Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca(2+) through a stretch-activated Ca(2+) channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca(2+) ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.     

Hormone release is tied to changes in cell size in the osmoreceptive prolactin cell of a euryhaline teleost fish, the tilapia, Oreochromis mossambicus

Prolactin (PRL) cells from a teleost fish, the tilapia, Oreochromis mossambicus, facilitate the direct study of osmoreception. The release of two prolactins, PRL(188) and PRL(177), which act in freshwater osmoregulation in teleost fish, rises in vitro within 5 min after extracellular osmolality falls. An increase in cell size accompanied this rise. Cell size and PRL release also increased, albeit more slowly, following the partial replacement of medium NaCl (55 mOsmolal) with an equivalent concentration of urea, a membrane-permeant molecule. Similar replacement using mannitol, which is membrane-impermeant, elicits no response. These findings suggest that osmoreception is linked to changes in cell volume rather than to extracellular osmolality per se.     

Effects of environmental osmolality on release of prolactin,growth hormone and ACTH from the tilapia pituitary

Prolactin (PRL) plays a central role in freshwater (FW) adaptation in teleost fish. Evidence now suggests that growth hormone (GH) acts in the seawater (SW) adaptation in at least some euryhaline fish. Reflecting its important role in FW adaptation, plasma levels of PRL(188) and PRL(177) are higher in tilapia (Oreochromis mossambicus) adapted to FW than in those adapted to SW. A transient but significant increase in plasma GH was observed 6h after transfer from FW to SW. Elevated plasma PRL levels were seen in association with reductions in plasma osmolality after blood withdrawal in FW fish whereas no significant change was seen in plasma GH levels. When pituitaries from FW tilapia were incubated for 7 days, secretion of both PRLs was significantly greater in hyposmotic medium than in hyperosmotic medium for the first 24h. Secretion of GH from the same pituitary was relatively low during this period compared with PRL secretion. No consistent effect of medium osmolality on GH release was seen for the first day, but its cumulative release was increased significantly in hyperosmotic medium after 2 days and thereafter. On the other hand, ACTH release was extremely low compared with the secretion of PRLs and GH and there was no consistent effect of medium osmolality. These results indicate that PRL release from the tilapia pituitary is stimulated both in vivo and in vitro as extracellular osmolality is reduced, whereas the secretion of GH increases temporarily when osmolality is increased. ACTH seems to be relatively insensitive to the changes in environmental osmolality.     

Disparate release of prolactin and growth hormone from the tilapia pituitary in response to osmotic stimulation

In most teleost fishes, prolactin (PRL) plays a key role in freshwater (FW) adaptation, whereas growth hormone (GH) is involved in seawater (SW) adaptation in salmonids and certain euryhaline species including the tilapia, Oreochromis mossambicus. Consistent with its osmoregulatory activity, PRL release increases in response to physiologically relevant reductions in extracellular osmolality. When dispersed PRL and GH cells from FW-acclimatized fish were incubated in media of varying osmolalities, PRL release increased significantly in response to a 12% reduction in medium osmolality during 1 and 4h of exposure. By contrast, cells from SW-acclimatized fish responded only to a 24% reduction in osmolality. Growth hormone release on the other hand increased whether medium osmolality was reduced or raised. Cell volume increased together with PRL release during the perifusion of dispersed PRL cells in direct proportion to the reduction in medium osmolality. Growth hormone release increased whether GH cell volume increased or decreased. In in vivo studies, circulating PRL levels increased as early as 1h after the transfer of fish from SW to FW, whereas GH levels remained unchanged during 24h of acclimatization. These results indicate that while PRL and GH cells are osmosensitive, the PRL cells respond to reductions in extracellular osmolality in a manner that is consistent with PRL's physiological role in the tilapia. While the rise in GH release following the reduction in osmolality is of uncertain physiological significance, the rise in GH release with the elevation of medium osmolality may be connected to its role in SW adaptation.     

Prolactin receptor and proliferating/apoptotic cells in esophagus of the Mozambique tilapia (Oreochromis mossambicus) in fresh water and in seawater

