N. S. Kavok

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

BackgroundNumerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC) is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNγ in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T4, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells.ResultsThe results obtained provide the first demonstration of phospholipase C, phospholipase D and protein kinase C nongenomic activation and diacylglycerol (DAG) accumulation by L-T4 in liver cells. The experiments were performed in either the [14C]CH3COOH-labeled rat liver slices or isolated hepatocytes pre-labeled by [14C]oleic acid. L-T4 activates the DAG production in a concentration- and time-dependent manner. DAG formation in stimulated cells is biphasic and short-lived event: there is an initial, rapid rise in DAG concentration and then a slower accumulation that can be sustained for a few minutes. The early phase of L-T4 generated DAG only is accompanied by phosphatidylinositol 4,5-bisphosphate level decrease and inositol 1,4,5-trisphosphate formation while the second phase is abolished by PKC inhibitor l,(5-isoquinolinesulphonyl)2methylpiperasine dihydrochloride (H7) and propranolol. The second phase of DAG production is accompanied by free choline release, phosphatidylcholine content drop and phosphatidylethanol (Peth) formation. Inhibitor of phospholipase-C-dependent phosphoinositide hydrolysis, neomycin sulfate, reduced the Peth as well as the DAG response to L-T4.ConclusionsThe present data have indicated the DAG signaling in thyroid hormone-stimulated liver cells. L-thyroxine activates a dual phospholipase pathway in a sequential and synchronized manner: phospholipase C initiates the DAG formation, and PKC mediates the integration of phospholipase D into the signaling response during the sustained phase of agonist stimulation.

Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes

BackgroundThyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T3) has been shown to prevent the α1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status.ResultsThe results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state sharply differed from PL. DAG was relatively enriched in [14C]oleic acid whereas PL were enriched in [3H]arachidonic acid.ConclusionsThe above data have indicated that thyroid hormones are important physiological modulators of DAG level in rat liver and cell plasma membranes. Age- and drug-induced malfunction of thyroid gland resulted in a prominent decrease of glycerolipid synthesis which may promote DAG accumulation in liver.

Size and shape influence of luminescent orthovanadate nanoparticles on their accumulation in nuclear compartments of rat hepatocytes

In this paper the process of nonfunctionalized negatively charged orthovanadate nanoparticle accumulation and redistribution in cells dependent on their shape and size was investigated. Aqueous colloidal solutions of nReVO4:Eu(3+) (Re=Gd, Y, La) luminescent nanocrystals of different sizes and shapes have been synthesized. The average sizes of spherical particles were 2, 20, and 300 nm, of spindle-like particles - 22×6.3 nm, and of rod-like particles - 57×4.4 nm. Luminescence of nReVO4:Eu(3+) nanocrystals was effectively excited by UV and visible irradiation. By means of luminescence microscopy and luminescence microspectroscopy, it has been revealed that spherical nanocrystals with an average diameter of 2 nm tend to accumulate mainly in the rat hepatocyte nuclei in situ and also in the isolated nuclei of these cells. An additional experiment has shown that nanoparticles reveal tropism to nuclear structural components. The penetration into nuclei does not require any modifications of the surface of nanoparticle and is governed by the shape and size of nanoparticle and also is determined by the cellular type.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

Nanotechniques Inactivate Cancer Stem Cells

One of the tasks of current oncology is identification of cancer stem cells and search of therapeutic means capable of their specific inhibition. The paper presents the data on phenotype characteristics of Ehrlich carcinoma cells as convenient and easy-to-follow model of tumor growth. The evidence of cancer stem cells as a part of Ehrlich carcinoma and significance of CD44+ and CD44– subpopulations in maintaining the growth of this type of tumor were demonstrated. A high (tenfold) tumorigenic activity of the Ehrlich carcinoma CD44+ cells if compared to CD44– cells was proven. In this pair of comparison, the CD44+ cells had a higher potential of generating in peritoneal cavity of CD44high, CD44+CD24–, CD44+CD24+ cell subpopulations, highlighting the presence of cancer stem cells in a pool of CD44+ cells.In this study, the ability of synthesized hybrid nanocomplexes, comprising the nanoparticles of rare earth orthovanadates GdYVO4:Eu3+ and cholesterol to inhibit the tumor growth and to increase the survival of the animals with tumors was established. A special contribution into tumor-inhibiting effect is made by each of its components. Treatment of Ehrlich carcinoma cells with two-component hybrid complex resulted in maximum reduction in the concentration of the most tumorigenic CD44high cells with simultaneous rise in the number of CD117+ cells that decreased an intensity of tumor growth by 74.70 ± 4.38% if compared with the control.

