Permeability properties and occludin expression in a primary cultured model gill epithelium from the stenohaline freshwater goldfish

Techniques for the primary culture of fish gill epithelia on permeable supports have provided ‘reconstructed’ gill models appropriate for the study of gill permeability characteristics in vitro. Models developed thus far have been derived from euryhaline fish species that can tolerate a wide range of environmental salinity. This study reports on procedures for the primary culture of a model gill epithelium derived from goldfish, a stenohaline freshwater (FW) fish that cannot tolerate high environmental salt concentrations. The reconstructed goldfish gill epithelium was cultured on permeable filter inserts and using electron microscopy and immunocytochemical techniques, was determined to be composed exclusively of gill pavement cells. When cultured under symmetrical conditions (i.e. with culture medium bathing both apical and basolateral surfaces), epithelial preparations generated appreciable transepithelial resistance (TER) (e.g. 1,150 ± 46 Ωcm²) within 36–42 h post-seeding in inserts. When apical medium was replaced with FW (asymmetrical conditions to mimic conditions that occur in vivo), epithelia exhibited increased TER and elevated paracellular permeability. Changes in permeability occurred in association with altered occludin-immunoreactive band position by western blot and no change in occludin mRNA abundance. We contend that the goldfish gill model will provide a useful in vitro tool for examining the molecular components of a stenohaline fish gill epithelium that participate in the regulation of gill permeability. The model will allow molecular observations to be made together with assessment of changing physiological properties that relate to permeability. Together, this will allow further insight into mechanisms that regulate gill permeability in fishes.     

Effect of cortisol on the physiology of cultured pavement cell epithelia from freshwater trout gills

Cortisol had dose-dependent effects on the electrophysiological, permeability, and ion-transporting properties of cultured pavement cell epithelia derived from freshwater rainbow trout gills and grown on cell culture filter supports. Under both symmetrical (L15 media apical/L15 media basolateral) and asymmetrical (freshwater apical/L15 media basolateral) culture conditions, cortisol treatment elevated transepithelial resistance, whereas permeability of epithelia to a paracellular permeability marker (polyethylene glycol-4000) decreased. Cortisol did not alter the Na(+)-K(+)-ATPase activity or the total protein content of the cultured preparations. During 24-h exposure to asymmetrical conditions, the net loss rates of both Na(+) and Cl(-) to the water decreased with increasing cortisol dose, an important adaptation to dilute media. Unidirectional Na(+) and Cl(-) flux measurements and the application of the Ussing flux-ratio criterion revealed cortisol-induced active uptake of both Na(+) and Cl(-) under symmetrical culture conditions together with an increase in transepithelial potential (positive on the basolateral side). Under asymmetrical conditions, cortisol did not promote active ion transport across the epithelium. These experiments provide evidence for the direct action of cortisol on cultured pavement cell epithelia and, in particular, emphasize the importance of cortisol for limiting epithelial permeability.     

Cultured trout gill epithelia enriched in pavement cells or in mitochondria-rich cells provides insights into Na<Superscript>+</Superscript> and Ca<Superscript>2+</Superscript> transport

Summary  The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kΩ cm²) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na⁺ fluxes and application of the Ussing flux ratio criterion demonstrated active Na⁺ uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca²⁺ uptake and Na⁺/K⁺-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.     

Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics

The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.     

Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

Effects of cortisol and prolactin on Na+ and Cl- transport in cultured branchial epithelia from FW rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of cortisol and prolactin as media supplements. Epithelia were of the double-seeded insert (DSI) type containing both pavement cells (PVCs) and mitochondria-rich cells (MRCs) and were grown in Leibovitz's L15 media on filters allowing exposure to different apical media conditions. Experiments were carried out in two series after 7-9 days symmetrical (L15 apical-L15 basolateral) culture. In both series, 100% L15 was maintained as the basolateral medium throughout and supplemented with physiologically relevant doses of either prolactin (50 ng/ml), cortisol (500 ng/ml), or cortisol + prolactin (500 + 50 ng/ml, respectively). In series 1, epithelia were exposed to progressively diluted apical media (100, 75, 50, 25, 12.5% L15, and FW) at 24-h intervals. The preparations retained integrity [high transepithelial resistance (TER); low ion efflux rates] during this prolonged dilution protocol. Cortisol, or cortisol + prolactin, resulted in a greater TER and reduced ion efflux rates during dilution, likely an effect on junctional permeability of PVCs, but did not promote active Na+ and Cl- uptake from apical FW. In series 2, epithelia were directly exposed to apical FW and ion fluxes measured over the first 6 h. Under these conditions, cortisol or cortisol + prolactin promoted active uptake of both Na+ and Cl- simultaneously from apical FW, probably attributable to actions on the MRCs. However, Na+-K+-ATPase activities were not significantly altered by any of the treatments in either series. Overall, prolactin alone did not appear to promote FW adaptation but exhibited synergism with cortisol. These results provide further support for the cultured DSI epithelium as an in vitro model for ion transport in FW fish.     

