Osmoreception: perspectives on signal transduction and environmental modulation

Osmoregulation is essential to life in vertebrates and osmoreception is a fundamental element in osmoregulation. Progress in characterizing the mechanisms that mediate osmoreception has been made possible by using a uniquely accessible cell model, the prolactin (PRL) cell of the euryhaline tilapia, Oreochromis mossambicus. In addition to a brief historical overview, we offer a summary of our recent progress on signal transduction and osmosensitivity in the tilapia PRL cell model. Prolactin is a central regulator of hydromineral balance in teleosts in freshwater (FW). Consistent with its essential role in FW osmoregulation, PRL release in tilapia is inversely related to extracellular osmolality, both in vivo and in vitro. Osmotically-driven changes in PRL cell volume control PRL release. A decrease in extracellular osmolality increases cell volume, leading to a rapid influx of Ca(2+) through stretch-activated channels followed by a sharp rise in PRL release. Our recent studies also suggest that cAMP is involved in the osmotic signal transduction, and that acclimation salinity can modulate PRL cell osmosensitivity. Prolactin cells from FW tilapia show a larger rise in PRL release after a reduction in medium osmolality than those from SW fish. Paradoxically, hyposmotically-induced increase in PRL mRNA was observed only in cells from SW fish. Our studies have revealed differences in the abundance of the water channel, aquaporin 3 (AQP3), and the stretch activated Ca(2+) channel, transient receptor potential vanilloid 4 (TRPV4) in PRL cells of FW and SW fish that may explain their differing osmosensitivity and osmoreceptive output in differing acclimation salinities.     

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.     

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.     

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.     

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge

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.     

Kinetic studies of growth hormone and prolactin during adaptation of coho salmon, Oncorhynchus kisutch, to different salinities.

The kinetics of growth hormone (GH) and prolactin (PRL) in coho salmon (Oncorhynchus kisutch) transferred from fresh water (FW) to seawater (SW) and vice versa were examined to help clarify the osmoregulatory roles of the two hormones during periods of migration to different salinities. Chum salmon GH or PRL was administered by a single injection intraarterially, and metabolic clearance rate (MCR) and secretion rate (SR) of injected hormones were calculated from the disappearance of the hormones from the plasma. When coho salmon smolts were acclimated to SW, MCR, SR, and plasma level of GH in SW-adapted (2-3 weeks) fish were twice as great as those in fish in FW. On the other hand, there was no difference in the kinetics of GH between the adult coho salmon in SW and those adapted to FW (2-3 weeks). The transfer of the adult coho salmon from SW to FW was followed after 2 days by a rise in plasma level and SR of PRL, which tended to stay at high levels after 2-3 weeks. The MCR of PRL increased significantly after 2-3 weeks in FW. These results support the likelihood of an important role of GH in SW adaptation and of PRL in FW adaptation in coho salmon.     

Expression of hormone genes and osmoregulation in homing chum salmon: a minireview

Pacific salmon migrate from ocean through the natal river for spawning. Information on expression of genes encoding osmoregulatory hormones and migratory behavior is important for understanding of molecular events that underlie osmoregulation of homing salmon. In the present article, regulation of gene expression for osmoregulatory hormones in pre-spawning salmon was briefly reviewed with special reference to neurohypophysial hormone, vasotocin (VT), and pituitary hormones, growth hormone (GH) and prolactin (PRL). Thereafter, we introduced recent data on migratory behavior from SW to FW environment. In pre-spawning chum salmon, the hypothalamic VT mRNA levels increased in the males, while decreased in the females with loss of salinity tolerance when they were kept in SW. The amounts of GH mRNA in the pituitary decreased during ocean migration prior to entrance into FW. Hypo-osmotic stimulation by SW-to-FW transfer did not significantly affect the amount of PRL mRNA, but it was elevated in both SW and FW environments along with progress in final maturation. Behaviorally, homing chum salmon continued vertical movement between SW and FW layers in the mouth of the natal river for about 12h prior to upstream migration. Pre-spawning chum salmon in an aquarium, which allowed fish free access to SW and FW, showed that individuals with the lower plasma testosterone (T) and higher estradiol-17beta (E2) levels spent longer time in FW when compared with the SW fish. Taken together, neuroendocrine mechanisms that underlie salt and water homeostasis and migratory behavior from SW to FW may be under the control of the hypothalamus-pituitary-gonadal axis in pre-spawning salmon.     

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.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.     

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.     

Prolactin gene expression and gill chloride cell activity in fugu Takifugu rubripes exposed to a hypoosmotic environment

To evaluate a possible involvement of prolactin (PRL) in low-salinity tolerance of a marine pufferfish Takifugu rubripes, or fugu, gene-expression profiles of PRL in the pituitary and PRL receptor (PRLR) in the osmoregulatory organs were investigated in fish exposed to 25%-dilute seawater (SW). Following transfer from full-strength (100%) SW to 25% SW, plasma osmolality and Na(+) and Cl(-) levels were slightly decreased on day 1, which were restored on days 3 and 7. Expression levels of PRL mRNA in the pituitary was significantly increased in response to 25% SW transfer, which was in sharp contrast with a remarkable decrease in growth hormone (GH) mRNA levels. These profiles suggest that PRL and GH are involved in hyper- and hypoosmoregulation, respectively, as is the case with euryhaline teleosts. Expression levels of PRLR mRNA in the gill and intestine were not significantly different from the initial levels, whereas, PRLR mRNA expression in the kidney was significantly higher on day 7 than the initial levels. Although transfer to 25% SW did not affect the average size of Na(+)/K(+)-ATPase-immunoreactive chloride cells in the gills, both size and density of apical openings of chloride cells became significantly smaller after transfer to 25% SW. These findings suggest that the possible hypoosmotic action of PRL is mediated by PRLR expressed in the osmoregulatory organs, and that low-salinity tolerance of fugu may involve reduction of an ion-secreting function of gill chloride cells. To further evaluate long-term effects of the low-salinity environment on growth and osmoregulation, fugu were raised in 25% and 100% SW for a prolonged period of 8 weeks. They grew similarly in 25% and 100% SW, and there was no significant difference in body weight and standard length at any weekly sampling point. The plasma osmolality was maintained at about 345mOsm/kg.H(2)O in both media, whereas the gill Na(+)/K(+)-ATPase activity was significantly lower in 25% SW than 100% SW. Gene expression of PRL in the pituitary was higher in 25% SW than in 100% SW; conversely, expression levels of GH were lower in 25% SW than in 100% SW. These findings support a hyperosmotic action of PRL and a hypoosmotic, rather than growth-promoting, action of GH in this marine teleost.     

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.