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- 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.
- Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptorPublication . Rotllant, J.; Redruello, Begoña; Guerreiro, P. M.; Fernandes, H.; Canario, Adelino V. M.; Power, DeborahThe scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1 – 35tyr)PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10 8 M) based on the N-terminal 1 – 34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3 H]myo-inositol incorporation in sea bream scales. However, peptides (10 8 M) with N-terminal deletions, such as (2 – 34), (3 – 34) and (7 – 34)PTHrP, were defective in stimulating cAMP production but stimulated [3 H]myo-inositol incorporation. (1 – 34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7 – 34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1 – 34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1 – 34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.
- 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.