We have previously shown that esophageal epithelium of a euryhaline goby displays elevated cell proliferation in freshwater (FW) fish, but undergo apoptosis during seawater (SW) acclimation. Prolactin (PRL) injection into the goby induced the cell proliferation, whereas cortisol treatment stimulated the cell proliferation and apoptosis [Takahashi, H., Sakamoto, T., Narita, K., 2006a. Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus). J. Comp. Physiol. B 176, 463-468, 2006). In the euryhaline tilapia (Oreochromis mossambicus), the dynamics of changes in cortisol-glucocorticoid receptors (GR) during acclimation to different salinities also suggests a role for glucocorticoid signaling in the esophageal cell turnover, but the mode of PRL action remains largely unclear. In the present study, we report on effects in the tilapia esophagus that result from changes in environmental salinity. Specifically, we assessed: (1) mRNA expression of PRL receptor (PRLR) using quantitative real-time RT-PCR; (2) esophageal cell proliferation and apoptosis, using immunohistochemistry of proliferating cells nuclear antigen (PCNA) and in situ nick end-labeling of genomic DNA (TUNEL); (3) the possible localization of immunoreactive PRLR on proliferating/apoptotic cells. Plasma PRL increased after FW acclimation; PRLR mRNA levels in the esophagus of FW fish were significantly higher than those in SW-acclimated fish. Cell proliferation was induced randomly throughout the esophageal epithelium after FW acclimation, while cell division and increased apoptosis were concentrated at the tips of esophageal epithelial folds in SW-acclimated fish. Immunoreactive PRLR appeared to be localized at proliferating cells and at certain apoptotic cells, whereas immunoreactive GR was observed over the whole epithelium including the apoptotic/proliferating cells. Thus, PRL appears to affect cell turnover directly in the esophageal epithelium of the euryhaline tilapia.     

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia,Oreochromis mossambicus

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.     

Immunomodulatory effects of prolactin and growth hormone in the tilapia,Oreochromis mossambicus

To clarify the roles of prolactin (PRL) and GH in the control of the immune system, the effects of environmental salinity, hypophysectomy, and PRL and GH administration on several immune functions were examined in tilapia (Oreochromis mossambicus). Transfer from fresh water (FW) to seawater (SW) did not alter plasma levels of immunoglobulin M (IgM) and lysozyme. The superoxide anion (O(2)(-)) production in head kidney leucocytes accompanied by phagocytosis was elevated in SW-acclimated fish over the levels observed in FW fish. Hypophysectomy of the fish in FW resulted in a reduction in O(2)(-) production in leucocytes isolated from the head kidney, whereas there was no significant change in plasma levels of IgM or lysozyme. Treatment with tilapia GH and PRLs (PRL(177) and PRL(188)) enhanced O(2)(-) production in vitro in head kidney leucocytes in a dose-related manner. Extrapituitary expression of two PRLs, GH and IGF-I mRNA was detected in lymphoid tissues and cells such as head kidney, spleen, intestine and leucocytes from peripheral blood and head kidney. PRL-receptor mRNA was detected in head kidney leucocytes, and the level of expression was higher in SW-acclimated fish than that in FW fish. Treatment with PRL(177) caused higher production of O(2)(-) in the head kidney leucocytes isolated from SW tilapia than that from FW fish. In view of the fact that PRL acts antagonistically to osmoregulation in SW, its immunomodulatory actions in this euryhaline fish would appear to be independent of its osmoregulatory action.     

Is the primitive regulation of pituitary prolactin (tPRL177 and tPRL188) secretion and gene expression in the euryhaline tilapia (Oreochromis mossambicus) hypothalamic or environmental?

We examined the effects of environmental salinity on circulating levels of the two prolactins (tPRL177 and tPRL188) and levels of pituitary tPRL177 and tPRL188 mRNA in the euryhaline tilapia, Oreochromis mossambicus. Fish were sham-operated or hypophysectomized and the rostral pars distalis (RPD) autotransplanted onto the optic nerve. Following post-operative recovery in (1/4) seawater, tilapia were transferred to fresh water (FW), (1/4) seawater (SW) or SW. Serum tPRL177 and tPRL188 levels in sham-operated and RPD-autotransplanted fish were highest in FW and decreased as salinity was increased. tPRL177 and tPRL188 mRNA levels in RPD implants as well as in pituitaries from the sham-operated fish were also highest in FW and decreased with increasing salinity. Serum osmolality increased with salinity, with the highest levels occurring in the seawater groups. We conclude that some plasma factor (probably plasma osmolality), in the absence of hypothalamic innervation, exerts a direct regulatory action on prolactin release and gene expression in the pituitary of O. mossambicus. This regulation is in accord with the actions of the two prolactins in the freshwater osmoregulation of the tilapia.     

