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- 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.
- 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.
- Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulationPublication . Carvalho, Edison Samir Mascarelhas; Gregorio, Silvia; Power, Deborah; Canario, Adelino V. M.; Fuentes, J.In the marine fish intestine luminal, HCO3 - can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO3 - secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH–Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 lM FK? 500 lM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO3 - results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 lM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen.