MO082THE EFFECTS OF DIFFERENT MICROMOLAR CONCENTRATIONS OF CADMIUM IONS ON THE PERITUBULAR MEMBRANE POTENTIAL OF PROXIMAL TUBULAR CELLS IN PERFUSED FROG KIDNEYS

Background and Aims Cadmium (Cd2+) is toxic metal and environmental pollutant. Accumulation of cadmium in the kidney results initially in proximal tubule dysfunction. Although Cd2+ toxicity is well documented, all mechanisms that are involved in the early stages of nephrotoxicity, especially considering low micromolar concentrations of Cd2+ ions are still unknown. The Aim of this study was to investigate the effects acute exposure to different peritubular micromolar concentrations of cadmium (0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) on the peritubular cell membrane potential in proximal tubular cells of frog kidney. Method The experiments were performed on isolated, doubly perfused kidneys of Rana esculenta of both sexes. Aortic and portal vein were cannulated in order to perfusate luminal and peritubular cell membraine. In controled conditions, Ringer solution was simultaneously used to perfusate both cell membraines. Cadmium chloride (different concentrations: 0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) were added to the peritubular perfusate separately, by switching the peritubular perfusate from the control Ringer solution to Ringer solution with addition of cadmium ions. Peritubular cell membrane potentials (PD) were measured with conventional 3 mol/L KCl microelectrodes. Results The peritubular application of different micromolar Cd2+ concentrations led to a rapid, sustained, reversible hyperpolarization of the peritubular cell membrane: 0.25 µmol/L, by −3.3±0.4 mV (n=8, p<0.001); 0.50 µmol/L, by −3.0±0.5 mV (n=11, p<0.001); 1.0 µmol/L, by −2.9±0.6 mV (n=8, p<0.01); 2.0 µmol/L, by −4.2±0.4 mV (n=13, p<0.01); 3.0 µmol/L, by −3.4±0.3 mV (n=14, p<0.001); 5.0 µmol/L, by −3.0±0.4 mV (n=10, p<0.001). After switching the perfusion from Ringer solution with addition of cadmium ions to control Ringer, the peritubular membraine potential returned to the average values that were maintained before the peritubular Cd2+ application (p>0.05). Comparing the effect of different Cd2+ concentrations, there was no difference in the hyperpolarization of the peritubular cell membrane (p>0.05).Each cell served as its own control. Conclusion Different low micromolar concentrations of Cd2+ provoked rapid and sustained hyperpolarization of peritubular membrane potential that did not show concentration-dependent response.

Ranacyclin-NF, a Novel Bowman–Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

Interspecies variation in DNA damage induced by pollution

The choice of a suitable species to translate pollution signals into a quantitative monitor is a fundamental step in biomonitoring plans. Here we present the results of three years of biomonitoring at a new coal power plant in central Italy using three different aquatic and terrestrial wildlife species in order to compare their reliability as sentinel organisms for genotoxicity. The comet assay was applied to the common land snail Helix spp., the lagoon fish Aphanius fasciatus, and the green frog Rana esculenta sampled in the area potentially exposed to the impact of the power station. The tissue concentration of some expected pollutants (As, Cd, Ni, Pb, Cr) was analysed in parallel samples collected in the same sampling sites. The three species showed different values in the comet assay (Tail Intensity) and different accumulation profiles of heavy metals. Aphanius fasciatus showed an increasing genotoxic effect over time that paralleled the temporal increase of the heavy metals, especially arsenic, and the highest correlation between heavy metals and DNA damage. Helix spp. showed levels of damage inversely related to the distance from the source of pollution and in partial accordance with the total accumulation of trace elements. On the contrary, Rana esculenta showed a low capability to accumulate metals and had inconsistent results in the comet test. The fish appeared to be the most efficient and sensitive species in detecting chemical pollution. Overall, both the fish and the snail reflected a trend of increasing pollution in the area surrounding the power plant across time and space.

Akt/eNOS signaling and PLN S-sulfhydration are involved in H2S-dependent cardiac effects in frog and rat

Hydrogen sulfide (H2S) has recently emerged as an important mediator of mammalian cardiovascular homeostasis. In nonmammalian vertebrates, little is known about the cardiac effects of H2S. This study aimed to evaluate, in the avascular heart of the frog, Rana esculenta, whether and to what extent H2S affects the cardiac performance, and what is the mechanism of action responsible for the observed effects. Results were analyzed in relation to those obtained in the rat heart, used as the mammalian model. Isolated and perfused (working and Langendorff) hearts, Western blot analysis, and modified biotin switch (S-sulfhydration) assay were used. In the frog heart, NaHS (used as H2S donor, 10−12/10−7 M) dose-dependently decreased inotropism. This effect was reduced by glibenclamide (KATP channels blocker), N G-monomethyl-l-arginine (NOS inhibitor), 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (guanylyl cyclase inhibitor), KT5823 (PKG inhibitor), and it was blocked by Akt1/2 (Akt inhibitor) and by detergent Triton X-100. In the rat, in addition to the classic negative inotropic effect, NaHS (10−12/10−7 M) exhibited negative lusitropism. In both frog and rat hearts, NaHS treatment induced Akt and eNOS phosphorylation and an increased cardiac protein S-sulfhydration that, in the rat heart, includes phospholamban. Our data suggest that H2S represents a phylogenetically conserved cardioactive molecule. Results obtained on the rat heart extend the role of H2S also to cardiac relaxation. H2S effects involve KATP channels, the Akt/NOS-cGMP/PKG pathway, and S-sulfhydration of cardiac proteins.