(2+)-activated photoprotein obelin is a convenient and perspective tool in studies of fast calcium fluxes.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

α2-Interferon- and oligoadenylate-induced morphological differentiation and modulation of the ion channels in neuroblastoma cells

The experiments were carried out on the IMR-32 human neuroblastoma and NIE-115 murine neuroblastoma cultured cells. Peculiarities of the ion channel expression and their correlation with the main morphological parameters characterizing neuronal differentiation were studied under conditions of incubation of the cells with 2-interferon and 2,5-oligoadenylate, 2–5A. Twenty-four hours after addition of 1000 U act./ml interferon to the culture medium, a 56% increase in the mean projective surface of the IMR-32 cells was observed, and after a nine-day-long exposition this increase was 132%, as compared with the control. Mean total length of the cellular protrusions nearly doubled after nine-day-long incubation. Morphological and electrophysiological properties of the N1E-115 murine neuroblastoma cells showed practically no changes after incubation with human 2-interferon. Cultivation of the IMR-32 human neuroblastoma cells in the presence of 2–5A evoked insignificant changes in their morphological parameters. By contrast, the mean total length of neurites of the N1E-115 neuroblastoma protrusion-supplied cells increased more than a factor of five after eight-day-long incubation with 2–5A in a 1.0 µM concentration, and at a 0.01 µM 2–5A concentration this increase was about fourfold. At the same time, the projective surface exhibited no significant changes either in the neurite-supplied or the neurite-free cellular subpopulations. Twenty-four hours after the incubation with human 2-interferon had been begun, the density of sodium current in the IMR-32 human neuroblastoma cells increased by 250% compared with the control. A similar effect was observed after the addition of 2–5A to the medium: the density of sodium current approximately doubled. Cultivation of the neurite-supplied N1E-115 murine neuroblastoma cells was followed by a gradual increase in the density of fast sodium current both in control and 2–5A-influenced samples, but in the latter case this increase was significantly faster. In the neurite-free cells, the density of sodium current was 27% higher after their 24-h-long incubation with 2–5A, as compared with the control values (11.05±0.9 and 8.7±0.9 pA/pF, respectively). Longer incubation resulted in a sharp decrease in the density of potassium current. The results of our study are in agreement with the data about species-related individuality of 2-interferon and different intensity of its effects on the cells passing different stages of cellular differentiation.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 199–207, May–June, 1995.