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- Cortisol and parathyroid hormone-related peptide are reciprocally modulated by negative feedbackPublication . Guerreiro, P. M.; Rotllant, J.; Fuentes, J.; Power, Deborah; Canario, Adelino V. M.In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or conWnement stress, suppressed circulating PTHrP 6 and 24-fold,respectively, by comparison to control Wsh.reduced cortisol levels, prevented the decrease of plasma PTHrP observed in conWned Wsh and raised plasma PTHrPrespectively, by comparison to control fish.
- 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.
- Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinitiesPublication . Guerreiro, P. M.; Fuentes, J.; Flik, G.; Rotllant, J.; Power, Deborah; Canario, Adelino V. M.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.
- Novel bioactive parathyroid hormone and related peptides in teleost fishPublication . Canario, Adelino V. M.; Rotllant, J.; Fuentes, J.; Guerreiro, P. M.; Teodósio, H. R.; Power, Deborah; Clark, M. S.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.
- Regulation of calcium balance in the sturgeon Acipenser naccarii: a role for PTHrPPublication . Fuentes, J.; Haond, Christophe; Guerreiro, P. M.; Silva, Nádia; Power, Deborah; Canario, Adelino V. M.Calcium regulation in sturgeon is of special interest because they are a representative of the ancient fishes possessing mainly cartilaginous skeletons and a supposedly low calcium demand. The present study aimed to characterize the effect of a chronic absence of dietary calcium and the effect of parathyroid hormone-related protein (PTHrPA) (1-34) (7) on calcium balance in juvenile sturgeon (Acipenser naccarii). At rest, sturgeon juveniles are in net positive calcium balance, since whole body calcium uptake is significantly higher than efflux and calcium accumulates in the body. To study the importance of dietary calcium, the sturgeon were kept on a calcium-free diet for 8 wk. This manipulation impaired growth as measured by failure to gain weight or increase in length and indicates that dietary calcium is important for growth in sturgeon. An increased whole body calcium uptake partially compensated dietary calcium deficiency and was associated with increased gill chloride cell number in lamellae and filaments in parallel with increased gill Na(+)K(+)-ATPase activity. In addition, a single injection of piscine PTHrP(1-34) significantly increased whole body calcium uptake and decreased whole body calcium efflux. Administration of PTHrP significantly increased circulating plasma calcium 4-24 h postinjection. The increase in net calcium transport and increased plasma levels of calcium is consistent with the actions of a hypercalcemic factor. It would appear that the sturgeon rely on calcium for growth and tightly regulate calcium transport. The action in calcium balance is consistent with PTHrP acting as a hypercalcemic factor in sturgeon.
- 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.
- Parathyroid hormone-related protein-stanniocalcin antagonism in regulation of bicarbonate secretion and calcium precipitation in a marine fish intestinePublication . Fuentes, J.; Power, Deborah; Canario, Adelino V. M.Parathyroid hormone-related protein-stanniocalcin antagonism in regulation of bicarbonate secretion and calcium precipitation in a marine fish intestine. Am J Physiol Regul Integr Comp Physiol 299: R150–R158, 2010. First published April 21, 2010; doi:10.1152/ajpregu.00378.2009.—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.
- Ca2+-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus)Publication . Martins, Rute S. T.; Fuentes, J.; Almeida, O. G.; Power, Deborah; Canario, Adelino V. M.The Ca2+-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca2+ and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80 lM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80 lM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20a-dihydroxy-4-pregnen- 3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11bhydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca2+-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca2+-dependent CaM and that possibly Ca2+-CaM determines the shift in steroidogenesis from C21 steroids to androgens.
- 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.
- Effects of salinity challenge on the endocrine control of osmoregulation and calcium homeostasis in the sea breamPublication . Guerreiro, P. M.; Fuentes, J.; Flik, G.; Canario, Adelino V. M.; Power, DeborahThe gilthead sea bream (Sparus auratu) is a marine species often found in coastal lagoons, experimenting episodic exposures to both brackish and hypersaline environments. However, little is known about the underlying endocrine mechanisms controlling osmoregulation in this and in most marine species. This study aimed at characterising some of the endocrine basis of sea bream osmoregulation, with emphasis on calcium homeostasis. Juvenile fish were exposed to different salinities, either by direct transfer or continuous adaptation over a short period of time. Salinities ranged from 0 to 55 p.p.t. and sampling was carried out 4, 24, 96 and 192 h after transfer. Six fish per group and per time point were sacrificed and plasma and tissue samples were collected. Osmolarity, osmolites and cortisol were measured in plasma. Prolactin, growth hormone, stanniocalcin, and calcitonin mRNA expressions were determined by PCR and northern blot. Mortality occurred after 4 hours in FW. Sea bream fry (2 month old, 20-60 may) were exposed to hypersaline and dilute seawater loaded with Ca and calcium fluxes were determined. Exposure of fry to lowered external salinity (50 and 25% SW) resulted in no mortality within 24 h and significantly decreased whole body calcium influx. Results will be discussed in relation to gene expression.
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