Choline and citicoline ameliorate oxidative stress in acute kidney injury in rats.

OBJECTIVES: The purpose of this study is to investigate the effects of cholinergic anti-inflammatory pathway (CAP)-activating drugs, choline and citicoline (Cytidinediphosphate-choline, CDP-choline), on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) parameters and the contribution of NADPH Oxidase4 (NOX4) p22phox. BACKGROUND: Endotoxemia induces a systemic inflammatory response characterized by the production of pro-inflammatory mediators and reactive oxygen species (ROS), which eventually develops acute kidney injury (AKI). NADPH Oxidase4 (NOX4) p22phox pathway contributes to the development of endotoxemia-induced AKI. Inflammatory response can be controlled by CAP. METHODS: Expressions levels of KIM-1, TNF-α, NOX4, p22phox and NFκB in the kidney tissues of rats were analyzed via RT-PCR in experimental groups; 1. Control, 2. LPS (10 mg/kg) + saline, 3. LPS + CDP-choline (375 mg/kg) and 4. LPS + choline (90 mg/kg). Choline and ROS levels in kidney tissues were also measured by a spectrofluorometric assay. RESULTS: LPS-induced elevations of ROS levels were decreased by CDP-choline or choline administration (p < 0.001). LPS-elevated KIM-1, TNFα, NOX4, p22 phox, and NFκB expressions were significantly decreased by choline or CDP-choline treatments (p < 0.001). CONCLUSION: Decreased ROS production in kidney tissues in treatment groups suggests that choline or CDP-choline may have therapeutic potential in endotoxemia-associated AKI via downregulating NOX4 and p22phox expressions (Tab. 1, Fig. 5, Ref. 45). Text in PDF www.elis.sk Keywords: endotoxemia, choline, cytidine diphosphate choline, acute kidney injury, reactive oxygen species.

Implications of Possible HBV-Driven Regulation of Gene Expression in Stem Cell-like Subpopulation of Huh-7 Hepatocellular Carcinoma Cell Line.

Elevated levels of STIM1, an endoplasmic reticulum Ca2+ sensor/buffering protein, appear to be correlated with poor cancer prognosis in which microRNAs are also known to play critical roles. The purpose of this study is to investigate possible HBV origins of specific microRNAs we identified in a stem cell-like subpopulation of Huh-7 hepatocellular carcinoma (HCC) cell lines with enhanced STIM1 and/or Orai1 expression that mimicked poor cancer prognosis. Computational strategies including phylogenetic analyses were performed on miRNome data we obtained from an EpCAM- and CD133-expressing Huh-7 HCC stem cell-like subpopulation with enhanced STIM1 and/or Orai1 expression originally cultured in the present work. Results revealed two putative regions in the HBV genome based on the apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions identified critical human genes, of which their transcripts are among the predicted targets of miR3653, which was increased significantly by STIM1 or Orai1 enhancement. Briefly, this study provides phylogenetic evidence for a possible HBV-driven epigenetic remodeling that alters the expression pattern of Ca2+ homeostasis-associated genes in STIM1- or Orai1 overexpressing liver cancer stem-like cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the viral genotype would have a clinical impact on prognosis of HBV-induced liver cancers.

Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages.

The cholinergic anti-inflammatory pathway plays an important role in controlling inflammation. This study investigated the effects of varenicline, an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, on inflammatory cytokine levels, cell proliferation, and migration rates in a lipopolysaccharide (LPS)-induced inflammation model in RAW 264.7 murine macrophage cell lines. The cells were treated with increasing concentrations of varenicline, followed by LPS incubation for 24 h. Prior to receptor-mediated events, anti-inflammatory effects of varenicline on different cytokines and chemokines were investigated using a cytokine array. Nicotinic AChR-mediated effects of varenicline were investigated by using a non-selective nAChR antagonist mecamylamine hydrochloride and a selective α7nAChR antagonist methyllycaconitine citrate. TNFα, IL-1ß, and IL-6 levels were determined by the ELISA test in cell media 24 h after LPS administration and compared with those of dexamethasone. The rates of cellular proliferation and migration were monitored for 24 h after drug treatment using a real-time cell analysis system. Varenicline decreased LPS-induced cytokines and chemokines including TNFα, IL-6, and IL-1ß via α7nAChRs to a similar level that observed with dexamethasone. Varenicline treatment decreased LPS-induced cell proliferation, without any nAChR involvement. On the other hand, the LPS-induced cell migration rate decreased with varenicline via α7nAChR. Our data suggest that varenicline inhibits LPS-induced inflammatory response by activating α7nAChRs within the cholinergic anti-inflammatory pathway, reducing the cytokine levels and cell migration.

