Search

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3(-) secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3(-) secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3(-) secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3(-) secretion rapidly decreased when PTHrP(1-34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1 ng ml(-1) compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) or the phospholipase C inhibitor (U73122, 10 μmol l(-1)) the effect of PTHrP(1-34) on HCO3(-) secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose-response curve in the range of 0.1-1000 ng ml(-1), achieves a maximal effect of 60-65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1 ng ml(-1). The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) and was insensitive to the phospholipase C inhibitor (U73122, 10 μmol l(-1)). In vivo injections of PTHrP(1-34) or the PTH/PTHrP receptor antagonist PTHrP(7-34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO3(-) secretion., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10(-6)M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose-response curve, with significant effects achieved at 10(-8)M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200μM) applied apically, but was completely abolished in the presence of apical bumetanide (200μM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The skin is the largest organ in the body and is a barrier between the internal and external environment. The present study evaluates how PTU, a goitrogen, that is used to treat hyperthyroidism affects the structure and electrical properties of the frog (Xenopus laevis) skin. The results are considered in the context of the two-membrane model established in the seminal work of Ussing and collegues in the 1940s and 1950s. In vitro experiments with skin from Xenopus adults revealed that PTU can act directly on skin and causes a significant increase (p<0.05, One-way ANOVA) in short circuit current (Isc) via an amiloride-insensitive mechanism. Juvenile Xenopus exposed to waterborne PTU (5 mg/L) had a significantly bigger and more active thyroid gland (p<0.01, Student's t-test) than control Xenopus. The bioelectric properties of skin taken from Xenopus juveniles treated with PTU in vivo had a lower Isc, (3.05±0.4, n=13) and Rt (288.2±39.5) than skin from control Xenopus (Isc, 4.19±1.14, n=14; Rt, 343.3±43.3). A histological assessment of skin from PTU treated Xenopus juveniles revealed the epidermis was significantly thicker (p<0.01, Student's t-test) and had a greater number of modified exocrine glands (p<0.01, Student's t-test) in the dermis compared to control skin. Modifications in skin structure are presumably the basis for its changed bioelectric properties and the study highlights a site of action for environmental chemicals which has been largely neglected., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The 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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

The Ca(2+)-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 Ca(2+) 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 80microM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80microM 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 17,20alpha-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11beta-hydroxysteroid 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 Ca(2+)-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 Ca(2+)-dependent CaM and that possibly Ca(2+)-CaM determines the shift in steroidogenesis from C21 steroids to androgens.

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 confinement stress, suppressed circulating PTHrP 6 and 24-fold, respectively, by comparison to control fish. Dexamethasone treatment reduced cortisol levels, prevented the decrease of plasma PTHrP observed in confined fish and raised plasma PTHrP levels in non-confined fish. In contrast, a single injection of (1-34) PTHrP caused a short-term (within 30 min and up to 2.5 h) decrease in plasma cortisol. The antagonistic effects between PTHrP and cortisol were substantiated by an overall (data pooled from all experiments) highly significant negative correlation (r0=-0.745, p<0.001, n=115) between the plasma levels of the two hormones. Although the underlying mechanism of the interaction still has to be determined, the high levels of PTHrP in circulation and the existence of systemic regulation favour the hypothesis that in fish PTHrP may act as an endocrine factor, although the gland that produces it still remains to be identified.

The effect of estradiol-17beta (E(2)) 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 (n=130, body weight 50-70 g) were randomly divided into 2 groups; control and E(2) 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 E(2) secreted significantly more PRL (P<0.05) in vitro than control fish. In E(2) 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 E(2) had a different effect during spring; PRL secretion was significantly decreased (P<0.01) compared with the control fish. Anatomical evidence of abundant VIP immunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIP in 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 E(2) primed pituitary glands.