Browsing by Author "Flik, G."
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- Calcium handling in Sparus auratus: effects of water and dietary calcium levels on mineral composition, cortisol and PTHrP levelsPublication . Abbink, W.; Bevelander, G. S.; Rotllant, J.; Canario, Adelino V. M.; Flik, G.Juvenile gilthead sea bream (Sparus auratus L.; 10–40·g body mass) were acclimatized in the laboratory to full strength (34‰) or dilute (2.5‰) seawater and fed normal, calcium-sufficient or calcium-deficient diet for nine weeks. Mean growth rate, whole-body calcium and phosphorus content and accumulation rates were determined, as well as plasma levels of ionic and total calcium, cortisol and parathyroid hormone related protein (PTHrP; a hypercalcemic hormone in fish). When confronted with limited calcium access (low salinity and calcium-deficient diet), sea bream show growth arrest. Both plasma cortisol and PTHrP increase when calcium is limited in water or diet, and a positive relationship was found between plasma PTHrP and plasma ionic calcium (R2=0.29, N=18, P<0.05). Furthermore, a strong correlation was found between net calcium and phosphorus accumulation (R2=0.92, N=16, P<0.01) and between body mass and whole-body calcium (R2=0.84, N=25, P<0.01) and phosphorus (R2=0.88, N=24, P<0.01) content. Phosphorus accumulation is strongly calcium dependent, as phosphorus accumulation decreases in parallel to calcium accumulation when the diet is calcium deficient but phosphorus sufficient. We conclude that PTHrP and cortisol are involved in the regulation of the hydromineral balance of these fish, with growthrelated calcium accumulation as an important target.
- Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinitiesPublication . Ruiz-Jarabo, I.; Klaren, P. H. M.; Louro, Bruno; Martos-Sitcha, J. A.; Pinto, P. I.; Vargas-Chacoff, L.; Flik, G.; Martinez-Rodriguez, G.; Power, Deborah; Mancera, J. M.; Arjona, F. J.Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, ff4), outer ring deiodination and Na+/K+ -ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and ff4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cadiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit 13 (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (diol and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity. (C) 2016 Elsevier Inc. All rights reserved.
- CYP27A1 expression in gilthead sea bream (Sparus auratus, L.): effects of calcitriol and parathyroid hormone-related proteinPublication . Bevelander, G. S.; Pinto, E. S. L. C.; Canario, Adelino V. M.; Spanings, T.; Flik, G.Little is known about vitamin D metabolism in fishes. Several reports have shown hydroxylase activities in various organs to produce vitamin D metabolites, but the enzymes involved have not been isolated or characterized. We isolated and characterized a renal mitochondrial hydroxylase, CYP27A1, that governs vitamin D metabolism in gilthead sea bream, Sparus auratus. The enzyme is highly expressed in kidney and to a far lesser extent in liver. When treated with 25-hydroxy vitamin D or calcitriol, the kidney responded differentially and time dependently with CYP27A1 mRNA expression levels. This response substantiates a role for CYP27A1 in fish vitamin D metabolism. This notion is strengthened by upregulation of CYP27A1 in sea bream treated with parathyroid hormone-related protein (PTHrP), and suggests an original role for PTHrP in calcitriol-regulated processes n fish similar to the role of PTH in mammalian vitamin D-dependent processes.
- 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.
- Endocrinology of calcium homeostasisPublication . Canario, Adelino V. M.; Flik, G.Michelle Wheatly reviewed the role of binding proteins in the regulation of calcium homeostasis during periods of net vectorial influx using the crayfish, Procambarus clarkii, as a model. The protein studied were the import and export proteins, epithelial Ca2+ channel (ECaC), sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA) and Na+/Ca2+ exchanger (NCX). The expression of these proteins is a coordinated process at multiple levels including the apical vs. basolateral domains, plasma membranes vs. organelle membranes and transmembrane Ca2+ transporters and binding proteins. An integrative model of cellular Ca2+ homeostasis was presented which took into account the spatial and temporal protein expression during crayfish molting.
- Measurement of PTHrP, PTHR1, and CaSR expression levels in tissues of sea bream (Sparus aurata) using quantitative PCRPublication . Hang, X. M.; Power, Deborah; Flik, G.; Balment, R.A quantitative PCR (Q-PCR) method has been established to measure the mRNA expression levels of parathyroid hormone–related protein (PTHrP), parathyroid hormone receptor type 1 (PTHR1), and calcium-sensing receptor (CaSR) in sea bream (Sparus aurata), using the housekeeping gene, - actin, as endogenous control. TaqMan primers and probes were designed using the Primer Express program, according to the published/unpublished sequences of the three target genes and -actin of sea bream. Different tissues including gill, kidney, duodenum, hindgut, rectum, liver, heart, brain, pituitary, skin, muscle, and gonad were removed and immediately snap-frozen from three juvenile sea bream (100–150 g) cultured in sea water. The mRNAs were extracted and reverse-transcribed into cDNAs, which were subsequently examined by the ABI 5700 system using an optimized Q-PCR method. Triplicate measures of each sample indicated consistency of the technique. However, the mRNA expression levels for each transcript in these tissues were variable between fish and also relatively low. Nevertheless, this methodology can be used in the future studies of factors that may alter gene expression in these tissues.