Glucocorticoid receptor upregulation during seawater adaptation in a euryhaline teleost,the tilapia (Oreochromis mossambicus)

Cortisol is an important seawater (SW) osmoregulatory hormone in the Mozambique tilapia (Oreochromis mossambicus), a highly euryhaline cichlid able to live in environments ranging from fresh water (FW) to salinities well in excess of full-strength seawater. Previous studies indicate that cortisol may promote SW adaptation by increasing gill chloride cell differentiation, Na(+)/K(+)-ATPase activity and subsequent excretion of excess salt following seawater acclimation. Despite cortisol's widely accepted role as a SW-adapting hormone, cortisol receptor regulation during SW acclimation is not well understood. The purpose of these studies was to determine whether the intracellular glucocorticoid receptor (GR) might be regulated in a manner consistent with cortisol's actions in SW adaptation. Saturation radioligand binding assays were conducted on gill cytoplasm preparations from fish sampled 4 and 24h and 4 and 14 days after transfer from FW to 2/3 SW or FW (control). Affinity (K(d)) of the gill GR remained constant over the timecourse, while numbers of receptors (B(max)) in SW fish were significantly elevated compared with controls at 24h and 4 days after transfer. Plasma osmolality was higher in fish transferred to SW for 24h, 4 days, and 14 days compared with those animals moved to FW. Plasma cortisol levels and hepatic cortisol binding remained constant between SW and FW fish throughout the timecourse of the salinity challenge. These studies indicate that seawater acclimation is accompanied by a specific upregulation of intracellular GR numbers in gill tissue. The lack of increase in circulating cortisol following SW adaptation may reflect enhancement of clearance of the steroid. It appears that an increase in cortisol receptors, which is closely associated with the rise in blood osmotic pressure that accompanies SW exposure, is an important component of cortisol's ability to promote SW adaptation in the tilapia.     

Effects of salinity, hypophysectomy, and prolactin on whole-animal transepithelial potential in the tilapia Oreochromis mossambicus

We have examined whether two recently isolated forms of tilapia (Oreochromis mossambicus) prolactin exert similar effects on osmoregulatory physiology. The effects of salinity, hypophysectomy, and replacement therapy with tilapia prolactins on whole-animal transepithelial potential (TEP), gill Na+, K+-ATPase activity, and plasma ions were determined. When intact fish adapted to 25% seawater (SW) were transferred to different salinities, TEP reached a steady state after 10 hr; TEP increased with increasing salinity from fresh water (FW) to 75% SW but was stable from 75 to 125% SW. Plasma osmolality, [Na+], and [Cl-] of these fish 24 hr after salinity change showed that fish in 100 and 125% SW had greater osmotic perturbation than those transferred to lower salinities. Following a 5-day recovery period in 25% SW, hypophysectomized fish transferred to FW for 10 hr had significantly lower TEP and plasma ion levels than either sham-operated fish or intact fish under the same conditions. Injection of hypophysectomized fish with "small" prolactin (tPRL177), "large" prolactin (tPRL188), or a combination of both (0.5 micrograms/g body weight) 22 hr and again 20 min prior to transfer from 25% SW to FW, restored TEP and plasma ion levels to those of sham-operated fish. Neither prolactin affected the TEP or plasma ions of sham-operated (intact) fish. Hypophysectomized fish had lower gill Na+,K+-ATPase activity than sham-operated fish in FW, but prolactin injections as described above did not affect gill Na+,K+-ATPase activity in either hypophysectomized or sham-operated fish. Our results indicate that the two forms of prolactin are indistinguishable with regard to several aspects of tilapia osmoregulation.     

Involvement of the cAMP messenger system and extracellular Ca(2+) during hyposmotically-induced prolactin release in the Mozambique tilapia

In accord with its role in freshwater osmoregulation, prolactin (PRL) release from the tilapia pituitary is stimulated by small, physiologically relevant reductions in plasma osmolality, a response that is mediated by an acute influx of intracellular Ca²⁺ through stretch-activated Ca²⁺channels. In the present study, the role of the calcium and cyclic AMP (cAMP) messenger system in the transduction of a response to a hyposmotic stimulus was examined using dispersed PRL cells and PRL cell membrane preparations from freshwater-acclimated tilapia. When PRL cells were treated with the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (100 μM), significant increases in cAMP levels and PRL release were observed at 1 h. Exposure to reduced medium osmolality (300 mOsmolal) in the presence of IBMX further augmented PRL release. Depletion of Ca²⁺ from the incubation medium blocked PRL release even in the presence of IBMX. By contrast, exposure of PRL cells to cholera toxin (CTX), an activator of adenylyl cyclase (AC), stimulated PRL release and cAMP accumulation in both the presence and absence of extracellular Ca²⁺. On the other hand, treatment with the Ca²⁺ ionophore A23187, which elicits a large rise in intracellular free Ca²⁺, reduced cAMP accumulation. Likewise, the AC activity of a PRL cell membrane preparation was reduced as extracellular Ca²⁺ concentration increased from 0.1 to 1 μM. These results indicate that: (1) the stimulation of PRL release and cAMP formation by a fall in extracellular osmolality are Ca²⁺-dependent; (2) large increases in intracellular Ca²⁺ attenuate cAMP formation; (3) direct agonists of cAMP messenger system, such as cholera toxin, however, stimulate PRL release independently of the extracellular Ca²⁺. These findings add to the evidence that the osmosensitive response of the tilapia PRL cell is mediated through a Ca²⁺-dependent mechanism. Nevertheless, the present findings also suggest that tilapia PRL cells have the ability to rapidly augment release PRL both via a Ca²⁺-dependent manner and via a cAMP-dependent pathway in the absence of extracellular Ca²⁺.     

