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Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3(-) secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3(-) secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3(-) secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3(-) secretion rapidly decreased when PTHrP(1-34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1 ng ml(-1) compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) or the phospholipase C inhibitor (U73122, 10 μmol l(-1)) the effect of PTHrP(1-34) on HCO3(-) secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose-response curve in the range of 0.1-1000 ng ml(-1), achieves a maximal effect of 60-65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1 ng ml(-1). The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) and was insensitive to the phospholipase C inhibitor (U73122, 10 μmol l(-1)). In vivo injections of PTHrP(1-34) or the PTH/PTHrP receptor antagonist PTHrP(7-34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO3(-) secretion.

The skin is the largest organ in the body and is a barrier between the internal and external environment. The present study evaluates how PTU, a goitrogen, that is used to treat hyperthyroidism affects the structure and electrical properties of the frog (Xenopus laevis) skin. The results are considered in the context of the two-membrane model established in the seminal work of Ussing and collegues in the 1940s and 1950s. In vitro experiments with skin from Xenopus adults revealed that PTU can act directly on skin and causes a significant increase (p < 0.05, One-way ANOVA) in short circuit current (Isc) via an amiloride-insensitive mechanism. Juvenile Xenopus exposed to waterborne PTU (5 mg/L) had a significantly bigger and more active thyroid gland (p < 0.01, Student’s t-test) than control Xenopus. The bioelectric properties of skin taken from Xenopus juveniles treated with PTU in vivo had a lower Isc, (3.05 ± 0.4, n = 13) and Rt (288.2 ± 39.5) than skin from control Xenopus (Isc, 4.19 ± 1.14, n = 14; Rt, 343.3 ± 43.3). A histological assessment of skin from PTU treated Xenopus juveniles revealed the epidermis was significantly thicker (p < 0.01, Student’s t-test) and had a greater number of modified exocrine glands (p < 0.01, Student’s t-test) in the dermis compared to control skin. Modifications in skin structure are presumably the basis for its changed bioelectric properties and the study highlights a site of action for environmental chemicals which has been largely neglected. The work was supported by POCI 2010 and the European Social Funds attributed by the Portuguese National Science Foundation (FCT) to CCMAR. Resources were used from the project ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192) financed by the Commission of the European Communities.

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. Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal and European Social Funds) [PTDC/MAR/104008/2008, PTDC/MAR-BIO/3811/2012, SFRH/BPD/66808/2009] info:eu-repo/semantics/publishedVersion

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 55p.p.t. Basolateral addition of AVT (10-6M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.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 10-8M, 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 (200μM) applied apically, but was completely abolished in the presence of apical bumetanide (200μM). 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. © 2013 Elsevier Inc., JAM-S is funded by Ministry of Education (Spain) through the program “Formación de Profesorado Universitario” (Ref: AP2008-01194). This work was partially supported by Ministry of Science and Education, Spain by Project AGL2010-14876 to JMM and by the Ministry of Science and Higher Education and European Social Funds through the Portuguese National Science Foundation by Project PTDC/MAR/104008/2008 to JF.

Summary In addition to high drinking rates, processing of intestinal fluid is essential for osmoregulation in marine fish. This study analyzed the long-term response of the sea bream (Sparus aurata L.) to relevant changes of external salinity 12, 35 and 55ppt focusing on the anterior intestine and in the less often studied rectum. Intestinal water absorption, epithelial HCO3- secretion and gene expression of the main molecular mechanisms (SLC26a6, SLC26a3, SLC4a4, atp6v1b, CFTR, NKCC1 and NKCC2) involved in Cl- and HCO3- movements were examined. The anion transporters SLC26a6 and SLC26a3 are several fold more expressed in the anterior intestine, while the expression of Atp6v1b (V-type H+-ATPase β subunit) several fold higher in the rectum. Prolonged exposure to altered external salinity was without effect on water absorption but associated with concomitant changes in intestinal fluid content, epithelial HCO3- secretion and salinity dependent expression of SLC26a6, SLC26a3 and SLC4a4 in the anterior intestine. However, the most striking response to external salinity was obtained in the rectum, where a 4-5-fold increase in water absorption was paralleled by a 2-3-fold increase in HCO3- secretion in response to 55 ppt. In addition, the rectum of high salinity acclimated fish shows a sustained (and enhanced) secretory current (Isc) identified in vitro in Ussing chambers, confirmed by the higher expression CFTR and NKCC1 and established by protein localization with immunohistochemistry. Taken together the present results suggest a functional anterior-posterior specialization with regards to intestinal fluid processing and subsequently to salinity adaptation of the sea bream. The rectum becomes more active at higher salinities and functions as the final controller of the intestinal function in osmoregulation.

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.

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 (20 min) 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 10 ng ml(-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 HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 μmol l(-1)), PI3K (LY-294002, 75 μmol l(-1)) or MEK (U-012610, 10 μmol l(-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 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) 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.