Effects of cell seeding density on real-time monitoring of anti-proliferative effects of transient gene silencing.

BACKGROUND: Real-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the real-time changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well-1, respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h. RESULTS: TRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well-1 seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well-1 density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well-1 for 72 h. CONCLUSIONS: Data suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells.

Effects of 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on receptor-independent and -dependent contractile responses in rat aorta.

BACKGROUND/AIM: This study investigates whether 1-(2-trifluoromethylphenyl)-imidazole (TRIM), originally proposed as a nitric oxide synthase inhibitor and also suggested to be an inhibitor of store-operated calcium entry in mouse anococcygeal muscle, inhibits receptor-independent and -dependent responses in rat thoracic aorta. MATERIALS AND METHODS: Cyclopiazonic acid- and serotonin-induced vascular responses were investigated in aortic segments isolated from male Sprague Dawley rats using isolated tissue experiments. Changes in intracellular calcium levels were also monitored via front surface fluorescence measurements in fura-2-loaded embryonic rat vascular smooth muscle cell line A7r5. RESULTS: TRIM inhibited serotonin-mediated vascular contractions without affecting cyclopiazonic acid-induced responses. In addition, TRIM caused a nonlinear rightward shift in the serotonin concentration-response curve, possibly via serotonin receptor modulation. CONCLUSION: TRIM may have an impact on investigation of tissue-specific receptor-independent and -dependent vascular responses. It may also be used as a lead compound in the development of selective serotonin receptor modulators.

Differential expression of store-operated calcium- and proliferation-related genes in hepatocellular carcinoma cells following TRPC1 ion channel silencing.

TRPC1 and store-operated Ca(2+) (SOC) entry have previously been associated with hepatocellular carcinoma cell proliferation. The aim of the study was to determine genes and processes associated with TRPC1 down-regulation and the resulting increase of SOC entry and decrease in hepatocellular carcinoma cell proliferation. For this purpose, transcriptome analysis was performed to determine differentially expressed genes in TRPC1-silenced Huh7 cells. SOC entry- and proliferation-related genes correlated with TRPC1 down-regulation were also examined. Changes in SOC entry and cell proliferation were monitored in the TRPC1-silenced and parental cells and found to be significantly increased and decreased, respectively, in TRPC1-silenced cells. A total of 71 genes were significantly differentially expressed (40 up- and 31 down-regulated), including four mitogen-activated protein kinase (MAPK) signalling-associated genes. STIM1 levels were significantly up-regulated and negatively correlated with TRPC1 levels. In addition, expression of two cell cycle regulation genes, CDK11A/11B and URGCP, was observed to decrease, whereas ERBB3 and FGFR4, pro-survival genes, increased significantly in TRPC1-silenced cells. In conclusion, these results suggest reciprocal alterations in TRPC1 and STIM1 levels and a role for STIM1 in the regulation of SOC entry in TRPC1-silenced Huh7 cells. In addition to TRPC1, STIM1 may participate in Huh7 cell proliferation by regulating SOC entry. Alterations in MAPK signalling genes may be involved in diminished cell proliferation in TRPC1-silenced Huh7 cells. Similarly, changes in cell cycle regulating genes in TRPC1-silenced cells indicate possible cell cycle arrest along with compensatory up-regulation of ERBB3 growth factor receptor-amongst others-to maintain hepatocellular carcinoma cell proliferation.

Effects of cyclopiazonic acid and dexamethasone on serotonin-induced calcium responses in vascular smooth muscle cells

We previously observed that sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) blockade by cyclopiazonic acid (CPA) significantly potentiates serotonin (5-hydroxytryptamine (5-HT))-induced vascular contractions. Furthermore, 5-HT receptor antagonist methysergide partially inhibited CPA-potentiated 5-HT contractions. In the present study, we further investigated whether SERCA inhibition potentiates 5-HT-induced Ca2+ responses along with attenuating the receptor antagonism by store-operated Ca2+ (SOC) entry and protein kinase C (PKC)-mediated mechanisms. The effects of dexamethasone that was previously shown to induce SOC entry and enhance 5-HT responses were also tested. For this purpose, intracellular Ca2+ levels were monitored in A7r5 embryonic rat vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. The results showed that CPA, although not dexamethasone, significantly potentiated 5-HT-induced Ca2+ elevations. Ketanserin partially decreased 5-HT-induced and CPA-potentiated Ca2+ elevations whereas both PKC inhibitor D-sphingosine and SOC entry blocker 2-aminoethoxydiphenyl borate (2-APB) abolished the remaining responses. The data suggests that diminished antagonistic effect on 5-HT-induced Ca2+ elevations in the presence of SERCA inhibition is induced by SOC entry and PKC activation (AU)

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Effects of cyclopiazonic acid and dexamethasone on serotonin-induced calcium responses in vascular smooth muscle cells.

