Cell volume increase and extracellular Ca2+ are needed for hyposmotically induced prolactin release in tilapia.

In the tilapia (Oreochromis mossambicus), as in many euryhaline teleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and water permeability and increase solute retention. Consistent with these actions, PRL release is stimulated as extracellular osmolality is reduced both in vivo and in vitro. In the current experiments, a perfusion system utilizing dispersed PRL cells was developed for permitting the simultaneous measurement of cell volume and PRL release. Intracellular Ca(2+) was monitored using fura 2-loaded cells under the same conditions. When PRL cells were exposed to hyposmotic medium, an increase in PRL cell volume preceded the increase in PRL release. Cell volume increased in proportion to decreases of 15 and 30% in osmolality. However, regulatory volume decrease was clearly seen only after a 30% reduction. The hyposmotically induced PRL release was sharply reduced in Ca(2+)-deleted hyposmotic medium, although cell volume changes were identical to those observed in normal hyposmotic medium. In most cells, a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) during hyposmotic stimulation was dependent on the availability of extracellular Ca(2+), although small transient increases in [Ca(2+)](i) were sometimes observed upon introduction of Ca(2+)-deleted media of the same or reduced osmolality. These results indicate that an increase in cell size is a critical step in the transduction of an osmotic signal into PRL release and that the hyposmotically induced increase in PRL release is greatly dependent on extracellular Ca(2+).

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

Prolactin (PRL) plays a central role in the freshwater osmoregulation of teleost fish, including the tilapia (Oreochromis mossambicus). Consistent with this action, PRL release from the tilapia pituitary increases as extracellular osmolality is reduced both in vitro and in vivo. Dispersed tilapia PRL cells were incubated in a perfusion chamber that allowed simultaneous measurements of cell volume and PRL release. Intracellular Ca(2+) concentrations were measured from fura 2-loaded PRL cells treated in a similar way. Gadolinium (Gd(3+)), known to block stretch-activated cation channels, inhibited hyposmotically induced PRL release in a dose-related manner without preventing cell swelling. Nifedipine, an L-type Ca(2+) channel blocker, did not prevent the increase in PRL release during hyposmotic stimulation. A high, depolarizing concentration of KCl induced a transient and marked increase of intracellular Ca(2+) and release of PRL but did not prevent the rise in intracellular Ca(2+) and PRL release evoked by exposure to hyposmotic medium. These findings suggest that a decrease in extracellular osmolality stimulates PRL release through the opening of stretch-activated ion channels, which allow extracellular Ca(2+) to enter the cell when it swells.

Osmoregulatory actions of growth hormone and prolactin in an advanced teleost.

To date, growth hormone (GH) is known to contribute to seawater adaptation only in salmonid fishes (primitive Euteleostei). Accordingly, the effects of homologous GH and two forms of homologous prolactin (PRL177 and PRL188) on hypoosmoregulatory ability and gill Na+,K(+)-ATPase activity in a more advanced euryhaline cichlid fish, the tilapia (Oreochromis mossambicus), were examined. Following adaptation of hypophysectomized fish to 25% seawater for 3 weeks, fish were given four injections of hormone or vehicle. They were then exposed to 100% seawater for 12 hr and examined for changes in plasma osmolality. Tilapia GH (0.02 and 0.2 microgram/g) significantly improved the ability of tilapia to decrease plasma osmolality following transfer to full-strength seawater, in a dose-related manner. Growth hormone treatment also significantly stimulated gill Na+,K(+)-ATPase activity (0.5 microgram/g). Both tilapia PRLs (PRL177 and PRL188) increased plasma osmolality in 100% seawater and reduced gill Na+,K(+)-ATPase activity, the effects induced by PRL188 being more significant than those by PRL177. Thus, GH may be involved in seawater adaptation of tilapia, a species belonging to the most advanced teleost super-order (Acanthopterygii), whereas both PRLs in tilapia are not involved in seawater adaptation.

Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia, Oreochromis mossambicus.

It is increasingly clear that growth hormone (GH) has growth-promoting effects in fishes, which are mediated in part by the insulin-like growth factor (IGF)-I. Growth-promoting actions of prolactin (PRL) have been reported in higher vertebrates, but are less well established in teleosts. We examined the effects of injecting homologous GH or the two homologous tilapia PRLs (tPRL177 and tPRL188) on the in vitro incorporation of [35S] sulfate (extracellular matrix synthesis) and [3H]thymidine (DNA synthesis) by ceratobranchial cartilage explants and on IGF-I mRNA levels in tilapia liver. Tilapia GH (tGH) and tPRL177 stimulated sulfate uptake at the highest doses examined. Thymidine incorporation was stimulated by tPRL177. tPRL188 was without these effects. Consistent with its somatotropic actions, tGH elevated IGF-I mRNA levels in the liver. tPRL177 also elevated liver IGF-I levels. Consistent with the previously described osmoregulatory actions of GH and PRL in teleosts, we observed that tGH elevated and tPRL177 and tPRL188 lowered levels of gill Na+,K+-ATPase activity. High-affinity, low-capacity binding sites for tGH in the tilapia liver were identified. tPRL177 binds with lower affinity than tGH to these sites but can displace 125I-labeled tGH from its receptor. The ability of tPRL177 to displace tGH was similar to that of ovine GH. tPRL188 did not displace 125I-labeled tGH binding. Collectively, this work suggests that tPRL177 may possess somatotropic actions similar to tGH, but only in freshwater tilapia where tPRL177 levels are sufficiently high for it to act as a competitive ligand for GH receptors.

