Juan Fuentes

Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution.

SUMMARY The aquaporins (formerly called the major intrinsic protein family) are transmembrane channel proteins. The family includes the CHIP group, which are functionally characterised as water channels and the GLP group, which are specialised for glycerol transport. The present study reports the identification and characterisation of a novel GLP family member in a teleost fish, the sea bream Sparus auratus. A sea bream aquaporin (sbAQP) cDNA of 1047 bp and encoding a protein of 298 amino acids was isolated from a kidney cDNA library. Functional characterization of the sbAQP using a Xenopus oocyte assay revealed that the isolated cDNA stimulated osmotic water permeability in a mercury-sensitive manner and also stimulated urea and glycerol uptake. Northern blotting demonstrated that sbAQP was expressed at high levels in the posterior region of the gut, where two transcripts were identified (1.6 kb and 2 kb), and in kidney, where a single transcript was present (2 kb). In situ hybridisation studies with a sbAQP riboprobe revealed its presence in the lamina propria and smooth muscle layer of the posterior region of the gut and in epithelial cells of some kidney tubules. sbAQP was also present in putative chloride cells of the gill. Phylogenetic analysis of sbAQP, including putative GLP genes from Fugu rubripes, revealed that it did not group with any of the previously isolated vertebrate GLPs and instead formed a separate group, suggesting that it may be a novel GLP member.

Novel bioactive parathyroid hormone and related peptides in teleost fish

We report the identification, gene expression and biological activity of two parathyroid hormones (PTH; PTHA and PTHB), two PTH‐related peptides (PTHrP; PTHrPA and PTHrPB) and a PTH‐like ligand (PTH‐L) with hybrid characteristics in puffer fishes (Takifugu rubripes and Tetraodon fluviatilis). Experimental data are consistent with PTH‐L and PTHrPA having calciotropic activities equivalent, respectively, to tetrapod PTH and PTHrP. We hypothesise on the basis of phylogenetic and functional analysis that PTH‐L could be a fish relic of an ancestral PTH/PTHrP gene.

Adaptation to different salinities exposes functional specialization in the intestine of the sea bream (Sparus aurata L.)

SUMMARY The processing of intestinal fluid, in addition to a high drinking rate, 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 55 p.p.t.), 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 expressed severalfold higher in the anterior intestine, while the expression of Atp6v1b (V-type H+-ATPase β-subunit) is severalfold higher in the rectum. Prolonged exposure to altered external salinity was without effect on water absorption but was 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- to 5-fold increase in water absorption was paralleled by a 2- to 3-fold increase in HCO3− secretion in response to a salinity of 55 p.p.t. In addition, the rectum of high salinity-acclimated fish shows a sustained (and enhanced) secretory current (Isc), identified in vitro in Ussing chambers and confirmed by the higher expression of CFTR and NKCC1 and by immunohistochemical protein localization. Taken together, the present results suggest a functional anterior–posterior specialization with regard 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 intestinal function in osmoregulation.

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1–34 N-terminal peptide

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.

Drinking in Atlantic salmon presmolts (Salmo salar L.) and juvenile rainbow trout (Oncorhynchus mykiss Walbaum) in response to cortisol and sea water challenge

Abstract Circulating levels of cortisol in freshwater Atlantic salmon presmolts (15–30 g) and juvenile rainbow trout (20–30 g) were significantly increased by cortisol implants (80 mg kg−1) over a 3-week period, compared to sham implanted fish. In freshwater fish, cortisol implants were without effect on blood ionic content and drinking rate, about 0.1 ml kg−1 h−1 in salmon presmolts and 0.25 ml kg−1 h−1 in juvenile rainbow trout. Transfer of cortisol implanted salmon presmolts to sea water (33 ppt) resulted in development of a more immediate and fuller drinking response and after 7 days they imbibed at about 3.81 ± 0.01 ml kg−1 h−1 compared to 1.89 ± 0.27 ml kg−1 h−1 for sham implanted fish. A similar response was noted in cortisol implanted juvenile rainbow trout transferred to 28 ppt sea water. In both species cortisol implants resulted in improved hypoosmoregulatory capacity especially with respect to blood plasma Cl−1 concentration. The osmoregulatory role of cortisol in control of drinking in salmonids is discussed.

Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken

BackgroundParathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.ResultsThe PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors.ConclusionsThe parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that genes of the PTH family appeared at approximately the same time during the vertebrate radiation and evolved via gene duplication/deletion events. PTH-L was lost from the genome of eutherian mammals and PTH, which has a paracrine distribution in lower vertebrates, became the product of a specific endocrine tissue in Amphibia, the parathyroid gland. The PTHrP gene organisation diverged and became more complex in vertebrates and retained its widespread tissue distribution which is congruent with its paracrine nature.

Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinities

SUMMARY Whole-body calcium uptake was studied in gilthead sea bream larvae (9–83 mg) in response to changing environmental salinity and [Ca2+]. Calcium uptake increased with increased fish size and salinity. Fish exposed to calcium-enriched, diluted seawater showed increased calcium uptake compared with fish in diluted seawater alone. Calcium uptake was unchanged in Na+-enriched, diluted seawater. Overall, [Ca2+], and not salinity/osmolarity per se, appears to be the main factor contributing to calcium uptake. By contrast, drinking was reduced by a decrease in salinity/osmolarity but was little affected by external [Ca2+]. Calculations of the maximum contribution from drinking-associated calcium uptake showed that it became almost insignificant (less than 10%) through a strong decrease in drinking rate at low salinities (0–8‰). Diluted seawater enriched in calcium to the concentration present in full-strength seawater (i.e. constant calcium, decreasing salinity) restored intestinal calcium uptake to normal. Extra-intestinal calcium uptake also benefited from calcium addition but to a lesser extent.

