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Caracterização funcional da microalga extremófila Coccomyxa sp.

Publication . Brás, Pedro Emídio; Fuentes, Juan; Fonseca, Filomena

As microalgas têm sido alvo de grande estudo devido ao seu potencial como fonte de novos compostos de interesse para a biotecnologia e também para compreender a sua sobrevivência em locais extremos. A microalga Coccomxya sp., estudada neste trabalho, é um poliextremófilo que foi isolado das águas ácidas da mina de S. Domingos. Este ambiente é caracterizado pelos valores baixos de pH (< 3) e a elevada concentração de iões metálicos, como o cobre e o ferro. Os principais objetivos deste trabalho foram: i) analisar as alterações metabólicas (tamanho e crescimento celulares e produção de pigmentos), ii) identificar e quantificar alterações de perfil de expressão genética em diferentes condições e iii) avaliar a potencial bioactividade num modelo animal ex-vivo de extratos celulares de culturas daquela microalga a diferentes pH, sem ou com exposição a cobre (0,6 mM). Quando expostas aos diferentes tratamentos as células de Coccomyxa sp. apresentaram três equilíbrios dinâmicos (pH 7, pH 7 exposto a cobre e pH 2,5). Pelos resultados do Real-Time PCR e pela produção de pigmentos (clorofila e carotenoides), o pH 7 demonstra ser o ambiente preferido da microalga. Os extratos de Coccomyxa sp., de culturas a pH 7 expostas a cobre, demonstram um grande potencial para serem utilizados como inibidores do NKCC presente no epitélio, revelando o seu potencial uso como diuréticos. Coccomyxa sp. apresenta ser uma fonte fiável de pigmentos e de extratos celulares com um grande potencial para serem usados na industria biotecnológica e farmacêutica.

2015-11-26Master thesis

Open Access

Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrP

Publication . Gregorio, Silvia F.; Carvalho, Edison S. M.; Campinho, Marco A.; Power, Deborah M.; Canario, Adelino V. M.; Fuentes, Juan

In 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.

2014-05Journal article

Open Access

Intestinal response to salinity challenge in the Senegalese sole (Solea senegalensis)

Publication . Ruiz-Jarabo, I.; Barany, A.; Jerez-Cepa, I.; Mancera, J. M.; Fuentes, Juan

Fish are continuously forced to actively absorb or expel water and ions through epithelia. Most studies have focused on the gill due to its role in Na+ and Cl- trafficking. However, comparatively few studies have focused on the changing function of the intestine in response to external salinity. Therefore, the present study investigated the main intestinal changes of long-term acclimation of the Senegalese sole (Solea senegalensis) to 5,15, 38 and 55 ppt. Through the measurement of short-circuit current (Isc) in Ussing chambers and biochemical approaches, we described a clear anterior/posterior functional regionalization of the intestine in response to salinity. The use of specific inhibitors in Ussing chamber experiments, revealed that the bumetanide-sensitive Na+/K+/Cl- co-transporters are the main effectors of Cl- uptake in both anterior intestine and rectum. Additionally, the use of the anion exchanger specific inhibitor, DIDS, showed a salinity/region dependency of anion exchanger function. Moreover, we also described ouabain-sensitive Na+/K+-ATPase (NKA) and Bafilomycin Al-sensitive H-ATPase activities (HA), which displayed changes related to salinity and intestinal region. However, the most striking result of the present study is the description of an omeprazole-sensitive H+/K+-ATPase (HKA) in the rectum of Senegalese sole. Its activity was consistently measurable and increased at lower salinities, reaching rates even higher than those of the NKA. Together our results provide new insights into the changing role of the intestine in response to external salinity in teleost fish. The rectal activity of HKA offers an alternative/cooperative mechanism with the HA in the final processing of intestinal water absorption by apical titration of secreted bicarbonate. (C) 2016 Elsevier Inc. All rights reserved.

2017-02Journal article

Restricted

Regulation of stanniocalcin secretion by calcium and PTHrP in Gilthead Seabream (Sparus aurata)

Publication . Ruiz-Jarabo, I.; Gregorio, Silvia; Fuentes, Xoan

Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP.

2022-06-04Journal article

Open Access

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

Publication . Martos-Sitcha, Juan Antonio; Campinho, Marco António; Miguel Mancera, Juan; Martinez-Rodriguez, Gonzalo; Fuentes, Juan

Aquaporins (AQPs) are specific transmembrane water channels with an important function in water homeostasis. In terrestrial vertebrates, AQP2 function is regulated by vasopressin (AVP) to accomplish key functions in osmoregulation. The endocrine control of aquaporin function in teleosts remains little studied. Therefore, in this study we investigated the regulatory role of vasotocin (AVTR) and isotocin (ITR) receptors in Aqp1 paralog gene function in the teleost gilthead sea bream (Sparus aurata). The complete coding regions of Aqp1a, Aqp1b, AVTR V1a2-type, AVTR V2-type and ITR from sea bream were isolated. A Xenopus oocyte-swelling assay was used to functionally characterize AQP1 function and regulation by AVT and IT through their cognate receptors. Microinjection of oocytes with Aqp1b mRNA revealed regulation of water transport via PKA (IBMX+forskolin sensitive), whereas Aqp1a mRNA injection had the same effect via PKC signaling (PDBU sensitive). In the absence of expressed receptors, AVT and IT (10(-8) mol l(-1)) were unable to modify AQP1 function. AVT regulated AQP1a and AQP1b function only when the AVTR V2-type was co-expressed. IT regulated AQP1a function, but not AQP1b, only when ITR was present. Considering that Aqp1a and Aqp1b gene expression in the sea bream intestine is highly salinity dependent in vivo, our results in ovo demonstrate a regulatory role for AVT and IT in AQP1 function in the sea bream in the processing of intestinal fluid to achieve osmoregulation.

2015-03Journal article

Open Access