The intestinal absorption of verbascoside (VB), a phenylpropanoid glycoside, extracted and purified from olive mill waste water (OMWW), was investigated using viable and healthy human colonic tissues, mounted in an Ussing chamber. Additionally, VB absorption and transport through the intestinal mucosa were quantified using permeability coefficients. VB absorption was time-dependent and varied in relation to the specific colonic segments considered. In particular, major uptake/absorption (0.50 μg/cm2) occurred between 5 and 15 min in the proximal tract of the colon, followed by descending colon (0.38 μg/cm2) between 30 and 60 min, and sigmoid–rectum colon (0.34 μg/cm2) at 60 min. Overall, VB was absorbed rapidly, with an average uptake of 0.29 μg VB per cm2, corresponding to a total accumulation efficiency of ~ 0.12%. Moreover, the presence of the VB in the basolateral side supported the hypothesis of its bioavailability in the extent of 0.1%. In addition, the permeability coefficient calculation has contributed in a deeper understanding of VB absorption and transport across the human intestinal barrier and could be utilized for other polyphenols present in food and in dietary supplements.

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (Isc), transepithelial potential (Vt) and resistance (Rt) were recorded in the continuous presence of cadmium. Addition of cadmium (20 μM to 1 mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in Isc cannot be explained by an action on: 1) H2 histamine receptor, 2) Ca2+ signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H+/K+-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H+/K+-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.

Fumonisins (FBs), Fusarium mycotoxins common food contaminant, are a potent inducer of oxidative stress and lipid peroxidation in intestinal cells. In order to verify this toxic effect in intestine tract, the aim was to assess lipid peroxidation (as malondialdehyde MDA increased levels) on intestine rat samples exposed to chyme samples from in vitro digestion of FBs contaminated corn samples. Naturally (9.61±3.2 μg/gr), artificially (726±94 μg/gr) and spiked corn samples at EU permitted FBs levels were digested and added to luminal side of Ussing chamber for 120 min. Fumonisins-free corn sample was used as control. The MDA increase was observed just in 83% of intestine samples exposed at EU FBs levels and the digestion process seems to reduce this incidence (50% of samples). Malondialdehyde levels were FBs dose- and subject-related and ranged from 0.07±0.01 to 3.59±0.6 nmol/mg. Highest incidence and MDA % increment (I) were found when intestine tracts were exposed to chymes from artificially corn sample. The induction of lipid peroxidation induced by FBs could be due to interactions between FBs and intestinal membranes, with consequent modifications in membrane permeability and oxygen diffusion-concentration, as suggested by other authors.

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors

The gut is a possible target toward mycotoxin Fumonisins (FBs) exposure. The study aims to investigate the effects induced by FBs contaminated-corn chyme samples on functional parameters of human and rat intestine by using Ussing chamber. Fumonisins-contaminated corn and processed corn samples were undergone to in vitro digestion process and then added to luminal side. A reduction (about 90%) of short circuit current (Isc μA/cm2) during exposure of human colon tissues to fumonisins-free corn chyme samples was observed, probably related to increased chyme osmolality. This hyperosmotic stress could drain water towards the luminal compartment, modifying Na+ and Cl- transports. The presence of FBs in corn chyme samples, independently to their concentration, did not affect significantly the Isc, probably related to their interference toward epithelial Na+ transport, as assessed by using a specific inhibitor (Amiloride). The rat colon tract represents a more accessible model to study FBs toxicity showing a similar functional response to human. In the rat small intestine a significant reduction (about 15%) of Isc parameter during exposure to uncontaminated or FBs contaminated corn chyme samples was observed; therefore such model was not suitable to assess the FBs toxicity, probably because the prevalent glucose and amino acids electrogenic absorption overwhelmed the FBs influence on ionic transport.

In the presence of risk factors, the anastomosis in the small intestine and in the colon are at risk for dehiscence and peritonitis. The apposition of a biological patch around the anastomosis might improve wound healing and therefore might prevent harmful, potentially life-threatening and costy complications. Aim: to verify if Tutomesh® facilitates the functional recovery of the intestinal anastomotic wound area (mucosa) in the pig ileum and colon. Methods: 24 Large White pigs (B.W. 25 kg; age 4-5 months) underwent ileal and colonic anastomosis with or without application of Tutomesh® and compared with healthy (intact) control intestinal segments. At days 2, 7, 14, 30 and 90 following surgery, ileal and colonic mucosa were isolated from similar anastomized and control tracts and mounted in Ussing chambers containing Krebs oxygenated solution at pH 7.4. Electrophysiological parameters, i.e. short circuit current (Isc) and transepithelial resistance (Rt), as markers of mucosal function, were continously measured by a digital voltage clamp system. Results: Ileal mucosa from control showed Isc of -17.10±4.72 μA/cm2 and Rt of 105.91±11.98 Ohm*cm2. In anastomized ileum Isc decreased by 52% and Rt increased by 58% (n=6 tissues); with Tutomesh® the Isc reduction was only 16.3% while Rt increased by 46% (for both n=6; p<0.001 vs. control). In colonic mucosa from 13 control tissues, Isc was -10.67±2.29 μA/ cm2, Rt 140.94±14.38 Ohm*cm2. Colonic Isc and Rt (n=6) remained stable with anastom- osis, while Tutomesh® significantly increased the current by 47.0% (n=7; p<0.001 vs. control). Conclusions: Our observations suggest that transport properties of intestinal mucosa improve significantly with Tutomesh® , a useful resorbable bio-patch which therefore helps the functional recovery of anastomoses, mainly in the ileum. Further studies are ongoing to assess the translational value of Tutomesh® in surgical patients.

Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a novel signaling mechanism in which depletion of Ca2+ in the endoplasmic reticulum (ER) leads to a STIM1- dependent (Stromal Interaction Molecule 1) increase in cAMP levels, independently of cytosolic Ca2+. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat ventricular myocytes (NRVM) and human "iCardiomyocytes" and exploit its potential role in cardiac cell hypertrophy. cAMP levels and ER [Ca2+]were monitored by live cell fluorescence imaging after transfection with the EPAC H30 and D1ER cameleon probes, respectively. The existence of SOcAMPS in NRVM was first assessed by using the low affinity Ca2+ chelator TPEN, able to induce a reduction of SR Ca2+ levels without affecting cytosolic [Ca2+]. TPEN (1mM) was shown to induce significant cAMP increases both in the absence and presence of 5 M Forskolin (FRSK). Depletion of SR by ionomycin (10 M) was found to exert similar effects. Similar data were obtained in human "iCardiomyocytes". The participation of STIM1 in the observed phenomenon was proven in NRVM by the 47% reduction of the [cAMP] response obtained after shRNA-mediated knockdown of STIM1. Interestingly, a significant increase of the TPEN+FRSK induced response was found after "in vitro" induced cell hypertrophy. These data establish, for the first time, the existence of SOcAMPS in the two cardiac cell models analyzed and suggest a potential role for this new signaling mechanism in cardiac cell hypertrophy.

The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 μM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 μM, followed by inhibition of cell proliferation (up to 8.6 μM), the immunomodulatory effect (up to 17.2 μM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 μM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.