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Carbonic anhydrase (CA) is a ubiquitous metalloenzyme involved in a number of physiological processes including respiration, pH homeostasis, electrolyte transport, calcification, biosynthetic reactions. The aim of this work was to explore a novel functional role of CA in animals: its involvement in the lysosomal system response to metal exposure. Mytilus galloprovincialis digestive gland was used as experimental model. The in vivo exposure of mussels to CdCl2 was able to evoke the induction of digestive gland CA. This response was paralleled by the activation of the lysosomal compartment (proliferation and/or increase in size of lysosomes), as assessed on LysoSensor Green marked cells by confocal microscopy and spectrofluorimetric analysis. When mussels were in vivo exposed to acetazolamide, specific CA inhibitor, the acidification of the lysosomal compartment was prevented. Moreover, during the co-exposure to cadmium and acetazolamide, the cadmium induced activation of the lysosomal compartment was completely inhibited. In conclusion, our results demonstrated in vivo the physiological contribution of CA to the acidification of the lysosomal compartment in digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction following cadmium exposure could physiologically sustain a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation.

The gastrointestinal tract is exposed to a number of pro-oxidant conditions and several gastrointestinal diseases are related to oxidative stress. The preservation of the gastrointestinal redox balance is of pivotal importance for the health status of the organism. It depends on a dynamic system involving antioxidant enzymes, as well as non-enzymatic molecules some of which derive from the diet. This work focuses on the intestinal epithelium and its aim was to explore the intracellular ROS scavenging activity of intracellularly absorbed food antioxidants. Freshly isolated intestinal explants obtained from different experimental animal models were used. They retain the functional and morphological characteristics of the epithelium in vivo. In situ confocal detection on CM-H2DCFDA charged explants experimentally exposed to pro-oxidant conditions was applied. Phenolic phytochemicals, such as hydroxytirosol or anthocyanins, commonly found in the Mediterranean diet, were demonstrated in surface enterocytes to cross the plasma membrane and exert a direct intracellular antioxidant activity. The effect was dose and time dependent and was compared with Trolox, synthetic analog of vitamin E. In conclusion, the gastrointestinal epithelium represents the first interface for the intracellular ROS scavenging activity of food antioxidants, and as such it can benefit directly and earlier than other body tissues of the protective action of food antioxidants against pro-oxidant conditions.

Chloride transport is involved in a wide variety of cellular functions, including regulation of the membrane potential, cell volume, acid base balance, and epithelial fluid absorption and secretion. Alterations in chloride transport mechanisms are related to a number of physio-pathological conditions. The aim of this study was to investigate chloride transport across the plasma membrane of cervical cancer cell lines (HeLa), relying on the halide binding properties of a green (GFP) fluorescent protein variant (YFP) that, if halide complexed, decreases the basal fluorescence intensity. The fluorescence changes of HeLa cells expressing YFP was analyzed by confocal and spectrofluorometric analysis. Our results highlights the presence of a transmembrane halide transport through fluorescence quenching when the transfected cells were exposed to NaI, used as a surrogate ion, under isotonic conditions. This quenching was inhibited by bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransporter, demonstrating the role of such transporter in intracellular halide absorption and allowing cellular fluorescence assay for a functional study of Na+-K+-2Cl-. The kinetic parameters of the transporter were quantified. The present work provides an alternative approach for the measurement and characterization of Cl- and halide transport in living cells expressing YFP-H148Q/I152L based on a non-invasive analysis method.

Colon epithelium is exposed to oxidative stress conditions via exogenous and endogenous routes and a number of colon diseases are associated with the imbalance of the cellular redox system. The aim of the present work was to study the intracellular antioxidant activity of red grape polyphenolic extracts on superficial enterocytes experimentally exposed to prooxidant conditions. In situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15min) was performed. First, the ability of the experimental set up to detect intracellular ROS scavenging activity by intracellularly absorbed antioxidants was demonstrated by a dose-response inhibition of the H2O2 induced intracellular probe fluorescence following pre-incubation with Trolox, synthetic analog of vitamin E. Then, the explants were incubated with grape skin polyphenolic extracts for 1 h producing a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. Results demonstrated that red grape polyphenolic extracts are able to exert an intracellular antioxidant activity on superficial colonocytes, inducing a protection action against pro-oxidant condition. Extracts obtained from mature grapes showed an increased protective activity

Lysosomes are intracellular organelles with an acidic internal pH maintained by a V-ATPase H+ pump. Lysosomal reactions (alterations in lysosomal number and size and lysosomal destabilization) are involved in a number of physiological responses, including xenobiotic and heavy metal sequestration and detoxification. The aim of the study was to investigate the role of carbonic anhydrase, an ubiquitous enzyme catalyzing the hydration of CO2 to HCO2- and H+, in the lysosomal response to heavy metal exposure by using mussel (Mytilus galloprovincialis) digestive cells, characterized by a well developed lysosomal system, as experimental model. CdCl2 was used as reference toxicant. A novel microplate fluorimetric assay was developed on digestive cells in suspension, charged with the fluorescent acidotropic probe Lysosensor™ Green DND-189, for quantitating the number of lysosomes and their pH. In parallel, single cell analysis was performed by confocal microscopy. Digestive cells from Cd exposed animals showed a time and dose dependent increase of the probe fluorescence, suggesting a proliferation of the lysosomal compartment and decrease of the internal pH. This was confirmed by single cell confocal analysis. In animals in vivo exposed to acetazolamide, specific inhibitor of carbonic anhydrase, the metal induced fluorescence increase was dramatically inhibited. In conclusion, results indicate a key role of carbonic anhydrase in the lysosomal responses to metal exposure.