We previously observed that sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) blockade by cyclopiazonic acid (CPA) significantly potentiates serotonin (5-hydroxytryptamine (5-HT))-induced vascular contractions. Furthermore, 5-HT receptor antagonist methysergide partially inhibited CPA-potentiated 5-HT contractions. In the present study, we further investigated whether SERCA inhibition potentiates 5-HT-induced Ca(2+) responses along with attenuating the receptor antagonism by store-operated Ca(2+) (SOC) entry and protein kinase C (PKC)-mediated mechanisms. The effects of dexamethasone that was previously shown to induce SOC entry and enhance 5-HT responses were also tested. For this purpose, intracellular Ca(2+) levels were monitored in A7r5 embryonic rat vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. The results showed that CPA, although not dexamethasone, significantly potentiated 5-HT-induced Ca(2+) elevations. Ketanserin partially decreased 5-HT-induced and CPA-potentiated Ca(2+) elevations whereas both PKC inhibitor D-sphingosine and SOC entry blocker 2-aminoethoxydiphenyl borate (2-APB) abolished the remaining responses. The data suggests that diminished antagonistic effect on 5-HT-induced Ca(2+) elevations in the presence of SERCA inhibition is induced by SOC entry and PKC activation.

Silencing of TRPC1 regulates store-operated calcium entry and proliferation in Huh7 hepatocellular carcinoma cells.

PURPOSE: Previously, we observed reciprocal changes in TRPC1 and TRPC6 expression levels in aging rat aorta and A7r5, rat embryonic vascular smooth muscle cells. Furthermore, downregulation of TRPC1 significantly elevated store-operated Ca(2+) entry suggesting the regulatory role of TRPC1 in A7r5 cells. Since TRPC6 upregulation shown to be associated with cell proliferation, the purpose of our study was to investigate the functional consequences of TRPC1 ion channel downregulation by RNA interference in Huh7 human hepatocellular carcinoma cell line. METHODS: Huh7 cells used in quantitative gene and protein expression as well as in functional analyses. To determine mRNA and protein levels, quantitative real-time RT-PCR and western blot analyses were performed, respectively. In functional analyses, real-time changes in proliferation, migration and intracellular Ca(2+) levels were monitored. RESULTS: In shTRPC1-transfected Huh7 cells, TRPC1 mRNA and protein levels significantly decreased whereas store-operated Ca(2+) entry significantly elevated. TRPC1-silencing suppressed cell proliferation without affecting cell migration in real-time cellular analyses. CONCLUSION: These results suggest that TRPC1 may take part both in regulation of store-operated Ca(2+) entry and proliferation of hepatocellular carcinoma cells.

Simultaneous measurement of cytosolic and mitochondrial calcium levels: observations in TRPC1-silenced hepatocellular carcinoma cells.

INTRODUCTION: The measurement of intracellular Ca(2+), cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca(2+) levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca(2+). In this study, we describe a Ca(2+) monitoring system that allows studying both adherent cells and tissues and discuss data obtained from hepatocellular carcinoma cells and rat thoracic aorta by using this system. METHODS: For this purpose, two apparatus, one for adherent cells and the other for intact rat aorta, were designed and produced. With this system, changes in cytosolic Ca(2+) levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca(2+) levels were simultaneously measured in TRPC1-silenced Huh7 hepatocellular carcinoma cells. In addition, the effects of trifluoromethylphenylimidazole (TRIM) on cyclopiazonic acid (CPA)-, serotonin (5-HT)-, and phenylephrine (PE)-induced changes in isometric force and cytosolic Ca(2+) levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated. RESULTS: After SOC entry activation, cytosolic Ca(2+) levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca(2+) levels following CPA-induced ER depletion were significantly (p<.05) diminished in TRPC1-silenced Huh7 cells. In addition, TRIM partially inhibited both 5-HT-induced contractions and cytosolic Ca(2+) levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca(2+) levels were similar in aorta from young and old (3 and 22 months, respectively) rats. DISCUSSION: We confirmed that the system provides valuable data about intracellular Ca(2+) dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca(2+) loading following CPA-induced ER depletion in TRPC1-silenced Huh7 cells suggests a possible role of TRPC1 in hepatocellular carcinoma cell apoptosis. The system also enables the simultaneous measurement of isometric force and cytosolic Ca(2+) levels and promotes understanding vascular physiology and disease.