Variations in the expression of vasotocin and isotocin receptor genes in the gilthead sea bream Sparus aurata during different osmotic challenges.

The dynamic changes in mRNA expression levels for vasotocin (AVT) and isotocin (IT) receptor gene levels were assessed in a time-course response study in immature male specimens of the gilthead sea bream (Sparus aurata) submitted to hyper- (55‰ salinity) and hypo-osmotic (5‰ salinity) challenges. Two different cDNAs for the AVT receptor and one for the IT receptor (V1a2-type and V2-type AVTR, and ITR, respectively) were cloned by screening an S. aurata brain cDNA library. Genes for these receptors were expressed differentially and is nearly ubiquitously in 26 of the examined tissues. In the gills, both environmental salinity challenges up-regulated AVTR V1a2-type gene expression concomitantly with mRNA expression protein activity of Na(+), K(+)-ATPase gene expression and protein, whereas the AVTR V2-type and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels were associated with mRNAs environmental salinity, indicating a possible connection between AVTRs and these transporters. In kidney, AVTR V1a2-type gene expression peaked rapidly and lasted only a short time (12-24h) in response to both osmotic challenges. In contrast, AVTR V2-type mRNA levels were enhanced in specimens exposed to hyperosmotic conditions, whereas they decreased under hypoosmotic environments, suggesting an antidiuretic role related to the vasoconstriction function. In the hypothalamus, only the expression of the AVTR V2-type gene was enhanced at 7 and 14 days under both experimental conditions. In the liver, both AVTRs had increased mRNA levels, with the upregulation of their AVTR V2-type gene increasing faster than the V1a2-type. The ITR gene was not sensitive to variations of external salinity in any of the analyzed tissues. Our results demonstrate the involvement of the vasotocinergic, but not the isotocinergic, pathway as well as the hypothalamic function, in the adjustments of both osmoregulatory and metabolic processes after osmotic challenges.

Parathyroid hormone-related protein-stanniocalcin antagonism in regulation of bicarbonate secretion and calcium precipitation in a marine fish intestine

Bicarbonate secretion in the intestine (duodenum) of marine fish has been suggested to play a major role in regulation of calcium availability for uptake. However, while the end process may lead to carbonate precipitation, regulation of transport of calcium and/or bicarbonate may actually result in fine-tuning of calcium availability for transport. To test this hypothesis, sea bream (Sparus auratus) duodenal preparations were mounted in Ussing-type chambers and the effect of parathyroid hormone-related protein (PTHrP) and stanniocalcin 1 (STC 1) on the control of intestinal bicarbonate secretion and calcium transport was analyzed. As expected, PTHrP increased net calcium uptake, as a result of an increase of calcium uptake without changes in calcium efflux. In contrast, purified sea bream STC 1 caused a minor decrease of calcium uptake and a two- to threefold increase in calcium efflux. As a result, STC 1 was able to invert the calcium flux from net calcium uptake to net calcium loss, which is in keeping with its known actions as a hypocalcemic factor. Furthermore, both PTHrP and STC 1 regulate intestinal bicarbonate secretion. PTHrP increased calcium uptake and simultaneously reduced the single factor that induces calcium precipitation, bicarbonate secretion. In contrast, STC 1, while reversing the calcium net flux to make it secretory, promoted intestinal bicarbonate secretion, both actions directed to decrease the calcium gradient across the epithelium and promote immobilization in the form of bicarbonate in the intestinal lumen. Together our results provide robust evidence to support an antagonistic action of PTHrP and STC 1 in the fine control of movements of both calcium and bicarbonate in the intestine of seawater fish.

The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata, L.)

The effect of estradiol-17beta (E(2)) implants on the in vitro secretion of prolactin (PRL) and its modulation by vasoactive intestinal peptide (VIP) in a marine teleost, sea bream (Sparus aurata L.), was determined. Experiments were conducted during winter and spring. During winter, fish (n=130, body weight 50-70 g) were randomly divided into 2 groups; control and E(2) treated (10 mg/kg, wet weight). Fish were sacrificed after 7 days treatment and in vitro pituitary cultures in Ringer bicarbonate supplemented with increasing doses (0-200 nM) of VIP were carried out for 18 h. Culture medium was analysed by PAGE and secreted PRL quantified by densitometry. Fish treated with E(2) secreted significantly more PRL (P<0.05) in vitro than control fish. In E(2) primed fish VIP caused a dose-dependent inhibition of PRL secretion in vitro. VIP had no detectable effect on the secretion of PRL from control pituitaries. Treatment with E(2) had a different effect during spring; PRL secretion was significantly decreased (P<0.01) compared with the control fish. Anatomical evidence of abundant VIP immunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIP in the regulation of PRL cells. In conclusion, the responsiveness of PRL in the pituitary gland varied with season. Moreover, in the sea bream VIP appears to modulate PRL secretion from E(2) primed pituitary glands.