Effect of blood withdrawal and angiotensin II on prolactin release in the tilapia,Oreochromis mossambicus

Repeated blood withdrawal (5% of estimated blood volume at 0, 1, 4, 8, 24, 48 and 76 h) from tilapia acclimated to fresh water (FW) resulted in a marked increase in plasma levels of prolactin (PRL) during the first 8 h, reaching a peak above 300 ng/ml after 4 h. The increase in plasma PRL levels was significant except for the level after 72 h. A slight but significant decrease in plasma osmolality was observed at all time points after the blood withdrawal. Repeated blood withdrawal from fish acclimated to seawater (SW) resulted in a marked increase in plasma osmolality after 4 and 8 h. A significant increase was observed in plasma growth hormone (GH) in the fish in SW until the end of the experiment, but there was no change in plasma PRL. Plasma levels of cortisol were significantly higher in the fish in SW than in those in FW during the first 24 h. Blood withdrawal resulted in a significant reduction in hematocrit values in both FW- and SW-adapted fish, suggesting hemodilution. In a separate experiment, a single blood withdrawal (20% of total blood) stimulated drinking after 5 h, regardless of whether the fish were held in FW or SW. Plasma PRL level was also elevated following a single blood withdrawal in the fish acclimated to FW, but not in the fish in SW. Intraperitoneal injection of ANG II (1.0 microg/g) into the fish in FW significantly increased plasma PRL levels after 1 h. Activation of the renin-angiotensin system after blood withdrawal and the dipsogenic action of angiotensin II (ANG II) are well established in fish. The reduction in plasma osmolality after repeated blood withdrawal in FW and the increased osmolality in SW suggest that blood volume is restored, at least in part, by drinking environmental water. These results suggest that the marked increase in PRL concentration after blood withdrawal from the fish in FW is due, at least in part, to a facilitative effect between ANG II and reduced plasma osmolality.     

Evidence that signal transduction for osmoreception is mediated by stretch-activated ion channels in tilapia

Prolactin (PRL) playsa central role in the freshwater osmoregulation of teleost fish,including the tilapia (Oreochromis mossambicus). Consistentwith this action, PRL release from the tilapia pituitary increases asextracellular osmolality is reduced both in vitro and in vivo.Dispersed tilapia PRL cells were incubated in a perfusion chamber thatallowed simultaneous measurements of cell volume and PRL release.Intracellular Ca²⁺ concentrations were measured from fura2-loaded PRL cells treated in a similar way. Gadolinium(Gd³⁺), known to block stretch-activated cation channels,inhibited hyposmotically induced PRL release in a dose-related mannerwithout preventing cell swelling. Nifedipine, an L-typeCa²⁺ channel blocker, did not prevent the increase in PRLrelease during hyposmotic stimulation. A high, depolarizingconcentration of KCl induced a transient and marked increase ofintracellular Ca²⁺ and release of PRL but did not preventthe rise in intracellular Ca²⁺ and PRL release evoked byexposure to hyposmotic medium. These findings suggest that a decreasein extracellular osmolality stimulates PRL release through the openingof stretch-activated ion channels, which allow extracellularCa²⁺ to enter the cell when it swells.

     

In vivo evidence for catecholaminergic inhibition of prolactin secretion in the teleostPoecilia latipinna

Summary Injections of L-dopa in freshwater (FW) fish reduced the size of the prolactin (PRL) cell nuclei, suggesting inhibition of PRL synthesis. A single injection of 6-hydroxydopamine (6-OHDA) in one-third seawater (1/3SW) fish reduced pituitary PRL content and increased PRL cell nuclear size at 6 h and 12 h, indicating stimulation of both synthesis and release of PRL. Two daily injections of 6-OHDA in 1/3SW fish led to PRL cell nuclear enlargement and elevated pituitary PRL content at 48 h after the second injection, indicating strong stimulation of PRL synthesis. Consideration of other parameters (plasma and body sodium levels, plasma osmotic pressure) suggests that the PRL cell responses to 6-OHDA were not mediated by internal osmotic changes. Pretreatment with 6-OHDA also appeared to accelerate the PRL cell activation induced by the transfer of fish from 1/3SW to FW.L-dopa opposed the enhancement of PRL release induced by a single injection of 6-OHDA. Injections of 3,4-dimethoxyphenylethylamine (DMPEA), a specific dopamine antagonist, caused short-term depletion of pituitary PRL, indicating enhanced PRL release.These results suggest that PRL secretion is subject to catecholaminergic inhibition, probably by dopamine. Considering these findings together with previous in vitro results (Wigham et al., 1975), it appears that the PRL cells are innervated by inhibitory catecholaminergic nerve fibres.Abbreviations DMPEA 3,4-dimethoxyphenylethylamine - L-dopa L-dihydroxyphenylalanine - FW freshwater - 6-OHDA 6-hydroxydopamine - PRL prolactin - 1/3SW one-third seawater     

