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- Regulation of bicarbonate secretion in marine fish intestine by the calcium-sensing receptorPublication . Gregorio, Silvia; Fuentes, JuanIn marine fish, high epithelial intestinal HCO3- secretion generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. The present study was designed to expose the putative role for calcium and the calcium-sensing receptor (CaSR) in the regulation of HCO3- secretion in the intestine of the sea bream (Sparus aurata L.). Effects on the expression of the CaSR in the intestine were evaluated by qPCR and an increase was observed in the anterior intestine in fed fish compared with unfed fish and with different regions of intestine. CaSR expression reflected intestinal fluid calcium concentration. In addition, anterior intestine tissue was mounted in Ussing chambers to test the putative regulation of HCO3- secretion in vitro using the anterior intestine. HCO3- secretion was sensitive to varying calcium levels in luminal saline and to calcimimetic compounds known to activate/block the CaSR i.e., R 568 and NPS-2143. Subsequent experiments were performed in intestinal sacs to measure water absorption and the sensitivity of water absorption to varying luminal levels of calcium and calcimimetics were exposed as well. It appears, that CaSR mediates HCO3- secretion and water absorption in marine fish as shown by responsiveness to calcium levels and calcimimetic compounds.
- Increased intestinal carbonate precipitate abundance in the sea bream (Sparus aurata L.) in response to ocean acidificationPublication . Gregorio, Silvia; Ruiz-Jarabo, Ignacio; Carvalho, Edison S. M.; Fuentes, JuanMarine fish contribute to the carbon cycle by producing mineralized intestinal precipitates generated as by-products of their osmoregulation. Here we aimed at characterizing the control of epithelial bicarbonate secretion and intestinal precipitate presence in the gilthead sea bream in response to predicted near future increases of environmental CO2. Our results demonstrate that hypercapnia (950 and 1800 μatm CO2) elicits higher intestine epithelial HCO3- secretion ex vivo and a subsequent parallel increase of intestinal precipitate presence in vivo when compared to present values (440 μatm CO2). Intestinal gene expression analysis in response to environmental hypercapnia revealed the up-regulation of transporters involved in the intestinal bicarbonate secretion cascade such as the basolateral sodium bicarbonate co-transporter slc4a4, and the apical anion transporters slc26a3 and slc26a6 of sea bream. In addition, other genes involved in intestinal ion uptake linked to water absorption such as the apical nkcc2 and aquaporin 1b expression, indicating that hypercapnia influences different levels of intestinal physiology. Taken together the current results are consistent with an intestinal physiological response leading to higher bicarbonate secretion in the intestine of the sea bream paralleled by increased luminal carbonate precipitate abundance and the main related transporters in response to ocean acidification.
- Intestinal response to ocean acidification in the European sea bass (Dicentrarchus labrax)Publication . Alves, Alexandra; Gregorio, Silvia F.; Ruiz-Jarabo, Ignacio; Fuentes, Juanthe intestine of marine fishes contributes to the ocean carbon cycle producing carbonate aggregates as part of the osmoregulatory process. Therefore, this study aimed to evaluate physiological adjustments of European sea bass (Dicentrarchus labrax) intestine to a higher pCO(2) environment likely in the near future (similar to 1700 mu atm). At the whole-body level, hypercapnia for 5 weeks resulted in fish having a significantly diminished specific growth rate, condition factor and hepatosomatic index. An increase in plasma osmolality and HCO3- concentration was detected, paralleled by decreased metabolites concentrations. In the intestine, high seawater pCO(2) was without effect on ouabain-sensitive ATPase activities, while Bafilomycin A1-sensitive ATPase activity significantly decreased in the anterior intestine. Anterior and mid intestine were mounted in Ussing chambers in order to measure bioelectrical parameters and bicarbonate secretion by pH-Stat ex-vivo. Hypercapnia induced a 2.3 and 2.8-fold increase in bicarbonate secretion rates in the anterior and mid intestine, respectively. In the intestinal fluid, HCO3- concentration increased 2.2-fold, and carbonate precipitates showed a 4.4-fold increase in response to hypercapnia, paralleled by a > 3-fold increase of drinking and a > 2-fold increase of intestinal volume at any given time. At the molecular level, hypercapnia elicited higher intestinal mRNA expression levels for atp6v1b (V-ATPase B subunit), slc4a4, slc26a3, and slc26a6, both in the anterior and mid intestine. As a whole, our results show that the intestine of sea bass responds to high seawater pCO(2), a response that comes at a cost at the whole-body level with an impact in the fish specific growth rate, condition factor, and hepatosomatic index.
- Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrPPublication . Gregorio, Silvia F.; Carvalho, Edison S. M.; Campinho, Marco A.; Power, Deborah M.; Canario, Adelino V. M.; Fuentes, JuanIn marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate cotransporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump beta-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC upregulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies downregulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h postinjection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3 equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance.