Pituitary and plasma arginine vasotocin levels in teleost fish

The role of arginine vasotocin (AVT) in teleostean osmoregulation is unclear. Its administration may produce dose-dependent effects on renal fluid loss though the physiological significance of such effects is unknown due to the paucity of data on the normal range in pituitary and plasma AVT levels. The present study addresses these questions by examining the pituitary AVT activity in the flounder and plasma AVT concentrations in the flounder, trout, and carp following alterations in environmental tonicity. Flounder pituitary AVT content, measured by bioassay, was elevated in freshwater (FW)-acclimated compared to seawater (SW)-acclimated fish and rapidly fell or rose, respectively, following transfer from FW to SW or from SW to FW to attain a new steady state. Plasma levels of AVT were higher in FW than in SW in both flounder and trout and it would, therefore, appear that the synthesis, storage, and secretion of AVT may be enhanced in FW. In the stenohaline carp plasma levels of AVT were unchanged on acclimation from FW to 40% SW. In SW flounder there was a significant positive correlation between plasma osmolality and plasma AVT concentration. Such a relationship was not apparent in FW fish, which may reflect differing roles for the hormone in these two extremes of osmotic environment.     

Effects of teleost urotensins on intestinal absorption of water and NaCl in tilapia, Sarotherodon mossambicus, adapted to fresh water or seawater

Urotensins I (UI) and II (UII), peptides released from the teleost urophysis, have been implicated in teleost osmoregulation. The effects of these peptides on intestinal absorption in tilapia are examined in vitro using anterior intestinal segments of freshwater (FW)- and seawater (SW)-adapted fish. Intestinal sacs are incubated with 10- and 20-mU/ml concentrations of semipurified Gillichthys UI or UII. UI causes significant decreases in water and NaCl absorption in FW fish but has no effect in SW fish. On the other hand, UII has no effect on FW fish but increases water and NaCl absorption in SW fish. The results suggest that the effects of UI on intestinal transport may be important in FW fish, whereas those of UII may be advantageous to SW fish. The anteriorintestine joins the urinary bladder, skin, and opercular membrane as a possible locus of action of the urotensins.     

Endocrine control of Na+,K+-ATPase and chloride cell development in brown trout (Salmo trutta): interaction of insulin-like growth factor-I with prolactin and growth hormone.

A 2-factorial (3x3) injection experiment was used to investigate the effect and interaction between different hormones on the initial phase of seawater (SW) acclimation in brown trout (Salmo trutta). Each fish was given 4 injections on alternate days in freshwater (FW). Factor 1 was either saline, 2 micrograms ovine prolactin (oPRL)/g, or 2 micrograms ovine growth hormone (oGH)/g. Factor 2 was either 0, 0. 01, or 0.1 mirograms recombinant human insulin-like growth factor-I (rhIGF-I)/g. In each of the 9 treatment groups, half of the fish were subjected to a 48-h SW-challenge test, and the remaining fish were sham-transferred to FW one day after the last injection. Hypo-osmoregulatory performance was increased by GH and impaired by PRL treatment as judged by changes in plasma osmolality, [Na+], [Cl-], total [Mg] and muscle water content (MWC) after SW transfer. IGF-I reduced plasma osmolality after transfer to SW but had no effect on plasma total [Mg] or MWC. The effects of the two factors on plasma osmolality, [Na+], [Cl-], and MWC were additive. In sham-transferred fish, GH and IGF-I, alone and in combination, stimulated Na+,K+-ATPase alpha-subunit mRNA (alpha-mRNA) content in the gill. This was paralleled by an overall increase in gill Na+, K+-ATPase activity in fish treated with 0.01 micrograms IGF-I/g. Simultaneous administration of PRL completely inhibited the increase in gill alpha-mRNA observed in the IGF-I-injected groups. Combination of GH and IGF-I did not further affect the alpha-mRNA level relative to the single hormone-injected groups. There was an overall decrease in Na+,K+-ATPase activity in pyloric caeca and middle intestine by the low dose and both doses of IGF-I respectively. No effect was observed in the posterior intestine. PRL and GH treatments did not affect enzyme activity in any intestinal segment. Both doses of IGF-I increased Na+,K+-ATPase-immunoreactive (NKIR) cell density in gill primary filaments. PRL and GH had no effect on primary filament NKIR cell density. GH and both doses of IGF-I reduced secondary lamellar NKIR cell density, whereas PRL had no effect. The main conclusion is that IGF-I and GH induce an overall redistribution of NKIR cells away from the secondary lamella onto the primary filament of FWacclimated trout. This is associated with an overall increased alpha-mRNA level in the gill, which may reflect an increased expression within individual NKIR cells in the primary filament. PRL completely abolished the IGF-I stimulation of alpha-mRNA levels, suggesting a desensitisation of the gill tissue to IGF-I, which may explain the overall anti-SW adaptive effect of PRL.     