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus)

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.     

Evidence that IP3 and ryanodine-sensitive intra-cellular Ca2+ stores are not involved in acute hyposmotically-induced prolactin release in Tilapia.

Prolactin (PRL) cells from the euryhaline tilapia, Oreochromis mossambicus, behave like osmoreceptors by responding directly to reductions in medium osmolality with increased secretion of the osmoregulatory hormone PRL. Extracellular Ca(2+) is essential for the transduction of a hyposmotic stimulus into PRL release. In the current study, the presence and possible role of intracellular Ca(2+) stores during hyposmotic stimulation was investigated using pharmacological approaches. Changes in intracellular Ca(2+) concentration were measured with fura-2 in isolated PRL cells. Intracellular Ca(2+) stores were depleted in dispersed PRL cells with thapsigargin (1 microM) or cyclopiazonic acid (CPA, 10 microM). Pre-incubation with thapsigargin prevented the rise in [Ca(2+)](i) induced by lysophosphatidic acid (LPA, 1 microM), an activator of the IP(3) signalling cascade, but did not prevent the hyposmotically-induced rise in [Ca(2+)](i) in medium with normal [Ca(2+)] (2mM). Pre-treatment with CPA produced similar results. Prolactin release from dispersed cells followed a pattern that paralleled observed changes in [Ca(2+)](i). CPA inhibited LPA-induced prolactin release but not hyposmotically-induced release. Xestospongin C (1microM), an inhibitor of IP(3) receptors, had no effect on hyposmotically-induced PRL release. Pre-exposure to caffeine (10mM) or ryanodine (1microM) did not prevent a hyposmotically-induced rise in [Ca(2+)](i). Taken together these results indicate the presence of IP(3) and ryanodine-sensitive Ca(2+) stores in tilapia PRL cells. However, the rapid rise in intracellular [Ca(2+)] needed for acute PRL release in response to hyposmotic medium can occur independently of these intracellular Ca(2+) stores.     

Signal transduction mechanisms mediating rapid, nongenomic effects of cortisol on prolactin release

While the mechanisms governing genomically mediated glucocorticoid actions are becoming increasingly understood, relatively little is known with regard to the cell signaling pathways that transduce rapid glucocorticoid actions. Studies of the cultured tilapia rostral pars distalis (RPD), a naturally segregated region of the fish pituitary gland that contains a 95-99% pure population of prolactin (PRL) cells and is easily dissected and maintained in a completely defined, serum-free media, indicate that physiological concentrations of cortisol rapidly inhibit PRL release. The attenuative action of cortisol on PRL release occurs within 10-20 min, is insensitive to the protein synthesis inhibitor, cycloheximide, and mimicked by its membrane impermeable analog, cortisol-21 hemisuccinate-conjugated bovine serum albumin (BSA). Cortisol and somatostatin, a peptide known to work through membrane receptors to inhibit PRL release, rapidly and reversibly reduces intracellular free Ca(2+) (Ca(i)(2+)), and inhibits 45Ca(2+) influx and BAYK-8644 induced PRL release. Preliminary investigations show cortisol, but not somatostatin, suppresses phospholipase C (PLC) activity in PRL cell membrane preparations. In addition, cortisol and somatostatin reduce intracellular cAMP and membrane adenylyl cyclase activity. These findings indicate that the acute inhibitory effects of cortisol on PRL release occur through a nongenomic mechanism involving interactions with the plasma membrane and inhibition of both the Ca(2+) and cAMP signal transduction pathways. Cortisol may reduce Ca(i)(2+) by inhibiting influx through L-type voltage-gated channels and possibly release through a PLC/inositol triphosphate sensitive intracellular Ca(2+) pool. In addition, it is also likely the steroid inhibits adenylyl cyclase activity in events leading to reduced cAMP production and the subsequent release of PRL.     