- Molecular and functional regionalization of bicarbonate secretion cascade in the intestine of the European sea bass (Dicentrarchus labrax)Publication . Alves, Alexandra; Gregorio, Silvia F.; Catão-Egger, Renata; Fuentes, JuanIn marine fish the intestinal HCO3-(-) secretion is the key mechanism to enable luminal aggregate formation and water absorption. Using the sea bass (Dicentrarchus labrax), the present study aimed at establishing the functional and molecular organization of different sections of the intestine concerning bicarbonate secretion and Cl- movements. The proximal intestinal regions presented similar HCO3-- secretion rates, while differences were detected in the molecular expression of the transporters involved and on regional HCO3- concentrations. The anterior region presented significantly higher Na /K+-ATPase activity, Cl transepithelial transport and basolateral slc4a4, apical slc26a6 and slc26a3 expression levels. In the mid intestine, the total HCO3- content was significantly increased in the fluid as in the carbonate aggregates. In the rectum no HCO3- secretion was observed and was characterized by the diminished HCO3- total content, residual molecular expression of slc4a4, slc26a6 and slc26a3, higher H+-ATPase activity and expression, suggesting the existence of a different bicarbonate handling mechanism. The possible regulation of HCO3- secretion by extracellular HCO3- and increased intracellular cAMP levels were also investigated. cAMP did not affect HCO3- secretion, although Cl- secretion was enhanced by cftr. HCO3- secretion rise due to the HCO3- basolateral increment showed that at resting levels slc4a4 was not a limiting step for secretion. The transcellular/intracellular dependence of apical HCO3- secretion differed between the proximal regions. In conclusion, intestinal HCO3- secretion has a functional region-dependent organization that was not reflected by the anterior-posterior regionalization on HCO3- secretion and expression profiles of chloride/water absorption related genes.
- Dysregulation of intestinal physiology by Aflatoxicosis in the Gilthead Seabream (Sparus aurata)Publication . Barany, Andre; Oliva, Milagrosa; Gregorio, Silvia; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel; Fuentes, JuanAflatoxin B1 (AFB1) is a mycotoxin often present in food. This study aimed to understand the physiological effects of AFB1 on the seabream (Sparus aurata) gastrointestinal system. In a first in vitro approach, we investigated ion transport using the short-circuit current (Isc) technique in Ussing chambers in the anterior intestine (AI). Application of apical/ luminal AFB1 concentrations of 8 and 16μM to healthy tissues was without effect on tissue transepithelial electrical resistance (TER), and apparent tissue permeability (Papp) was measured using fluorescein FITC (4 kD). However, it resulted in dose-related effects on Isc. In a second approach, seabream juveniles fed with different AFB1 concentrations (1 and 2mg AFB1 kg−1 fish feed) for 85days showed significantly reduced gill Na+ /K+-ATPase (NKA) and H+-ATPase (HA) activities in the posterior intestine (PI). Moreover, dietary AFB1 modified Isc in the AI and PI, significantly affecting TER in the AI. To understand this effect on TER, we analyzed the expression of nine claudins and three occludins as markers of intestinal architecture and permeability using qPCR. Around 80% of the genes presented significantly different relative mRNA expression between AI and PI and had concomitant sensitivity to dietary AFB1. Based on the results of our in vitro, in vivo, and molecular approaches, we conclude that the effects of dietary AFB1 in the gastrointestinal system are at the base of the previously reported growth impairment caused by AFB1 in fish.
- Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus aurata L.)Publication . Ferlazzo, A.; Carvalho, Edison Samir Mascarelhas; Gregorio, Silvia; Power, Deborah; Canario, Adelino V. M.; Tischitta, F.; Fuentes, J.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 (<20min) 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 10ngml–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, 50mmoll–1), PI3K (LY-294002, 75mmoll–1) or MEK (U-012610, 10mmoll–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 3h 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.
- Ocean acidification compromises energy management in Sparus aurata (Pisces: Teleostei)Publication . Ruiz-Jarabo, I.; Gregorio, SF; Alves, Alexandra; Mancera, J. M.; Fuentes, JuanThe effects of ocean acidification mediated by an increase in water pCO(2) levels on marine organisms are currently under debate. Elevated CO2 concentrations in the seawater induce several physiological responses in teleost fish, including acid-base imbalances and osmoregulatory changes. However, the consequences of CO2 levels enhancement on energy metabolism are mostly unknown. Here we show that 5 weeks of exposure to hypercapnia (950 and 1800 mu atm CO2) altered intermediary metabolism of gilthead seabream (Sparus aurata) compared to fish acclimated to current ocean values (440 mu atm CO2). We found that seabream compromises its physiological acid-base balance with increasing water CO2 levels and the subsequent acidification. Intestinal regions (anterior, mid, and rectum) engaged in maintaining this balance are thus altered, as seen for Na+/K+ ATPase and the vacuolar-type H+-ATPase activities. Moreover, liver and muscle counteracted these effects by increasing catabolic routes e.g., glycogenolysis, glycolysis, amino acid turnover, and lipid catabolism, and plasma energy metabolites were altered. Our results demonstrate how a relatively short period of 5 weeks of water hypercapnia is likely to disrupt the acid-base balance, osmoregulatory capacity and intermediary metabolism in S. aurata. However, long-term studies are necessary to fully understand the consequences of ocean acidification on growth and other energy-demanding activities, such as reproduction.
- Agouti overexpression in a transgenic model regulates integrity, permeability and electrogenic amino acid transport in zebrafish intestinePublication . Leal, Esther; Angotzi, Anna Rita; Godino-Gimeno, Alejandra; Gregorio, Silvia F.; Rotllant, Josep; Saera-Vila, Alfonso; Fuentes, Juan; Cerdá-Reverter, José MiguelOverexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to elucidate the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity. In addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic analysis reveal that amino acid, monocarboxylates, ionic and vitamin transmembrane transporters were substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting augmented intestinal laxity that may enhance paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membraned tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.
- AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata)Publication . Martos-Sitcha, J. A.; Gregorio, Silvia; Carvalho, Edison Samir Mascarelhas; Canario, Adelino V. M.; Power, Deborah; Mancera, J. M.; Martínez-Rodriguez, G.; Fuentes, J.The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55 p.p.t. Basolateral addition of AVT (10 6 M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55 p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose–response curve, with significant effects achieved at 10 8 M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200 lM) applied apically, but was completely abolished in the presence of apical bumetanide (200 lM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.
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