Mitochondrial potential (ΔΨm changes in single rat hepatocytes: The effect of orthovanadate nanoparticles doped with rare-earth elements

Rare-earth-based nanoparticles (NPs) are widely used as fluorescent probes for imaging in vitro and in vivo. One of the challenges that restrain NPs applications in biomedical research is their effect on subcellular structures. In this paper, the ability of lanthanide NPs to affect the cellular oxidative balance and alter the mitochondrial function was analyzed. Since size and shape mutually affect the cellular internalization and intracellular distribution of NPs, the investigations were performed with NPs of spherical (GdYVO4:Eu3+, spindle-(GdVO4: Eu3+ and rod-like (LaVO4: Eu3+ shapes. Quantitative microfluorimetry with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide) as a mitochondrial probe was used for monitoring of the mitochondrial transmembrane potential (ΔΨm) in single living cells. Changes in the ratio of the JC-1 probe fluorescence were used to analyze the NPs effect on ΔΨm. The fastest suppressive effect (within 1 hour) was found for spherical NPs. Gradual lowering of ΔΨm was observed at the exposure of cells within 24 hours for all types of NPs. Exogenous thiols were required for ΔΨm protection. The protective role of exogenous glutathione (GSH) proves that the increase of reactive oxygen species (ROS) formation with depletion of GSH can mediate NPs toxicity. The dynamics of the shape-dependent effect can be explained by the features of NPs transportation into cells.Graphical abstract

Reactive oxygen species generation in aqueous solutions containing GdVO 4 :Eu 3+ nanoparticles and their complexes with methylene blue

It this letter, we report the study of free radicals and reactive oxygen species (ROS) generation in water solutions containing gadolinium orthovanadate GdVO4:Eu3+ nanoparticles (VNPs) and their complexes with methylene blue (MB) photosensitizer. The catalytic activity was studied under UV-Vis and X-ray irradiation by three methods (conjugated dienes test, OH· radical, and singlet oxygen detection). It has been shown that the VNPs–MB complexes reveal high efficiency of ROS generation under UV-Vis irradiation associated with both high efficiency of OH· radicals generation by VNPs and singlet oxygen generation by MB due to nonradiative excitation energy transfer from VNPs to MB molecules. Contrary to that under X-ray irradiation, the strong OH. radicals scavenging by VNPs has been observed.

Estimation of luminescent properties of the derivatives of polymethine probes on their interaction with cells of different types

The spectral and luminescent properties of the derivatives of polymethine probes based on 3,3′-dialkyloxacarbocyanine bromide (H-510) and 1,1′-dialkyl-3,3,3′,3′-tetramethylindodicarbocyanine bromide (D-307) (where alkyl is ethyl or octadecyl groups) on accumulation in bone marrow and liver cells of rats have been compared. The general regularities in the interaction between the probes and the membrane microenvironment have been revealed for each type of the cells. It was found that the structural features of membranes and the length of the chains of alkyl substitutes essentially affect the redistribution of dye molecules in the binding centers of particular types, as evidenced by changes in the spectral characteristics of probes. It has been shown by microfluorimetry that the accumulation of short-chain derivatives in single cells is representative of the distinctions in the parameters of their functional metabolic state, which coincides with the present view of the heterogeneous structure of these cellular populations. The hormonal action and the influence of incubation without serum on the intensity of cellular processes were accompanied by changes in the fluorescence of cells stained with the ethyl derivatives of the probes H-510 and D-307. This fact allows us to consider them as optical indicators of the cellular activity. The dye D-307 was found to be more resistant to photodestruction than H-510 both in single cells and model system.

A study of the effect of adrenaline on the transmembrane potential of the plasma membrane of hepatocytes from rats of different age using fluorescent probes

The resting membrane potential of isolated hepatocytes from 2- and 20-month-old rats and its changes upon activation of cells by adrenaline have been studied by the method of quantitative microfluorimetry using diethyl derivatives of polymethine probes (H-510 and D-307). The potential was estimated by the Nernst equation adapted to lipophilic cationic probes. It was shown using both probes that the transmembrane potential of hepatocytes decreases with age. The microfluorimetry data were confirmed by the results of spectrofluorimetric measurements in a cell suspension. Changes in fluorescence occurring in adrenaline-activated single cells and suspensions were unidirectional, the effect of the hormone on the cells of old animals being less pronounced. The results indicate that the potential of the plasma membrane of individual hepatocytes can be adequately estimated by microfluorimetry, which can be used in metabolic and toxicologic investigations.