Osmoregulatory effects of hypophysectomy and homologous prolactin replacement in hybrid striped bass

The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na(+),K(+)-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilisxMorone chrysops). They were kept in dilute (isoosmotic) seawater for about 10 days after surgery. Seven days after transfer to FW, Hx fish had lower plasma osmolality and lower levels of Na(+), Cl(-), and Ca(2+) than sham-operated and intact fish. Fish were injected four times with oPRL (1, 5, or 20 microg/g body mass), sbPRL (10 or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na(+), Cl(-), and Ca(2+) above values seen in saline-injected controls. Hypophysectomy did not affect branchial Na(+),K(+)-ATPase activity, but enzyme activity was significantly reduced in Hx fish receiving oPRL (20 mug/g) or sbPRL (10 or 100 ng/g). These results indicate that PRL acts to maintain plasma osmotic and ionic balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na(+),K(+)-ATPase activity.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

Verapamil: influence upon basal and stimulated rat growth hormone and prolactin release in vitro

Verapamil is an organic calcium antagonist which is believed to prevent the passage of calcium (Ca2+) across the cell membrane into the cell. In a rat pituitary perifusion-immunoprecipitation system, verapamil (50 microM) prevents the inhibitory effect of increased extracellular Ca2+ (5.4 mM) on basal and stimulated release of stored, prelabeled [3H]GH and [3H]PRL. [3H]GH release from pituitary explants perifused in standard medium (GIBCO Minimum Essential Medium: 1.8 mM Ca2+) is transiently increased by 50 microM verapamil while [3H]PRL release is suppressed. With continued exposure to 50 microM verapamil, [3H]GH release rates fall below (89.8 +/- 2.1% of base) preverapamil levels while [3H]PRL release rates simply remain suppressed (48.2 +/- 7.3% of base). With 250 microM verapamil, poststimulatory inhibition of [3H]GH release occurs more quickly, and after its withdrawal rebound release of both GH and PRL occur. Inhibition of [3H]GH release by 25 nM somatostatin (SRIF) and post-SRIF rebound [3H]GH release is not prevented by 50 microM verapamil. The early, rapid [3H]GH release phase of 1 mM dibutyryl cyclic AMP (dbcAMP) stimulation is potentiated by verapamil pretreatment, but only if the verapamil is continued during dbcAMP stimulation. Potassium (21 mM K+)-stimulated release of both 3H-labeled hormones is inhibited after similar pretreatment 50 microM verapamil. Conclusions: (a) verapamil antagonizes the inhibitory effects of increased extracellular Ca2+ on basal or dbcAMP-stimulated [3H]GH and [3H]PRL release; (b) in standard medium (1.8 mM Ca2+), 50 microM verapamil increases basal [3H]GH release suggesting either a direct effect or an antagonism of 1.8 mM extracellular Ca2+; (c) although verapamil-sensitive Ca2+ movement is not necessary for dbcAMP stimulation of [3H]GH release, verapamil potentiates dbcAMP-stimulated release; (d) because verapamil also inhibits K+-stimulated [3H]GH and [3H]PRL release, these observations support previous suggestions that K+- and dbcAMP-stimulated rapid hormone release occurs from different intracellular sites; and (e) because verapamil does not prevent any phase of SRIF action and since these two agents differentially alter K+- and cAMP-stimulated release, their mechanisms of action must partially differ.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchus labrax: a candidate protein for osmosensing

The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the TRP ion channels superfamily that has been proposed as a potential fish osmosensor in previous studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing the hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 min to 30 days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24 h to 30 d. In contrast, in the kidney, the TRPV4 expression decreased from 10 min to 7d of exposure to FW and then it increased at 30 d. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membranes in branchial lamellae, the cartilage of gills, the posterior pituitary gland and in the collecting ducts. Possible roles of TRPV4 are discussed.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Quantitative ultrastructural evidence of alterations in prolactin secretion related to external salinity in a teleost fish (Poecilia latipinna)