Patterns of thyroxine and triiodothyronine in serum and follicle-bound oocytes of the tilapia, Oreochromis mossambicus, during oogenesis.

This study describes simultaneous measurements of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), in the oocytes and serum of a female teleost fish over a complete reproductive cycle. We have identified patterns in circulating T4 and T3 levels as well as their accumulation into oocytes during the reproductive cycle of the tilapia (Oreochromis mossambicus). This is the first description of the patterns with which thyroid hormones accumulate in teleost oocytes. The sampling strategy used in the study eliminated the possible influences of covarying environmental factors that may affect thyroid hormone levels independently of reproductive events. Hormones in serum and oocytes were measured by radioimmunoassay utilizing miniature Sephadex columns. The total content of both thyroid hormones in the oocytes increased throughout most of the ovarian cycle as the oocytes increased in size from less than 2 mg to approximately 6.5 mg by ovulation. By contrast, concentrations of thyroid hormones in the oocytes rose only during the first third of post-spawning oocyte growth (up to approximately 2 mg) before attaining plateaus at approximately 6 ng/g for T4 and 13 ng/g for T3. Serum concentrations of T4 and T3 varied in cyclical patterns during oogenesis, dropping to lows of 3.4 ng/ml (T4) and 2.7 ng/ml (T3) when the oocytes were 1.5 and 2 mg, respectively, and then increasing to 6.5 ng/ml (T4) and 4.8 ng/ml (T3) when the oocytes reach approximately 6 mg. The concentrations of both hormones decreased shortly before spawning. Maximum concentrations of thyroid hormones in the oocytes were reached approximately 10 days prior to those in the serum. Although the serum levels of T4 were greater than those of T3, the reverse was found in the oocytes. Triiodothyronine appears to be accumulated selectively over T4 and the patterns with which both thyroid hormones accumulate in the oocytes of the tilapia do not appear to be tied to serum levels.

The loss of 45Ca2+ associated with prolactin release from the tilapia (Oreochromis mossambicus) rostral pars distalis.

The relationship between tritium 3H-labeled prolactin (PRL) release and the loss of tissue-associated 45Ca2+ was examined in the tilapia rostral pars distalis (RPD) using perifusion incubation under conditions which inhibit or stimulate PRL release. Depolarizing [K+] (56 mM) and hyposmotic medium (280 mOsmolal) increased both the release of [3H]PRL and the loss of 45Ca2+. The responses to high [K+] were faster and shorter in duration than those produced by reduced osmotic pressure. The depletion of Ca2+ from the incubation medium with 2 mM EGTA suppressed the [3H]PRL response evoked by high [K+] or reduced osmotic pressure. Exposing the tissues to Ca(2+)-depleted medium in the absence of high [K+] or reduced osmotic pressure produced a sharp, but brief, increase in 45Ca2+ loss. Cobalt (10(-3) M), a competitive inhibitor of calcium-mediated processes, inhibited the [3H]PRL response to hyposmotic medium and to high [K+]. Cobalt also diminished the increased loss of 45Ca2+ evoked by exposure to reduced osmotic pressure, but was ineffective in altering responses to high [K+]. Methoxyverapamil (D600; 10(-5) M), a blocker of certain voltage-sensitive Ca2+ channels, did not alter either the [3H]PRL or the 45Ca2+ responses to high [K+] and reduced osmotic pressure. Taken together with our earlier studies, the present findings suggest that exposure to high [K+] or hyposmotic medium produces rapid changes in the Ca2+ metabolism of the tilapia RPD that are linked to the stimulation of PRL secretion. Nevertheless, the increased 45Ca2+ loss, but not [3H]PRL release, upon exposure to Ca(2+)-depleted media suggests that Ca2+ loss may not always reflect intracellular events that lead to PRL release.

Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro.

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [3H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45Ca2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. Our studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca2+ metabolism.

Effects of depolarizing concentrations of K+ and reduced osmotic pressure on 45Ca2+ accumulation by the rostral pars distalis of the tilapia (Oreochromis mossambicus).