Increased urotensin I and II immunoreactivity in the urophysis of Gillichthys mirabilis transferred to low salinity water

The function of the fish caudal neurosecretory system is uncertain, but a role in osmoregulation has been suggested by many investigators. Our objective was to determine if acclimation to water of different salinity has an effect on immunoreactive patterns and staining intensities of the two caudal neuropeptides, urotensins I (UI) and II (UII), in Gillichthys mirabilis. Five fish, originally maintained in seawater, were transferred to deionized fresh water (FW), and five were transferred to new seawater (SW). After 24 hr spinal cords were removed and fixed, FW and SW spinal cords were paired in blocks to receive identical treatment, and cryostat sections were double immunostained for both peptides using a double sequential immunofluorescence procedure. FW spinal cord exhibited increased staining intensities for both UI and UII in their urophyses (the neurohemal organ) compared to the SW spinal cords. The magnitude of intensity difference appeared greater for UI than for UII. In addition, the FW urophyses had more loci displaying intense, perivascular UI immunoreactivity than the SW urophyses. Thus, it appears that environmental salinity has an effect on the urophysial content of UI and UII in this euryhaline fish. The increased immunoreactivity in FW fish could reflect increased synthesis and storage, decreased release of the stored peptides, or decreased peptide degradation.     

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+.     

Physiological concentrations of ouabain rapidly inhibit prolactin release from the tilapia pituitary

Ouabain, a cardiac glycoside and inhibitor of Na(+), K(+)-ATPase, is now believed to be a steroid hormone in mammals. We have recently identified ouabain immunoreactivity in the plasma of the tilapia, a euryhaline teleost. Changes in plasma concentrations of immunoreactive ouabain (20-40 pM) in response to salinity change were well correlated with the changes in plasma osmolality and cortisol. Our previous studies have shown that cortisol rapidly inhibits prolactin (PRL) release from the tilapia pituitary by suppressing intracellular Ca(2+) ([Ca(2+)]i) and cAMP. In the present study, low doses of ouabain (10-1000 pM) inhibited PRL release dose-dependently during 2-24 h of incubation. There was no effect on growth hormone (GH) release, except for a significant increase at 1000 pM during 8-24 h of incubation. Significant dose-related increases in PRL release were observed at higher doses of ouabain (100-1000 nM), whereas significant inhibition was seen in GH release at 1000 nM during 2-24h of incubation. Ouabain at 1-100 pM had no effect on Na(+), K(+)-ATPase activity of the pituitary homogenate. The enzyme activity was inhibited by higher concentrations of ouabain, 10% at 1 nM, 15% at 10 nM, 28% at 100 nM, and 45% at 1000 nM. Ouabain also attenuated stimulation of PRL release by the Ca(2+) ionophore, A23187, and by a combination of dibutyryl cAMP and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthin. Intracellular Ca(2+) concentrations were monitored in the dispersed PRL cells with the Ca(2+)-sensitive dye, fura-2. Ouabain at 1 nM reversibly reduced [Ca(2+)]i within seconds, whereas 1 microM ouabain increased [Ca(2+)]i. A rapid reduction in [Ca(2+)]i was also observed when PRL cells were exposed to 1 microM cortisol, whereas there was no consistent effect at 1 nM. These results suggest that ouabain at physiological concentrations rapidly inhibits PRL release from the tilapia pituitary by suppressing intracellular Ca(2+) and cAMP metabolism. The stimulation of PRL release by high concentrations of ouabain (100-1000 nM) may result from an increase in [Ca(2+)]i, and subsequent depolarization due to the inhibition of Na(+), K(+)-ATPase activity.