Response of developing cultured freshwater gill epithelia to gradual apical media dilution and hormone supplementation

We investigated gradual dilution of the apical medium (Leibovitz's L15 to fresh water [FW], analogous to gradual reduction in environmental salinity) and basolateral hormone support on the electrophysiological and ion-transporting properties of "developing" FW trout gill epithelia cultured on filter inserts. Epithelia were of the double-seeded type, containing both pavement cells and mitochondria-rich cells. In these experiments we were able to circumvent "symmetrical development" (typically L15 apical/L15 basolateral for 6-9 days) by commencing dilution of apical media (unchanged L15 basolateral, i.e., asymmetrical conditions) at culture-day 3, the time when transepithelial resistance (TER) and potential (TEP) would normally be increasing rapidly under symmetrical conditions. In Series 1 (without basolateral hormone support), epithelia were exposed to progressively diluted apical media (100%, 75%, 50% L15) at 24-hr intervals, thereafter cultured in 50% L15 apical media for 4 days, and then in apical FW. In Series 2, epithelia were exposed to progressively diluted apical media (100%, 75%, 50%, 25%, 12.5% L15, and FW) at 24-hr intervals with physiologically relevant doses of cortisol (500 ng ml(-1)), prolactin (50 ng ml(-1)), or cortisol + prolactin (500 ng ml(-1) + 50 ng ml(-1), respectively) added to basolateral media (100% L15). In Series 1, TER reached a plateau phase over 25 kohms cm2 under 50% L15/L15 culture conditions (after 4 days of culture) but fell to approximately 6 kohms cm2 after 24 hr in FW/L15 conditions. In Series 2, TER stabilized at 4-11 kohms cm2 depending on treatment. In general, apical media dilution during epithelial development was well tolerated. Preparations exhibited continued integrity right down to apical FW, indicated by only modest increases in net ion losses (i.e., basolateral to apical movement of ions), relatively stable TER values, and the expected changeover from positive to negative TEP in FW. Cortisol was clearly beneficial to FW adaptation, promoting greater TER, reduced unidirectional and net Na+ and Cl- flux rates, and elevated Na+, K+ -ATPase activity. Prolactin also offered some support, where its actions on TER were less than but additive to those of cortisol. There was no direct evidence that prolactin limited ion movements during gradual dilution. These in vitro studies demonstrate that "developing epithelia" were able to tolerate gradual dilution of apical media, the remarkable barrier properties of gill epithelia, and the importance of cortisol and prolactin in promoting integrity of this barrier during FW adaptation.     

Improved Cell Line IPEC-J2, Characterized as a Model for Porcine Jejunal Epithelium

Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line’s initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.     

The effect of chronic cortisol elevation on urea metabolism and excretion in the rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

The objective of this study was to investigate the possible involvement of cortisol in controlling urea metabolism and excretion in the ammoniotelic rainbow trout (Oncorhynchus mykiss). Trout fitted with dorsal aortic and internal urinary catheters received either no implant (control), or were implanted with coconut oil (sham), cortisol in coconut oil, RU486, a glucocorticoid receptor blocker, in coconut oil, or cortisol+RU486 in coconut oil, and monitored over 72 h. Rainbow trout treated with cortisol (±RU486) had similarly elevated plasma cortisol concentrations that were six fold greater than in control and sham fish. Elevated circulating cortisol concentrations caused a three-fold rise in plasma and urine urea concentrations, which was blocked by RU486. Similarly, a positive correlation between plasma cortisol and plasma urea concentrations was observed in fish treated with cortisol alone but not in fish treated with cortisol+RU486. Cortisol treatment caused an elevation in branchial (two fold higher) and urinary (three fold higher) excretion rates of urea compared to sham-implanted fish, which was prevented by treatment with RU486. However, as branchial and renal clearance were unaffected, there appears to be no stimulation or inhibition of urea excretion mechanisms in the gill or kidney separate from effects due to changes in plasma urea concentrations. Thus, cortisol and glucocorticoid receptors appear to be involved in the regulation of endogenous urea production but not in the control of urea excretory mechanisms in the ammoniotelic trout.Abbreviations GFR glomerular filtration rate - GS glutamine synthetase - O-UC ornithine urea cycle - PEG polyethylene glycol - UFR urine flow rate Communicated by: G. Heldmaier     

Cortisol stimulates calcium transport across cultured gill epithelia from freshwater rainbow trout

