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G. Flik and S. F. Perry

ABSTRACT

Exposure of rainbow trout to a reduced ambient calcium level (from 490 to 25 μmol Ca2+/l) caused hypocalcaemia and induced a rapid increase (within 1 h) in systemic cortisol levels. Under conditions of low environmental calcium concentrations, cortisol levels remained increased for at least 8 days. After this time the in-vitro Ca2+-transport capacity of branchial basolateral membrane vesicles was increased due to stimulation of Ca2+-ATPase activity, presumably as a result of chloride cell proliferation. Pituitary prolactin cells were unaffected by low ambient calcium levels. Fish kept in water containing 490 μmol Ca2+/l and treated with cortisol for 7 days displayed an increase in whole body calcium uptake and an enhancement of the branchial calcium transport capacity; concomitantly, hypercalcaemia was observed. We conclude that, in the rainbow trout, cortisol exerts hypercalcaemic effects by stimulating Ca2+ uptake from the water and that this effect forms an intrinsic part of the established mineralocorticoid action of cortisol in fish.

Journal of Endocrinology (1989) 120, 75–82

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R Ørnsrud, E J Lock, C N Glover and G Flik

Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA–VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA–CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.

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E J Lock, R Ørnsrud, L Aksnes, F A T Spanings, R Waagbø and G Flik

Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr–smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (<1 mM Ca2+) to the plasma (~1.25 mM Ca2+) and to strongly hypercalcic (8–12 mM Ca2+). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D3 and 24,25(OH)2D3 did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr–smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

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Metz JR, MO Huising, J Meek, AJ Taverne-Thiele, SE Wendelaar Bonga and G Flik

Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.

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RJ Arends, R van der Gaag, GJ Martens, SE Wendelaar Bonga and G Flik

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.

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RJ Arends, JM Mancera, JL Munoz, SE Wendelaar Bonga and G Flik

We investigated short-term effects (up to 24 h) of air exposure and confinement, and long-term effects (up to 11 days) of confinement, to elucidate signalling pathways in the stress response of gilthead sea bream Sparus aurata L. Plasma glucose and lactate were taken as indicators of sympathetic activation, and alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotrophic hormone (ACTH) and cortisol as indicators of activation of the brain-pituitary-interrenal (BPI) axis. Air exposure for 3 min resulted, within 30 min, in an increase in plasma concentrations of cortisol, alpha-MSH, glucose, lactate, osmolality and plasma Na, Cl and Mg. Plasma ACTH and beta-endorphin and plasma K, Ca and P did not change. We conclude that air exposure mainly activates the brain-sympathetic-chromaffin cell (BSC) axis. In fish confined at a density of 70 kg/m(3) (compared with 4 kg/m(3) in controls), cortisol, ACTH and alpha-MSH increased within 1 h, indicating activation of the BPI axis. Plasma glucose, Na, Cl and Mg increased with an 8 h delay compared with the response to air exposure. No changes in plasma lactate, osmolality, K, Ca and P were observed. Long-term confinement induced a biphasic cortisol response with peaks at 1 h and at 2 and 3 days. A gradual increase in plasma beta-endorphin concentrations peaked at 7 days; the concentration of alpha-MSH increased rapidly within 1 h and then declined to control values 4 days after the onset of confinement. No changes in ACTH were detected. Our data provide evidence that a stressor-specific activation of the BSC and BPI axes may occur in Sparus aurata.

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RJ Arends, J Rotllant, Metz JR, JM Mancera, SE Wendelaar Bonga and G Flik

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.

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S Varsamos, SE Wendelaar Bonga, G Flik, R Quere and T Commes

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.

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A. E. Lamers, G. Flik, W. Atsma and S. E. Wendelaar Bonga

ABSTRACT

In stressed tilapia, Oreochromis mossambicus, total α-melanocyte-stimulating hormone (α-MSH) levels and di-acetyl α-MSH/mono-acetyl α-MSH (di:mono) ratios are elevated. We therefore investigated the role of α-MSH in the regulation of the pituitary–interrenal axis. The corticotrophic activities of des-acetyl α-MSH, mono-acetyl α-MSH and di-acetyl α-MSH were compared. These forms of α-MSH were isolated from neurointermediate lobes and tested in a superfusion experiment with homologous interrenal tissue. The corticotrophic activity of di-acetyl α-MSH was the highest, followed by that of des-acetyl α-MSH and mono-acetyl α-MSH. Apparently, acetylation of α-MSH is of functional significance for corticotrophic action. Di-acetyl α-MSH proved to be about 100 times less potent than ACTH(1–39): the half-maximal stimulating concentrations for ACTH and di-acetyl α-MSH were 0·89 nmol/l and 110 nmol/l respectively. Surprisingly, a superfusate from neurointermediate lobes proved to be only about three times less active than a superfusate from the pituitary pars distalis, in which the corticotrophic activity is attributable to its ACTH content. When selectively stripped of all forms of α-MSH by passage through a Sepharose column coated with an antiserum against α-MSH the neurointermediate lobe superfusate was devoid of corticotrophic activity. Thus α-MSH appears to be the corticotrophic factor in the superfusate of the neurointermediate lobe. After the same treatment, the corticotrophic activity of the pars distalis superfusate was not affected. We conclude that (in vivo) an as yet unidentified factor is co-released with α-MSH from the neurointermediate lobe, and that this potentiates its corticotrophic activity.

Journal of Endocrinology (1992) 135, 285–292

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D. Swennen, F. Rentier-Delrue, B. Auperin, P. Prunet, G. Flik, S. E. Wendelaar Bonga, M. Lion and J. A. Martial

ABSTRACT

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/1. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Journal of Endocrinology (1991) 131, 219–227