- Parathyroid hormone-related protein and calcium regulation in vitamin D-deficient sea bream (Sparus auratus)Publication . Abbink, W.; Hang, X. M.; Guerreiro, P. M.; Spanings, T.; Ross, H. A.; Canario, Adelino V. M.; Flik, G.Gilthead sea bream (Sparus auratus L.) were fed a vitamin D-deficient diet for 22 weeks. Growth rate, whole body mineral pools and calcium balance were determined. Plasma parathyroid hormone-related protein (PTHrP) and calcitriol levels were assessed. Expression of mRNA for pthrp and pth1r was quantified in gills and hypophysis. Fish on vitamin D-deficient diet (D− fish) showed reduced growth and lower calcium turnover (calcium influx, efflux and accumulation rates decreased) and unaltered plasma calcium levels. Plasma calcitriol levels became undetectable, PTHrP levels decreased in the D− fish. In controls, a significant increase in plasma PTHrP level over time was seen, i.e. it increased with body mass. Relationships were found between plasma PTHrP and the whole body pools of calcium, phosphorus and magnesium, indicative of a role for PTHrP in bone development. Expression of pthrp and pth1r mRNA was down-regulated in the hypophysis of D−fish, whereas in gill tissue, pthrp and pth1r mRNA were up-regulated. We conclude that lower pthrp mRNA expression and plasma values in D− fish reflect lower turnover of PTHrP under conditions of hampered growth; up-regulation of pthrp mRNA in gills indicate compensatory paracrine activity of PTHrP during calcitriol deficiency to guarantee well-regulated branchial calcium uptake. This is the first report to document a relation between PTHrP and calcitriol in fish.
- Parathyroid hormone-related protein: a calcium regulatory factor in sea bream (Sparus aurata L.) larvaePublication . Guerreiro, P. M.; Fuentes, T.; Power, Deborah; Ingleton, P. M.; Flik, G.; Canario, Adelino V. M.The effects of an N-terminal peptide (amino acids 1-38) of Fugu parathyroid hormone-related protein (PTHrP 1-38) on calcium regulation of larval sea bream were investigated in seawater (36 per thousand) and after transfer to dilute seawater (12 per thousand). Exposure to PTHrP 1-38 evoked a 1.5-fold increase in calcium influx in both full-strength and dilute seawater. Calcium influx in dilute seawater-adapted larvae was roughly one-half that observed in full-strength seawater controls. PTHrP 1-38 also reduced drinking of fish in seawater but, at all concentrations tested, was without effect in dilute seawater. The amount of water imbibed was 55% lower in dilute seawater than in seawater. PTHrP 1-38 exposure affected the calcium influx route: the main contribution of calcium uptake shifted from intestinal absorption to extraintestinal uptake, probably by the induction of a dose-dependent increase in branchial (active) transport. Moreover, seawater-adapted fish exposed to 1 nM and 10 mM PTHrP 1-38 experienced a 2.5-fold reduction in overall calcium efflux. Overall, the calciotropic action of PTHrP 1-38 resulted in a dose-dependent increase in net calcium balance.
- PTHrP potentiating estradiol-induced vitellogenesis in sea bream (Sparus auratus, L.)Publication . Bevelander, G. S.; Hang, X. M.; Abbink, W.; Spanings, T.; Canario, Adelino V. M.; Flik, G.n fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E2-naïve and E2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E2 in combination with sbPTHrP a higher vitellogenin production was seen than with E2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E2-naïve liver cells, both in vivo and in vitro.
- PTHrP regulation and calcium balance in sea bream (Sparus auratus L.) under calcium constraintPublication . Abbink, W.; Bevelander, G. S.; Hang, X. M.; Lu, W. Q.; Guerreiro, P. M.; Spanings, T.; Canario, Adelino V. M.; Flik, G.Juvenile gilthead sea bream were exposed to diluted seawater (2.5‰ salinity; DSW) for 3·h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30·days. Branchial Ca2+ influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca2+ and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca2+ (R2=0.30, N=39, P<0.05), and plasma sPTHrP and body mass of the fish (R2=0.37, N=148, P<0.001) were found. Immunoreactive sPTHrP was demonstrated in pituitary gland pars intermedia cells that border the pars nervosa and co-localises with somatolactin. In the pituitary gland, pthrp, pth1r and casr mRNAs were downregulated after both short- and long-term exposure to DSW. A correlation between pituitary gland pthrp mRNA expression and plasma Ca2+ (R2=0.71, N=7, P<0.01) was observed. In gill tissue, pthrp and pth1r mRNAs were significantly upregulated after 30·days exposure to DSW, whereas no effect was found for casr mRNA expression. We conclude that in water of low salinity, declining pituitary gland pthrp mRNA expression accompanied by constant plasma sPTHrP levels points to a reduced sPTHrP turnover and that sPTHrP, through paracrine interaction, is involved in the regulation of branchial calcium handling, independently of endocrine pituitary gland sPTHrP.