Showing papers by "Sabina Passamonti published in 2013"

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins.

Abstract: Background and aims Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. Methods and results Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. Conclusion Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Role of endothelial cell membrane transport in red wine polyphenols-induced coronary vasorelaxation: involvement of bilitranslocase

Abstract: Red wine polyphenols (RWP) induce nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-mediated coronary vasodilatation involving the redox-sensitive PI3-kinase/Akt-dependent pathway in the endothelium. However, there is a gap of knowledge in explaining how bioactive polyphenols initialize their signalling pathway in endothelial cells. Here, we investigated the hypothesis that flavonoids act subsequently to their entry into the endothelium via the flavonoid membrane transporter bilitranslocase (TC 2.A.65.1.1). Thus, vascular reactivity studies were performed using isolated porcine coronary artery rings. We separately determined the NO- and EDH-mediated components of the relaxation in the presence of specific inhibitors. In either case, bilitranslocase antibodies significantly reduced the relaxations of coronary artery rings induced by RWP. Furthermore, bilitranslocase antibodies significantly reduced RWP-induced phosphorylation levels of Akt and eNOS, assessed in cultured endothelial cells from porcine coronary arteries by Western blot analysis. The present findings indicate that bilitranslocase-mediated membrane transport substantially contributes to the initial step of RWP-induced coronary vasodilatation.

Metabonomic investigation of rat tissues following intravenous administration of cyanidin 3-glucoside at a physiologically relevant dose

Abstract: Anthocyanins, which are dietary flavonoids occurring in fruit and beverages, are reported to have a beneficial impact on a wide range of chronic diseases, such as cardiovascular, neurodegenerative and neoplastic diseases. To understand the underlying mechanisms, a biochemical description of the changes in cell metabolism caused by anthocyanins can be provided by metabonomic studies. The aim of this study was to detect changes in the profiles of metabolites induced by the administration of cyanidin 3-glucoside to adult male rats. A physiological dose of cyanidin 3-glucoside was intravenously administered, and blood, kidneys and liver were collected after 5 min. The tissues were rapidly frozen in liquid nitrogen, stored briefly at −80 °C, homogenised under cryogenic conditions and extracted in ice-cold methanol:water (95:5, v/v). The extracts were then analysed using UPLC/QTOF-MS. Multivariate statistical analysis of the data was performed using orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Discriminating variables were compared to the in-house standard database, considering matches in retention times, parent mass ions, mass fragment patterns and isotopic patterns. This metabolomic approach made it possible to identify as many as eight metabolite markers, including bile acids, reduced and oxidised glutathione and some lipids. Such changes suggest that cyanidin 3-glucoside has a major effect on tissue antioxidant status as well as on energy and glucose metabolism.

Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma.

Abstract: Background Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues.

Bilitranslocase is involved in the uptake of bromosulfophthalein in rat and human liver

Abstract: Hepatic disposition of bromosulfophthalein (BSP), bilirubin and bile salts partially overlap, as these anions share both uptake and excretion mechanisms. Multiple organic anion transporters mediate hepatic BSP uptake, i.e. members of the SLCO and SLC22 gene families and bilitranslocase (TCDB #2.A.65.1.1). This study aimed at evaluating the relative contribution of bilitranslocase in BSP uptake in precision-cut human and rat liver slices. To this purpose, two different anti-sequence bilitranslocase antibodies were used as specific, functional inhibitors of bilitranslocase. The intact liver physiology was accurately reproduced in this BSP uptake assay, since uptake was strongly temperature-dependentand inhibited by hepatotropic organic anions, such as 50 nM bilirubin, 1 μM nicotinic acid, 2 μM digoxin, 5 μMindocyanine green and 100 μM taurocholate. The bilitranslocase antibodies inhibited BSP uptake both in rat and human liver slices. The combined use of bilitranslocase antibodies and taurocholate caused additive-type inhibition, confirming that bilitranslocase is not a bile salt transporter; by contrast, bilirubin caused no additive-type inhibition. In conclusion this data, indicate the role of the bilirubin transporter bilitranslocase as one of the transporters involved in the uptake of anions like BSP in parallel with other organic anion carriers. Moreover this data indicate the value of precision-cut liver slices for phenotypic drug uptake studies.

Cytotoxicity and uptake of archaeosomes prepared from Aeropyrum pernix lipids.

Abstract: Archaeon Aeropyrum pernix K1 is an obligate aerobic hyperthermophilic organism with C25,25-archeol membrane lipids with head groups containing inositol. Interactions of archaeosomes, liposomes prepared from lipids of A. pernix, with mammalian cells in vitro were studied. In vitro cytotoxicity was tested on five different cell lines: rodent mouse melanoma cells (B16-F1) and Chinese hamster ovary (CHO) cells, and three human cell lines-epithelial colorectal adenocarcinoma cells (CACO-2), liver hepatocellular carcinoma cell line (Hep G2) and endothelial umbilical vein cell line (EA.hy926). Archaeosomes were nontoxic to human Hep G2, CACO-2 and mildly toxic to rodent CHO and B16-F1 cells but showed strong cytotoxic effect on EA.hy926 cells. Confocal microscopy revealed that archaeosomes are taken up by endocytosis. The uptake of archaeosomes and the release of loaded calcein are more prominent in EA.hy926 cells, which is in line with high toxicity toward these cells. The mechanisms of uptake, release and action in these cells as well as in vivo functioning have to be further studied for possible targeted drug delivery.