ERK1/2 and p38 MAPKs Are Complementarily Involved in Estradiol 17ß-d-Glucuronide-Induced Cholestasis: Crosstalk with cPKC and PI3K

Objective: The endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E217G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect. Design: ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E217G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis. Results: E217G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E217G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E217G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2. Conclusions: cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E217G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.Fil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Zucchetti, Andrés Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Toledo, Flavia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-D-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.Fil: Miszczuk, Gisel Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Larocca, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Marrone, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Marinelli, Raul Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentin

Adaptive downregulation of Cl- /HCO3 - exchange activity in rat hepatocytes under experimental obstructive cholestasis

In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (J(OH)-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 +/- 0.3 vs. 3.1 +/- 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 +/- 0.3 vs. 3.5 +/- 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.This work was supported by grants from Spanish Carlos III Health Institute (ISCIII) [J. M. Banales (FIS PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129) cofinanced by "Fondo Europeo de Desarrollo Regional" (FEDER); "Instituto de Salud Carlos III" [CIBERehd: J. M. Banales], Spain; BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J. M. Banales), Department of Health of the Basque Country (J. M. Banales: 2017111010) and Euskadi RIS3 (J. M. Banales: 2016222001, 2017222014, 2018222029). "Fundacion Cientifica de la Asociacion Espanola Contra el Cancer" (AECC Scientific Foundation, to J. M. Banales). F. A. Crocenzi was recipient of a Young Investigator Scholarship from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Critical population status of the jaguar panthera onca in the Argentine Chaco: camera-trap surveys suggest recent collapse and imminent regional extinction

The population of jaguars in the semi-arid Chaco is the least well-known in Argentina. Its status in the region is described only from interviews confirming presence through 2003. To update distribution and population density information for the Chaco region, we undertook three camera trap surveys, combined with searches for sign at sites across latitudinal and protection gradients and 156 interviews with local inhabitants across three larger areas. The sites for camera trap surveys were located in areas with the highest density of records in the Argentine Chaco: Copo National Park (1204 trap-days, 24 stations, 344 km of transects); Aborigen Reserve (1993 trap-days, 30 stations, 251 km of transects); El Cantor (2129 trap-days, 35 stations, 297 km of transects). We did not record a single camera trap photograph of jaguars. We recorded very few jaguar tracks and only in the Aborigen Reserve (n=3) and El Cantor (n=1). The map of distribution points confirmed through interviews suggests that the jaguar range has not changed significantly in the past 10 years, however, the camera trap and sign surveys suggest that densities are extremely low. Before our study, the Chaco population was thought to be the largest in Argentina. This perception was wrong: the Chaco jaguar population is the most threatened in the country. In order to determine the status of endangered species, systematic and intensive studies are essential to provide the information necessary for decision-making to manage and conserve these species.Fil: Quiroga, Verónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazu | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazu; Argentina. Asociación Civil Centro de Investigaciones del Bosque Atlántico; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Forestales; Argentina. Asociación para la Conservación y el Estudio de la Naturaleza; ArgentinaFil: Boaglio, Gabriel Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Asociación para la Conservación y el Estudio de la Naturaleza; Argentina. Secretaría de Ambiente y Desarrallo Sustentable de la Nación; ArgentinaFil: Noss, Andrew. University of Florida; Estados UnidosFil: Di Bitetti, Mario Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina. Asociación Civil Centro de Investigaciones del Bosque Atlántico; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Forestales; Argentin

Local and continental determinants of giant anteater (Myrmecophaga tridactyla) abundance: Biome, human and jaguar roles in population regulation

The giant anteater (Myrmecophaga tridactyla) is currently found in a wide variety of habitats from Honduras to Argentina. Across this wide range, researchers have postulated that anteater populations arenegatively affected by several factors, including hunting, habitat loss and fragmentation, fire, vehicle collisions, and predation by jaguars. But no studies to date have evaluated the relative importance of thesefactors across sites, either at a regional or continental scale. We used camera traps to analyze variationin giant anteater abundance at two spatial scales. At a regional scale, we conducted camera trap surveysin the dry Chaco of Argentina and used occupancy models to explore the effect of protection status andhuman accessibility on giant anteaters? relative abundance. At a continental scale, we used data from 40camera trap studies (representing 42 different locations) and Generalized Linear Models (GLM) to assessthe potential relation of biome, human disturbance and the presence of jaguars on giant anteater cameratrap records. In the Argentine Chaco, protection and human disturbance do not significantly affect theproportion of area used by the species. The average anteater records/100 camera days and the proportionof sites used are very high across the study area. At a continental scale, anteaters are more frequentlyrecorded in dry forests than in moist forests. Locations with very high human disturbance have cameratrap rates that are 5?10 times lower compared to intermediate or low disturbance locations. Finally, giantanteater capture frequency increases up to 70% where jaguars are absent. Dry biomes and intermediatelevels of human disturbance may favor anteaters by providing greater habitat heterogeneity coupledwith a lower jaguar abundance. This may explain the relatively high abundance of giant anteaters in theArgentine Chaco. However, growing human populations, the advancing agriculture-livestock frontier,and an expanding road network may in time eliminate giant anteaters from most of this region.Fil: Quiroga, Verónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; ArgentinaFil: Noss, Andrew Jay. University of Florida; Estados UnidosFil: Boaglio, Gabriel Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Di Bitetti, Mario Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; Argentin