The accumulation of 45Ca2+ into tilapia prolactin (PRL) tissue was examined under conditions which alter prolactin release. In initial experiments, PRL tissue was incubated in medium containing 12 microCi/ml 45Ca2+ in hyperosmotic medium (355 mOsmolal). Under these conditions, 45Ca2+ accumulated steadily, reaching a plateau within 15-20 min. Subsequent exposure to La3+, which displaces Ca2+ from superficial pools in a wide variety of tissues, rapidly (within 5 min) removed nearly 70% of the 45Ca2+ associated with the tissue. Following this initial removal of 45Ca2+, the level of 45Ca2+ in the PRL tissue remained constant, and is referred to as the La3(+)-resistant pool of Ca2+. This pool of Ca2+ is thought to reflect the entry rate of Ca2+ from extracellular sources. Prolactin tissue exposed to hyposmotic medium or to depolarizing [K+], which stimulates PRL release, significantly increased 45Ca2+ accumulation in this La3(+)-resistant pool. These results indicate that reduced osmotic pressure and depolarization may alter release from tilapia PRL cells, in part, through their ability to increase the entry of extracellular Ca2+.

Effects of cortisol and growth hormone replacement on osmoregulation in hypophysectomized coho salmon (Oncorhynchus kisutch).

Both cortisol and GH were able to reverse partially the effects of hypophysectomy on coho salmon (Oncorhynchus kisutch) as judged by improved seawater (SW) tolerance after long-term treatment; however, neither hormone significantly restored gill Na+, Na+,K+-ATPase activity. In the first experiment, gill Na+,K+-ATPase activity remained low in hypophysectomized (Hx) coho treated with cortisol (15 micrograms/g body wt, suspended in cocoa butter) for 96 hr (48 hr in fresh water followed by 48 hr in seawater). In addition, plasma sodium levels were higher in the cortisol-treated Hx animals compared with those in untreated controls. In the second experiment, treatment with cortisol-filled Silastic capsules and implants of bovine GH (mixed with cholesterol in a ratio calculated to yield a dose of 1.5 microgram/g body wt/week) for 12 days also failed to alter gill Na+,K+-ATPase activity, but did reduce plasma sodium levels in animals transferred to SW for 48 hr. These results suggest that these hormones may be involved in the development of hypoosmoregulatory ability in coho salmon. However, the failure of either hormone to restore gill Na+,K+-ATPase activity suggests that a combination of these hormones and/or an additional hormone(s) acting in a synchronized fashion may be required for full effectiveness.

Effects of cortisol and growth hormone on osmoregulation in pre- and desmoltified coho salmon (Oncorhynchus kisutch).

Salmonid species which undergo smoltification show a concurrent enhancement in saltwater (SW) osmoregulatory ability. This developmental change is marked by an increase in SW tolerance and gill Na+,K+-ATPase activity which appears to result, in part, from an increase in gill chloride cell density. Previous studies have suggested that cortisol and growth hormone (GH) may stimulate SW osmoregulatory mechanisms in salmonids. In this study, these hormones were examined for their ability to induce smoltification-associated osmoregulatory changes in pre- and desmoltified coho salmon (Oncorhynchus kisutch). Cortisol treatment for 12 days increased gill Na+,K+-ATPase activity in presmolts and gill residual (Na+,K+-independent) ATPase activity in both groups. Chloride cell density in presmolt primary and secondary lamellae and in desmolt secondary lamellae was increased as well. The rise in plasma sodium levels in fish transferred to SW was reduced only in desmolts. Treatment with bovine GH for 12-13 days increased gill Na+,K+-ATPase activity in presmolts and in desmolts. However, GH treatment in either group did not increase gill residual ATPase activity or alter plasma sodium levels in SW-transferred animals. Gill chloride cell density in presmolts also was unaffected (desmolts were not examined). Thus, both cortisol and GH are partially able to produce changes similar to those observed during smoltification. The contrasting effects of these hormones on gill chloride cell density and gill residual ATPase activity suggest that cortisol may stimulate chloride cell proliferation and/or differentiation, whereas GH may act specifically to increase gill Na+,K+-ATPase activity.

Environmental salinity and the thyroidal response to thyrotropin in juvenile coho salmon (Oncorhynchus kisutch).

The effect of environmental salinity on the thyroidal response, measured by plasma concentrations of thyroxine (T4), to exogenous bovine thyrotropin (bTSH) was assessed early and late in the smoltification of coho salmon (Oncorhynchus kisutch). A single injection of 100 mIU bTSH significantly increased plasma T4 concentrations in fish in both fresh water and seawater, and both early (March 19) and late (May 24) in smoltification. In fish transferred to seawater, the increase in T4 titers was somewhat earlier and its duration shorter, suggesting an environmentally induced change in the kinetics of T4 entry and exit from the blood stream. The overall response to TSH was greater near the beginning (March 19) of the smoltification-associated T4 increase than near the end (May 24), suggesting a development-related change in thyroid function.