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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.
- 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.
- Expression of pituitary prolactin, growth hormone and somatolactin is modified in response to different stressors (salinity, crowding and food-deprivation) in gilthead sea bream Sparus auratusPublication . Laiz-Carrión, R.; Fuentes, J.; Redruello, Begoña; Guzmán, José M.; Río, María P. Martín del; Power, Deborah; Mancera, J. M.Prolactin (PRL), growth hormone (GH) and somatolactin (SL) expression was studied in gilthead sea bream (Sparus auratus) in response to several different stressors (salinity, food deprivation or stocking density). In the first experiment, specimens were acclimated during 100 days at three different environmental salinities: low salinity water (LSW, 6 ppt), brackish water (BW, 12 ppt) and seawater (SW, 38 ppt). Osmoregulatory parameters corresponded to those previously reported for this species under similar osmotic conditions. Pituitary PRL expression increased with decreasing environmental salinity, and was significantly different between SW- and LSW-acclimated fish. Pituitary GH expression was similar between SW- and BW-acclimated fish but decreased in LSW-acclimated specimens. Pituitary SL expression had a ‘‘U-shaped” relationship to environmental salinity with the lowest expression in BW-acclimated fish. In a second experiment SW-acclimated specimens were randomly assigned to one of four treatments and maintained for 14 days: (1) fed fish under low density (LD, 4 kg m!3); (2) fed fish under high density (HD, 70 kg m!3); (3) food deprived fish under LD; and (4) food deprived fish under HD. Plasma glucose and cortisol levels corresponded to those previously reported in S. auratus under similar experimental conditions. Pituitary PRL and SL expression increased in fish maintained under HD and decreased in food deprived fish. In conclusion, an effect of environmental salinity on pituitary PRL and GH expression has been demonstrated. In addition, crowding stress seems to interact with food deprivation in S. auratus and this is reflected by changes in pituitary PRL, GH and SL expression levels.
- 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.
- 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.
- Branchial osmoregulatory response to salinity in the gilthead sea bream,Sparus auratusPublication . Laiz-Carrión, R.; Guerreiro, P. M.; Fuentes, J.; Canario, Adelino V. M.; Martín Del Río, María P.; Mancera, J. M.The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2–192 hr) and long-term (2 weeks) exposure to different environmental salinities (5%, 15%, 25%, 38% and 60%) was investigated. A ‘‘U-shaped’’ relationship was observed between environmental salinity and gill Naþ,Kþ-ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (Po0.05) in fish adapted to 60% salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5% and 60%. Muscle water content (MWC) was significantly lower in fish adapted to 60%. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5% and 60%), whereas fish exposed to intermediate salinities (15% and 25%) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Naþ,Kþ-ATPase. However, at very low salinities both haemodilution and up-regulation of gill Naþ,Kþ-ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion uptake.
- Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1–34 N-terminal peptidePublication . Rotllant, J.; Worthington, G. P.; Fuentes, J.; Guerreiro, P. M.; Teitsma, C. A.; Ingleton, P. M.; Balment, R.; Canario, Adelino V. M.; Power, DeborahA 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– 35Tyr) 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 ¼ 20) for flounder and 2.5 0.29 ng/ml (n ¼ 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.
- The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata, L.)Publication . Brinca, Lilia; Fuentes, J.; Power, DeborahThe effect of estradiol-17b (E2) 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 (n¼130, body weight 50–70 g) were randomly divided into 2 groups; control and E2 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 E2 secreted significantly more PRL ðP < 0:05Þ in vitro than control fish. In E2 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 E2 had a different effect during spring; PRL secretion was significantly decreased ðP < 0:01Þ compared with the control fish. Anatomical evidence of abundant VIPimmunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIPin 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 E2 primed pituitary glands.