Summary Quantitative ultrastructural morphometric studies were made on the prolactin cells of Poecilia latipinna adapted to freshwater (FW), one-third seawater (1/3 SW) and full-strength seawater (SW), and at various times after transfers between 1/3 SW and FW.In fully-adapted fish the rates of prolactin (PRL) synthesis and PRL release are inversely related to environmental salinity. During adaptation to a new salinity the two rates are temporarily uncoordinated, with release increasing or decreasing more readily than synthesis. Synthesis appears to take 30 h or longer to come into balance with the increased release rate following transfer from 1/3 SW to FW, and 72 h or longer to adjust to the reduction in release rate that follows the reverse transfer. The excess PRL granules that accumulate in the latter situation appear to be removed by lysosomal digestion. As in other teleosts, in fish adapted to the external medium the size of the stored PRL granules is inversely related to external salinity, but this relationship breaks down during adaptation to a new salinity.The stellate cells which penetrate between the PRL cells are more prominent, more extensively ramified, and appear more metabolically active in FW-adapted fish than in the other groups. These cells seem to be closely related in function to the secretory activity of the PRL cells.We thank Mr. W.A. Thomson and Mr. D.I. Hollingworth for technical assistance and Dr. D.I.C. Pearson (Department of Physics, University of Nancy, Nancy, France) for advice on mathematical analysis and computer programs. The work was carried out during tenure of an S.R.C. Studentship by T.F.C. Batten     

Response of prolactin cells to environmental calcium in the eel (Anguilla anguilla L.)

Prolactin (PRL) cell activity was investigated in eels kept in fresh water (FW), deionized water (DW) supplemented or not with Ca (2 mM), in Ca-enriched FW (10 mM), in normal (Ca 3.4 mM) or Ca-free 1/3 sea water (SW), and in SW (Ca 10.2 mM) or Ca-free SW (Ca 0.15 mM). Light-microscopic studies, including measurement of the nuclear area and cell height, showed that PRL cell activity, reduced in DW, is not affected by Ca supplementation. Activity is reduced in Ca-enriched FW, in 1/3 SW and in SW, conditions inducing an increase in the plasma sodium level. The lack of calcium in saline environments partly suppresses the nuclear atrophy occurring in SW. There is no significant correlation between external or total plasma calcium concentration and PRL cell activity. In artificial Ca-free SW, eels show a rapid increase in plasma osmolarity and sodium levels; there is a significant negative correlation between these two plasma values and the nuclear area or cell height of PRL cells. As in some other teleosts, plasma osmolarity and plasma sodium seem to play a more important role than external or internal calcium in controlling PRL secretion. This correlation is not apparent in eels kept in SW, having unstimulated PRL cells but active calcium-sensitive (Ca-s) cells in the pars intermedia.     

Water and NaCl absorption by the intestine of the tilapiaSarotherodon mossambicus adapted to fresh water or seawater and the possible role of prolactin and cortisol

Summary Rates of intestinal water, sodium and chloride absorption in tilapia, adapted to fresh water (FW) and seawater (SW), were measured in vitro, using noneverted sacs made from the anterior, middle and posterior intestinal regions. The anterior intestine from SW fish showed considerably less water, sodium and chloride absorption compared with that seen in FW fish. The middle intestine showed either minimal absorption or some secretion in both FW and SW. In the posterior intestine, water absorption was only limitedly affected by SW-adaptation, but sodium and chloride absorption rates were significantly lower in SW fish. Reductions in water absorption were already evident in the anterior intestine 24 h after transfer to 1/3 SW but reached lower levels 3 to 5 days following transfer to 100% SW. Thus, the anterior intestine of tilapia responds to increased environmental salinity by decreasing uptake of ions, whereas the posterior intestine maintains similar water absorption in both FW and SW, although ion absorption is lower in SW.Prolactin administration to SW fish augmented sodium and water absorption in the anterior intestine but had no effect on chloride absorption. In contrast, cortisol administration to FW fish decreased absorption of sodium, chloride and water to levels usually seen in SW fish. The observed effects of these hormones in tilapia intestinal absorption may be confined to the specialized anterior intestinal region in this species; hormonal effects on the rest of the intestine were not examined.     

Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.     

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.     

Interaction of calcium and cyclic nucleotides in thyroliberin-stimulated prolactin release

The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH₃ and GH₄ rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH₃ and GH₄ cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH₃ or GH₄ cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH₃ or GH₄ cells.