Mitogen-activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 μM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.Fil: Toledo, Flavia D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Barosso, Ismael R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Zucchetti, Andrés E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sanchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

Puma density, habitat use and conflict with humans in the Argentine Chaco

The puma Puma concolor is the most widely distributed felid in the Americas. Although it utilizes human-modified landscapes, its extensive territorial requirements, trophic needs, and real or perceived threats tolivestock render the puma susceptible to conflict with humans. Our objectives were to evaluate the popu-lation density, habitat use, and puma-human conflict in the Argentine Chaco. We conducted camera-trapsurveys and interviews over a three-year period, at three sites with different levels of legal protection andwith different ranch outpost and livestock densities: Copo National Park (1204 trap days, 24 stations, 17 interviews, national park, lowest ranch/livestock density), Aborigen Reserve (1993 trap days, 29 stations,13 interviews, indigenous reserve, medium ranch/livestock density) and El Cantor (2129 trap days, 35stations, 11 interviews, no protection, highest ranch/livestock density). Puma population density waslow (<1 individual/100 km2) and we found no significant differences in puma density across the threesites. Occupancy models show a positive relationship between puma detectability and the distances fromvehicle roads. Legal protection status of the área does not positively affect puma density, probably due tothe large edge effect, and weak law enforcement capacity at Copo National Park. Low density of pumasat the three sites could result primarily from retaliation killing of pumas by local ranchers in responseto predation on goats. Pumas in the Chaco require effectively managed protected areas, regulation ofwildlife hunting and livestock management practices to minimize depredation.Fil: Quiroga, Verónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; ArgentinaFil: Noss, Andrew. University of Florida; Estados UnidosFil: Paviolo, Agustin Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; ArgentinaFil: Boaglio, Gabriel Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Di Bitetti, Mario Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentin

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2). He re, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB) to limit OS and the impairment in biliary secretory function induced by the model pro-oxidant agent, tert-butylhydroperoxide (tBuOOH). UB fully prevented the formation of reactive oxygen species (ROS) and membrane lipid peroxidation induced by tBuOOH in isolated rat hepatocytes. In the isolated rat hepatocyte couplet model, UB (17.1 µM) prevented the endocytic internalization of Bsep and Mrp2 and the impairment in their secretory function induced by tBuOOH. UB also prevented actin disarrangement, as evaluated by both plasma membrane bleb formation and actin fluorescent staining. Finally, UB prevented tBuOOH-induced cPKC activation. Experiments in isolated perfused rat livers showed that UB prevents the increase in oxidized glutathione biliary excretion and the drop in bile flow and the biliary excretion of specific Bsep and Mrp2 substrates. We conclude that physiological concentrations of UB are sufficient to prevent the biliary secretory failure induced by OS, by counteracting actin disarrangement and the consequent internalization of canalicular transporters relevant to normal bile formation. This reveals an important role for UB in preserving biliary secretory function under OS conditions.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Toledo, Flavia D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Arriaga, Sandra Mónica María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Bioquímica Clínica; Argentina.Fil: Ochoa, Justina E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sánchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Mottino, Aldo D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

Effects of ploidy and nutritional conditions on muscle morphology, proliferation and myogenic proteins expression in Rhamdia quelen larvae

Although Rhamdia quelen is a promising species for farming in South America, many aspects of its development and optimal conditions of culture remain unknown. In this sense, we explore muscle development and some proteins related to myogenic process in diploid and triploid larvae submitted to fasting-refeeding. Regarding muscle morphology, within diploid groups, fasted larvae (FD) showed a significant decrease in white muscle fiber area compared with control (CD) and refed (RD-1) groups. Then, when food was provided, area values restored nearly to control. Based on these results, it is probable that temporary muscle fiber atrophy takes place in fasted diploid fish. Conversely, no significant morphological changes were observed among triploid groups. When the effects of ploidy on somatic growth were assessed, FD and RT-1 groups registered a significantly higher percentage of fibers with an area smaller than 500 μm2 compared with fasted triploid larvae (FT) and RD-1 groups, respectively. Additionally, immunolocalization of the proliferating cell nuclear antigen (PCNA) decreased during starvation in fish of both ploidies and only recovered to normal after refeeding in triploid fish. An increase in PCNA related to ploidy was detected in CD and RD-1 compared with their triploid counterparts. In relation to myogenic proteins, Myog showed a significant increase expression in diploid larvae during starvation. Mstn was not affected by ploidy or alimentary variations. Our results show that diploid fish are more affected by short-term starvation than triploid fish, which could be indicative of differential physiological responses of diploid and triploid larvae to alimentary changes in culture. This might be relevant to optimize conditions of culture for both diploid and triploid fish.Fil: Boaglio, Andrea Carolina. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Histología y Embriología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mendía Broda, Andrea Cecilia. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Histología y Embriología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muñoz, Lautaro H.. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Mascambroni, Maria Belen. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: López, Pablo A.. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Morón Alcain, Esteban. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Arranz, Silvia Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Vigliano, Fabricio Andrés. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Histología y Embriología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.Fil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Toledo, Flavia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Basiglio, Cecilia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin