F. Trischitta

Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1 mg/l to 1 mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (p<0.05). Consequently, two enzymes involved in ROS scavenging, superoxide dismutase (SOD) and catalase (CAT) have higher activities and the proportion of oxidized glutathione (GSSG) is higher in hepatopancreas and mantle of treated animals, compared to untreated animals (p<0.05). Furthermore Hsp70 demonstrates an up-regulation in all the analyzed tissues of exposed animals, attesting the stress status induced by the surfactant with respect to the unexposed animals. The results highlight that SDS, under the tested concentrations, exerts a toxic effect in mussels in which the disruption of the osmotic balance follows the induction of oxidative stress.

Evaluation of Functionality and Biological Responses of Mytilus galloprovincialis after Exposure to Quaternium-15 (Methenamine 3-Chloroallylochloride).

Although the irritant effects of quaternium-15 have been established, little is known about the toxicological consequences induced by this xenobiotic on aquatic invertebrates. The present article reports toxicological, histological and physiological effects of quaternium-15 following the exposure of Mytilus galloprovincialis for 18 days at three different concentrations (0.1, 1.0 and 2.0 mg/L). The results demonstrate that at higher concentrations histological damages to M. galloprovincialis gills occur, like melanosis, light exfoliations, increase of mucus production and infiltrative inflammation. In addition digestive gland cells of M. galloprovincialis, were not able to perform the regulation volume decrease (RVD) owing to osmotic stress following the exposure to the preservative. Overall, this first study on quaternium-15 highlights that it can jeopardize both the morphology and vital physiological processes in marine invertebrates, depending on the duration of exposure and the concentration of the preservative, indicating that further studies are necessary to increase our knowledge about the effects of this substance, commonly added to our products of daily use.

Effects of palmitoylethanolamide on intestinal injury and inflammation caused by ischemia‐reperfusion in mice

Our primary aim in this study was to test the hypothesis that PEA, a member of the fatty acid ethanolamide family and an endogenous PPAR‐α ligand, exerts anti‐inflammatory effects on SAO shock, causing a severe form of circulatory shock and enhanced formation of ROS. SAO shock was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. In this study, we demonstrated that the administration of PEA, 5 min before reperfusion, significantly reduced all of the parameters involved during inflammation, such as proinflammatory cytokine production (TNF‐α, IL‐1β), adhesion molecules (ICAM‐1, P‐selectin) expression, NF‐κB expression, and apoptosis (Bax, Bcl‐2, TUNEL assay) activation. In addition, to study whether the protective action of PEA on SAO shock is also related to the activation of PPAR‐α, we have investigated the effect of PEA in PPAR‐α KO mice subjected to SAO shock. Our study clearly demonstrates that PEA significantly attenuated the degree of intestinal injury and inflammation caused by I/R injury. Moreover, the positive effects of PEA were at least in part dependent on the PPAR‐α pathway. The results clearly indicate that PEA exerts an anti‐inflammatory effect, also in a SAO shock model, which could imply a future use of PEA in the treatment of I/R shock.

Effect of CdCl2 on Regulatory Volume Decrease (RVD) in Mytilus galloprovincialis digestive cells.

This study investigated the role of cadmium, a widespread heavy metal in the aquatic environment, on cell volume regulation of digestive cells isolated from the digestive gland of Mytilus galloprovincialis. These cells when exposed to a rapid change (from 1100 to 800 mOsm/kg) of the bathing solution osmolality swelled but thereafter underwent a Regulatory Volume Decrease (RVD), tending to recover the original size. This homeostatic response is altered by cadmium, as suggested by experiments performed both on isolated cells pre-incubated with cadmium (10(-5)M) and on cells isolated from animals exposed to sub-lethal concentrations of the metal (40 μg/l for 21 days). It is suggested that cytoskeleton and Na(+)/K(+)ATPase are the possible targets of cadmium which impairment is responsible of the altered homeostatic response.

Digestive cells from Mytilus galloprovincialis show a partial regulatory volume decrease following acute hypotonic stress through mechanisms involving inorganic ions.

The response of isolated digestive cells of the digestive gland of Mytilus galloprovincialis to hypotonic shock was studied using videometric methods. The isolated cells exposed to a rapid change (from 1100 to 800 mosmol kg−1) of the bathing solution osmolality swelled but thereafter underwent a regulatory volume decrease (RVD), tending to recover the original size. When the hypotonic stress was applied in the presence of quinine and glibenclamide, known inhibitors of swelling activated ion channels, the cells did not exhibit an RVD response; in addition, they showed a larger increase in size in respect to control cells. These observations suggest that the digestive cells of the digestive gland have the machinery to cope with the hyposmotic shock allowing them to exhibit a small but significant RVD preventing an excessive increase in cell size. The pharmacological treatment of digestive cells during the RVD experiments suggests that cell volume is regulated by K+ and Cl− efflux followed by an obliged water efflux from the cell. The involvement of organic osmolytes such as taurine and betaine seems to be excluded by NMR measurement on digestive cells. Copyright

Effects of CdCl2 on electrophysiological parameters in the intestine of the teleost fish, Anguilla anguilla

