Bilitranslocase is a rat liver plasma membrane carrier, displaying a high‐affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono‐ and di‐glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein‐based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (KM = 5.3 µm), which is competitively inhibited by cyanidine 3‐glucoside (Ki = 51.6 µm) and mainly noncompetitively by cyanidin (Ki = 88.3 µm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin‐binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high‐affinity binding site for cyanidine 3‐glucoside (Kd = 1.7 µm) on the carnation carrier only, and a high‐affinity bilirubin‐binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabelled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high‐affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of ≈ 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti‐sequence bilitranslocase antibodies, the protein‐modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

Mast cells (MCs) reside in tissues that are common targets of Candida spp. infections, and can exert bactericidal activity, but little is known about their fungicidal activity. MCs purified from rat peritoneum (RPMC) and a clinical isolate of C. albicans, were employed. Ingestion was evaluated by flow cytometry (FACS) and optical microscopy. The killing activity was assayed by FACS analysis and by colony forming unit method. RPMC degranulation was evaluated by β-hexosaminidase assay and phosphatidylserine externalization by FACS. Phagocytosing RPMC were also analyzed by transmission electron microscopy. Herein, we show that the killing of C. albicans by RPMC takes place in the extracellular environment, very likely through secreted granular components. Ultrastructural analysis of the ingestion process revealed an unusual RPMC-C. albicans interaction that could allow fungal survival. Our findings indicate that MCs have a positive role in the defense mechanism against Candida infections and should be included among the cell types involved in host-defense against this pathogen.

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Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1−/− and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1−/− mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1−/− mice. Furthermore, contrary to expectations, Trpc1−/− BMMCs responded to antigen with enhanced calcium signalling but with little defect in degranulation or associated signalling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1−/− BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1−/− mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.