Effects of passage number on proliferation and store-operated calcium entry in A7r5 vascular smooth muscle cells.

INTRODUCTION: Embryonic rat aortic smooth muscle cells, A7r5, have been used extensively as an in vitro vascular smooth muscle cell model. They are usually provided at 11th passage by supplier and generally used before 25th passage. However, the exact passage number (P#) used is not reported in general. METHODS: In this study, A7r5 cells (P#<18 and P#>23) were used in quantitative gene (5-HT2A and SERCA2b) expression as well as in functional analyses including measurements of real-time changes in cell proliferation and in 5-HT- and CPA-induced intracellular Ca(2+) levels. RESULTS: CPA-induced SR Ca(2+) release and store-operated Ca(2+) entry significantly increased (p<.05) while cell proliferation decreased in P#>23 cells (p<.01). Furthermore, 5-HT-induced Ca(2+) elevations and 5-HT2A mRNA levels did not change whereas SERCA2b mRNA levels and CPA-induced [Ca(2+)]i levels were significantly elevated in P#>23 cells (p<.05, p<.01, respectively). DISCUSSION: Changes in SERCA2b expression and SOCE may contribute to suppression of cell proliferation during A7r5 subculturing. Therefore, passage numbers and subculturing procedure should be reported and taken into account during expressional and functional analyses for an accurate comparison of published data.

Introducing basic molecular biology to Turkish rural and urban primary school children via hands-on PCR and gel electrophoresis activities.

This study includes the results of a 2-day education project titled "Molecular Biology Laboratory Summer School, MoBiLYO." The project was held at a University Research Center by scientists from Department of Pharmacology and graduate students. The project was composed of introductory lectures, model construction, DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis. The participants were 13-year-old eighth-graders attending primary schools affiliated with Ministry of National Education in urban and rural areas of Izmir, Turkey. The purpose of this study was to introduce basic molecular biology concepts through individually performed experiments such as PCR and gel electrophoresis integrated with creative drama. The students were assessed at the beginning and the end of each project day via mini-tests, experimental and presentation skills evaluation forms. Data showed that students' knowledge about DNA structure and basic molecular biology techniques significantly increased. On the basis of experimental and presentational skills, there was no significant difference between kids from urban and rural schools or between public and boarding public schools, whereas the average score of girls was significantly higher than that of boys. In conclusion, individually performed experiments integrated with creative drama significantly increased students' perception of complex experimental procedures on basic molecular biology concepts. Data suggests that integration of these concepts into the science and technology curriculum of Turkish primary education may support the recruitment of future scientists who can handle rapidly developing genomic techniques that will affect our everyday life.

Na(+)-K(+)-ATPase and Ca(2+) clearance proteins in smooth muscle: a functional unit.

The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.

Expression levels of TRPC1 and TRPC6 ion channels are reciprocally altered in aging rat aorta: implications for age-related vasospastic disorders.

We previously showed that the expression of transient receptor potential canonical (TRPC)6 ion channel elevated when TRPC1 was knocked down in A7r5 cultured vascular smooth muscle cells. Therefore, the purpose of this study was to explore whether TRPC6 is also upregulated in aging rat aorta comparable to that of TRPC1 in longitudinal in vivo aging model. We further investigated a possible causal relationship between altered phenylephrine-induced contractions and the expression levels of TRPC6, a purported essential component of alpha-adrenergic receptor signaling in aging aorta. Immunoblot analysis showed that TRPC1 protein levels significantly decreased whereas TRPC6 increased drastically in aorta from 16- to 20-month-old rats compared to that from 2 to 4 months. Immunohistochemical data demonstrated spatial changes in TRPC6 expression within the smooth muscle layers along with increased detection in the adventitia of the aged rat aorta. The phenylephrine-induced contractions were potentiated in aging aorta. In conclusion, based on this aging model, TRPC6 overexpression could be related with TRPC1 downregulation and might be responsible for the increased adrenoceptor sensitivity which contributes to the development of age-related vasospastic disorders.

Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca2+ entry in A7r5 vascular smooth muscle cells.

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca2+ entry and store-operated Ca2+ entry following Ca2+ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca2+ entry in vascular smooth muscle cells.