Abstract In the present study the action of cadmium, a well known environmental pollutant, on the salt absorptive function of the eel, Anguilla anguilla , intestine was evaluated by estimating the rate of net Cl − absorption expressed as short circuit current ( I sc , μA cm −2 ). In tissues mounted on Ussing chamber, I sc and transepithelial potential difference ( V t , mV) responded in a concentration dependent manner to the addition of CdCl 2 to the serosal or mucosal bathing solutions. The dose response curve indicated that the maximal inhibition of I sc and V t (90% inhibition) after 1 h of incubation was obtained at a concentration of 100 μM from the serosal side and 5 mM from the mucosal one. To understand the nature of I sc inhibition induced by cadmium, the response of electrical parameters to the heavy metal exposure was examined in tissues exposed to different substances known to block Cl − transport at different cellular levels. Hence, bumetanide, which is known to inhibit Cl − transport by blocking the luminal Na + –K + –2Cl − cotransporter and Cl − -conductive channels at the basolateral membrane of the enterocyte, added to the mucosal (10 μM) or the serosal solution (100 μM), abolished the response of I sc and V t to CdCl 2 treatment. Similar results were obtained when tissues were pre-treated with ouabain (serosal, 100 μM) or when Na + or Cl − were omitted from the bathing media. Furthermore, cadmium alters the permselectivity of the tight junctions since the magnitude of the diffusion potential evoked by an imposed serosa–mucosa NaCl gradient (2:1) was strongly reduced by addition of CdCl 2 to either serosal or mucosal solution. Microelectrode experiments point to a relative impermeability of the luminal membrane to the heavy metal since the luminal membrane potential ( V m ) remains unchanged during perfusion with CdCl 2 . The results suggest that cadmium acts on the transport process responsible for Cl − -absorption in eel intestine. Therefore, one of the factors contributing to the toxic effect of cadmium on fish could be related to the ion-balance disturbances of body fluids subsequent to the altered osmoregulatory function of the intestine in marine teleosts.

Different effects of cGMP and cAMP in the intestine of the European eel, Anguilla anguilla

The regulation of salt absorption in the sea water cell intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E1 on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increase the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying Cl− transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine vasoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream ( Sparus aurata L.)

SUMMARY The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose–response curve was obtained, with a maximal inhibition of 60–65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml−1, which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO3−/CO2 buffer. Specific inhibitors of JAK2 (AG-490, 50 μmol l−1), PI3K (LY-294002, 75 μmol l−1) or MEK (U-012610, 10 μmol l−1) caused a 50–70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na+–HCO3− co-transporter, but not on the Slc26a6A or Slc26a3B Cl−/HCO3− exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Requirement of HCO3- for Cl(-)-absorption in seawater-adapted eel intestine.

The role of HCO3−/CO2 buffer in Cl− absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl− absorption, expressed as short/circuit current (Isc), was measured in either 20 mM HCO3−/1% CO2 Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO3−/CO2 with HEPES/O2 was without effect on Isc and transepithelial voltage (Vt), whereas bilateral removal of HCO3−/CO2 reduced Isc and Vt by 50%, indicating that the presence of HCO3−/CO2 buffer at one side of the epithelium is sufficient to keep Cl− absorption at the maximum rate. We examined in further detail the individual components of the HCO3−/CO2 system that stimulates Cl− absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO2 to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on Isc and Vt, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO3−/1% CO2 Ringer or 20 mM HCO3−alone. Stimulation of Isc induced by unilateral (luminal or serosal) HCO3−/CO2 was inhibited by luminal or serosal 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS) (0,25 mM) or by serosal Na+ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of Isc induced by luminal addition of HCO3−/CO2, whereas it was without effect when HCO3−/CO2 was added serosally or bilaterally. We reached the following conclusions, (a) Cl− absorption is stimulated by HCO3−/CO2 buffer via an increase in intracellular HCO3−concentration and/or pHi changes consequent to the HCO3−uptake mediated by HCO3−transport systems operating on both cell membranes, (b) A Na+-dependent SITS-inhibitable HCO3−transport mechanism operates at the basolateral membrane, (c) The transfer of HCO3−through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO3−, via a SITS-sensitive transport system, occurs most likely in form of OH−, which originates from the dehydration reaction of HCO3−catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na+/H+ antiporter on either cell membrane.

Cell volume regulation following hypotonic shock in hepatocytes isolated from Sparus aurata

The response of isolated hepatocytes of Sparus aurata to hypotonic shock was studied by the aid of videometric and light scattering methods. The isolated cells exposed to a rapid change (from 370 to 260 mOsm/kg) of the osmolarity of the bathing solution swelled but thereafter underwent a decrease of cell volume tending to recovery the original size. This homeostatic response RVD (regulatory volume decrease) was inhibited in the absence of extracellular Ca²+ and in the presence of TMB8, an inhibitor of Ca²+ release from intracellular stores. It is likely that Ca²+ entry through verapamil sensitive Ca²+-channels, probably leading to a release of Ca²+ from intracellular stores, is responsible for RVD since the blocker impaired the ability of the cell to recover its volume after the hypotonic shock. RVD tests performed in the presence of various inhibitors of different transport mechanisms, such as BaCl₂, quinine, glybenclamide and bumetanide as well as in the presence of a KCl activator, NEM, led us to suggest that the recovery of cell volume in hypotonic solution is accomplished by an efflux of K+ and Cl⁻ through conductive pathways paralleled by the operation of the KCl cotransport, followed by an obliged water efflux from the cells.