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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.
- PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challengePublication . Fuentes, J.; Brinca, Lilia; Guerreiro, P. M.; Power, DeborahThe endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p < 0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p < 0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p < 0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na+/K+-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.
- 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.
- 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.
- A PTH/PTHrP receptor antagonist blocks the hypercalcemic response to estradiol-17bPublication . Fuentes, J.; Guerreiro, P. M.; Modesto, Teresa; Rotllant, J.; Canario, Adelino V. M.; Power, DeborahEstradiol (E2) increases circulating calcium and phosphate levels in fish, thus acting as a hypercalcemic and hyperphosphatemic factor during periods of high calcium requirements, such as during vitellogenesis. Since parathyroid hormone (PTH)-related protein (PTHrP) has been shown to be calciotropic in fish, we hypothesized that the two hormones could be mediating the same process. Sea bream (Sparus auratus) juveniles receiving a single intraperitoneal injection of piscine PTHrP(1-34) showed an elevation in calcium plasma levels within 24 h. In contrast, injections of the PTH/PTHrP receptor antagonist PTHrP(7-34) decreased circulating levels of calcium in the same period. Intraperitoneal implants of estradiol-17 (E2; 10 g/g) evoked significant increases of circulating plasma levels of calcium and phosphorus and a sustained increases of circulating plasma levels of PTHrP. However, a combined treatment of E2 and PTHrP(7-34) evoked a markedly lower calcium response compared with E2 alone. We conclude that PTHrP or a related peptide that binds the PTH/PTHrP receptor mediates, at least in part, the hypercalcemic effect of E2 in calcium and phosphate balance in fish.
- Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related proteinPublication . Flanagan, J. A.; Power, Deborah; Bendell, L. A.; Guerreiro, P. M.; Fuentes, J.; Clark, M. S.; Canario, Adelino V. M.; Danks, J. A.; Brown, B. L.; Ingleton, P. M.This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96–117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.