Nicotinic acetylcholine receptor-mediated effects of varenicline on LPS-elevated prostaglandin and cyclooxygenase levels in RAW 264.7 macrophages

Introduction: The purpose of this study is to delineate anti-inflammatory and antioxidant potential of varenicline, a cigarette smoking cessation aid, on decreasing lipopolysaccharide (LPS)-elevated proinflammatory cytokines in RAW 264.7 murine macrophage cultures which we showed earlier to occur via cholinergic anti-inflammatory pathway (CAP) activation. To this end, we investigated the possible suppressive capacity of varenicline on LPS-regulated cyclooxygenase (COX-1 and COX-2) via α7 nicotinic acetylcholine receptor (α7nAChR) activation using the same in vitro model.Materials and Methods: In order to test anti-inflammatory effectiveness of varenicline, the levels of COX isoforms and products (PGE2, 6-keto PGF1α, a stable analog of PGI2, and TXA2) altered after LPS administration were determined by Enzyme Linked Immunosorbent Assay (ELISA). The antioxidant effects of varenicline were assessed by measuring reductions in reactive oxygen species (ROS) using a fluorometric intracellular ROS assay kit. We further investigated the contribution of nAChR subtypes by using non-selective and/or selective α7nAChR antagonists. The results were compared with that of conventional anti-inflammatory medications, such as ibuprofen, celecoxib and dexamethasone.Results: Varenicline significantly reduced LPS-induced COX-1, COX-2 and prostaglandin levels and ROS to an extent similar to that observed with anti-inflammatory agents used.Discussion: Significant downregulation in LPS-induced COX isoforms and associated decreases in PGE2, 6-keto PGF1α, and TXA2 levels along with reduction in ROS may be partly mediated via varenicline-activated α7nAChRs

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

TRPC1 and store-operated Ca2+ (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, inTRPC1-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

Bazı kalsiyum giriş blokörü ilaçların tavşan tüm kulak preparatı üzerindeki etkilerinin araştırılması

Bu tezin, veri tabanı üzerinden yayınlanma izni bulunmamaktadır. Yayınlanma izni olmayan tezlerin basılı kopyalarına Üniversite kütüphaneniz aracılığıyla (TÜBESS üzerinden) erişebilirsiniz.ÖZET Temel kullanımları periferik vazodilatör etkinliklerine dayanan, klinikte geniş kullanım alanı bulan kalsiyum giriş blokörü ilaçlardan verapamil, sinnarizin ve diltiazemin, kalsiyum iyonunun hücre içine girişi üzerindeki inhibitor etkinlikleri, periferik vasküler sistemi temsil eden tavşan tüm kulak preparatında incelenmiştir. Çalışmada direkt etkili düz kas gevşetici bir ilaç olan papaverin de kontrol amacıyla kullanılmıştır. ECsso(M) ve EDsao(m) değerleri karşılaştırıldığında verapamil, sinnarizin, diltiazem ve papaverinin reseptöre bağımlı ve voltaja bağımlı kalsiyum girişine karşı farklı güçte etki gösterdikleri saptanmıştır. Etkinin zamana karşı seyri incelendiğinde; sinnarizin ve verapamilin geç başlayıp uzun süren, diltiazem ve papaverinin ise çabuk başlayıp kısa süren bir etki profiline sahip oldukları gözlenmiştir. Elde edilen bulgular tavşan tüm kulak preparatının kalsiyum giriş blokörü ilaçların periferik etkilerinin incelenmesi ve karşılaştırılması için basit, etkin ve güvenilir bir preparat olduğunu ortaya koymuştu

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

WOS: 000376479400010PubMed ID: 26944908We 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.Ege UniversityEge University [BAP-14ECZ022]This work was supported by the Ege University Research Project [BAP-14ECZ022] to Cigdem Selli

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

WOS: 000378646700040PubMed ID: 27513427Background/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.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [104S568]; Ege UniversityEge University [BAP-02ECZ006]This work was supported in part by the Scientific and Technological Research Council of Turkey (TUBITAK, 104S568, to MT) and Ege University (BAP-02ECZ006, to MT)

TRPC1 ion channel gene regulates store-operated calcium entry and proliferation in human aortic smooth muscle cells

WOS: 000391393100024This study investigates whether the reciprocal changes in transient receptor potential canonical (TRPC) 1 and TRPC6 expressions, which have previously been observed in aging rat aorta, are functional in store-operated calcium (SOC) entry and proliferation in human vascular smooth muscle cells. TRPC1 levels were modulated via silencing and overexpression vectors in human primary aortic smooth muscle cells. Following TRPC1 gene modulation, TRPC1 and TRPC6 expression levels were measured using quantitative real-time RT-PCR. In functional analyses, real-time changes in intracellular calcium levels and cell proliferation were determined. Microarray analysis was performed to identify genes associated with functional alterations following TRPC1 silencing. TRPC1 expression was significantly increased in TRPC1-overexpressing cells and inhibited in TRPC1-silenced cells, as expected. TRPC6 expression was significantly decreased in TRPC1-overexpressing cells but not affected by TRPC1 silencing. SOC entry was significantly enhanced in TRPC1-silenced cells but not altered by TRPC1-overexpression. Furthermore, cell proliferation was correlated with changes in TRPC1 expression. Microarray analysis revealed that cell cycle-associated genes were significantly differentially expressed in TRPC1-silenced cells. In addition, STIM1 levels were downregulated significantly following TRPC1 silencing. Data suggest that TRPC1 has a functional role in SOC entry regulation as well as in human aortic smooth muscle cell proliferation.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110S096]; Ege UniversityEge University [EBILTEM 11BIL004]; European Molecular Biology Organization (EMBO) Short-Term Fellowship [ASTF 115-2013]; State Planning Organization Infrastructure ProjectTurkiye Cumhuriyeti Kalkinma Bakanligi [DPT 2009K120640]This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, 110S096 to MT), and in part by Ege University (EBILTEM 11BIL004 to MT). Support to conduct bioinformatics analyses at the Edinburgh Cancer Research Centre was provided by a European Molecular Biology Organization (EMBO) Short-Term Fellowship (ASTF 115-2013 to CS). CS would like to thank Dr Andrew H Sims, Edinburgh University, UK, for his supervision during the EMBO Fellowship. Research activities were performed at the Pharmaceutical Sciences Research Laboratory (FABAL) with the support of the State Planning Organization Infrastructure Project (DPT 2009K120640 to Faculty of Pharmacy, Ege University). The authors would like to thank Dr Petek Ballar Kirmizibayrak (Department of Biochemistry, Faculty of Pharmacy, Ege University) and Dr Buket Kosova (Department of Medical Biology, Faculty of Medicine, Ege University) for their comments on the study

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

WOS: 000351020100005PubMed ID: 25535724Introduction: The measurement of intracellular Ca2+, cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca2+ levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca2+. In this study, we describe a Ca2+ 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 Ca2+ levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca2+ 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 Ca2+ levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca2+ 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 Ca2+ dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca2+ 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 Ca2+ levels and promotes understanding vascular physiology and disease. (C) 2015 Elsevier Inc. All rights reserved.Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TUBITAK 103S176, 104S568, 108S072]; Ege UniversityEge University [02ECZ006, 04ECZ011, 05BIL016]This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK 103S176, 104S568, 108S072 to M.T.) and in part by the Ege University (02ECZ006, 04ECZ011 and 05BIL016 to M.T.). Authors thank Dr. R.J. Paul, University of Cincinnati, for his comments on both apparatus design and the manuscript, and Dr. G. Gonul for making the precise wire attachments of the apparatus