Summary The effect of cortisol on calcium (Ca²⁺) transport across cultured rainbow trout gill epithelia composed of both pavement cells (PVCs) and mitochondria-rich cells (MRCs) was examined. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), cortisol had subtle effects on gill epithelial preparations. Both control and cortisol treated epithelia exhibited Ca²⁺ influx and efflux rates (measured radioisotopically using ⁴⁵Ca) that were approximately balanced, with a slight inwardly directed net Ca²⁺ flux. Ussing flux ratio analysis indicated active Ca²⁺ transport in the inward direction across epithelia bathed symmetrically regardless of hormone treatment. In contrast, under asymmetrical conditions (freshwater apical/L15 media basolateral) control epithelia exhibited active Ca²⁺ transport in the outward direction (basolateral to apical) throughout experiments conducted over a 24-h period, whereas cortisol-treated preparations exhibited active transport in the inward direction (apical to basolateral) during the early stages of an asymmetrical culture period (e.g., T0–6 h) and passive transport during the later stages (e.g., T18–24 h). When soft freshwater (with tenfold lower [Ca²⁺]) was used for asymmetrical culture instead of freshwater, control epithelia developed outwardly directed active Ca²⁺ transport properties, whereas cortisol-treated preparations did not. The results of this study support a hypercalcemic role for cortisol in rainbow trout and demonstrate that treating cultured gill epithelia composed of both PVCs and MRCs with cortisol can stimulate active Ca²⁺ uptake under circumstances that more closely resemble natural conditions for fish gills (i.e., freshwater bathing the apical side of the epithelium).     

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.     

Blockade of the Glucocorticoid Receptor with RU 486: Effects In Vitro and In Vivo on Human Adipose Tissue Lipoprotein Lipase Activity

Cortisol is known to induce lipoprotein lipase (LPL) activity in human adipose tissue in vitro and in vivo such as in Cushing's syndrome. The significance of the glucocorticoid receptor (GR) for this induction was evaluated in the present study. The synthetic steroid molecule RU 486, a potent glucocorticoid antagonist, was used as a tool to block the GR, in vitro and in vivo. In addition to LPL activity, glucose tolerance, blood pressure and plasma lipids, all variables influenced by Cortisol, were studied in order to evaluate the peripheral antiglucocorticoid activity of RU 486 in vivo, in man. Addition of both Cortisol and RU 486 to incubations of human adipose tissue pieces significantly inhibited the increase in LPL activity that could be induced by Cortisol alone (p<0.01). In a ten-fold molarity excess RU 486 totally abolished Cortisol action (p<0.01). With Cortisol and RU 486 in equimolar concentrations the RU 486 blockade was probably incomplete and LPL activity induced (p<0.05). The results imply that the stimulating effect of Cortisol on LPL activity in human adipose tissue is mediated via the GR. Administration of 400 mg RU 486 at 2200 hours on two consecutive days to healthy men caused a significant rise in serum Cortisol levels measured at 0800 hours (p<0.05). The mean LPL activity in the subcutaneous abdominal adipose tissue remained unchanged. The mean level of serum triglycerides decreased significantly (p<0.01) and there was a negative correlation between change in LPL activity and change in triglyceride levels (r=-0.73, p<0.05). Glucose tolerance and blood pressure were not affected. In conclusion, a total blockade of the GR with RU 486 can be achieved in human adipose tissue in vitro. The blockade abolishes the stimulating effect of Cortisol on LPL activity suggesting that the stimulation is GR dependent. In vivo, with the dose used, the negative pituitary feedback regulation probably compensates for the blockade, at least during the morning hours, thus obviating any peripheral effect of blockade of the GR.     

Does RU486 modify hormonal responses to handling stressor and cortisol treatment in fed and fasted rainbow trout?

This study examines the effect of the steroid analogue, RU486, on the physiological responses of fed and chronically fasted rainbow trout to an acute handling stressor. This potent ligand of the glucocorticoid receptor was administered as a slow-release implant either alone, or in combination with cortisol. There were temporal changes in plasma cortisol concentrations following administration of cortisol implants in both fed and fasted trout. By day 14, plasma cortisol levels in fed fish were similar in all treatment groups, but in fasted fish, the effect of cortisol administration on plasma cortisol concentrations was still evident; RU486 administered with cortisol, did not affect this response. Cortisol administration also elicited a small, but significant increase in plasma GH concentrations in fed rainbow trout and in plasma glucose concentrations in fasted animals. RU486-treatment prevented these responses. Conversely, whereas RU486 alone had no effect on hepatic 5'-monodeiodinase activity, when administered with cortisol it enhanced the marked suppressive effect of cortisol evident in both fed and fasted groups, suggesting that it may exert an interactive effect with cortisol on this process. Stressor-related changes in plasma cortisol, glucose, GH and thyroid hormone concentrations were evident in both fed and fasted groups; however, there was no evidence of a suppressive effect of RU486 treatment on any of the measured plasma parameters. Although RU486 did not prevent the stressor-related changes, the post-stressor cortisol profiles in RU-treated trout were extremely erratic compared with the oil-treated controls. This implies a disturbance of the normal interactions of the components of the hypothalamus-pituitary-interrenal tissue axis.     

Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus

When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals (“chirps”). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7 d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5 h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.     

Effects of progesterone, promegestone and RU 486 on glucocorticoid receptor levels in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain glucocorticoid receptors whose levels are modulated by a variety of steroids. In the absence of any added steroid to the cell culture medium, the levels of glucocorticoid receptors in the cells decline during culture, which is counteracted by the addition of a variety of glucocorticoid agonists. The effectiveness of the glucocorticoid in preventing the loss of glucocorticoid receptors is in turn counteracted by the addition of the synthetic progestin promegestone and the synthetic antiglucocorticoid RU 486. Of the two, RU 486 is the most potent in antagonizing the effect of cortisol on the GR levels. Promegestone antagonizes the effect of cortisol, too, although higher concentrations are necessary. Progesterone was without a clear effect either as a glucocorticoid agonist or an antagonist. Progesterone, however, was extensively metabolized by mammary epithelial cells in culture. Based on these observations we conclude that in mammary epithelial cells glucocorticoids positively regulate the metabolism of their own receptors and that antiglucocorticoids, such as RU 486 and progestins, can antagonize that effect.     

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.     

Glucocorticoid receptors are involved in the regulation of pulsatile urea excretion in toadfish

The objectives of this study were to characterize the pattern of pulsatile urea excretion in the gulf toadfish in the wake of exogenous cortisol loading and to determine the receptors involved in the regulation of this mechanism. Toadfish were fitted with indwelling arterial catheters and were infused with isosmotic NaCl for 48 h after which fish were treated with cortisol alone, cortisol+peanut oil, cortisol+RU486 (a glucocorticoid receptor antagonist) or cortisol+spironolactone (a mineralocorticoid receptor antagonist). Upon cortisol loading, fish treated with cortisol alone, cortisol+oil or cortisol+spironolactone experienced a two- to threefold reduction in pulsatile urea excretion. This reduction was due to a decrease in urea pulse size with no effect on pulse frequency compared to values measured during the control NaCl infusion period. In addition, these fish showed an increase in plasma urea concentrations upon treatment. These apparent effects of cortisol treatment were abolished in fish treated with cortisol+RU486. In contrast, these fish showed an increase in pulsatile urea excretion mediated by a twofold increase in pulse size with no change in frequency. Likewise, fish treated with cortisol+RU486 showed a significant decrease in plasma urea concentrations over the course of the experiment. The findings of this study indicate that high levels of cortisol reduce pulsatile urea excretion by decreasing pulse size. In addition, it appears that glucocorticoid receptors and not mineralocorticoid receptors are involved in the regulation of the toadfish pulsatile urea excretion mechanism.Communicated by G. Heldmaier     

Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats.

We have examined the role of glucocorticoids in the stress-induced inhibition of testicular steroidogenesis. Immobilization (3 hr) reduced plasma testosterone (T) levels to 24% of control values but did not affect plasma LH levels. This reduction was partially reversed by in vivo injections of the antiglucocorticoid, RU486, prior to the stress session at a dose of 10 mg/kg BW, but not at 1.0 or 50 mg/kg BW. Stressed rats that were treated with 10 mg/kg BW RU486 had twofold higher plasma T levels than vehicle-treated stressed animals. Injections of RU486 did not affect plasma LH levels in control or stressed rats and did not affect T levels of unstressed rats. Stressed rats had eightfold higher plasma corticosterone levels than controls, and RU486 had no effect on control or stress levels of corticosterone. The possible role of glucocorticoids in mediating the effect of stress on testicular T production was investigated also in vitro by incubating testicular interstitial cells from unstressed rats for 3 hr with corticosterone (0, 0.01, 0.1, or 1.0 microM) or dexamethasone (0, 0.001, 0.01, or 0.1 microM), followed by an additional 2 hr with hCG (0, 25, 50, or 100 microIU). Both corticosterone and dexamethasone inhibited hCG-stimulated T production in a dose-dependent manner. Cells incubated with the highest concentration of either of the glucocorticoids showed significantly reduced responses to hCG stimulation. In the absence of hCG, in vitro T production was not affected by dexamethasone or 0.01 and 0.1 microM corticosterone. However, the highest dose of corticosterone (1.0 microM) produced a 63% elevation in basal T production. Coincubation of testicular interstitial cells with corticosterone (1.0 microM) or dexamethasone (0.1 microM) and RU486 (0.01, 0.1, and 1.0 microM) reversed the glucocorticoid-induced suppressions of T production in a dose-dependent manner. Our results suggest that during stress increases in plasma levels of glucocorticoids in male rats act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins, and that the decline of testosterone production during immobilization stress is in part mediated by a direct action of glucocorticoids on the testis.