Changes in lactate content in the gills of the mudskippers Periophthalmus chrysospilos and Boleophthalmus boddaerti in response to environmental hypoxia

The gills of Periophthalmus chrysospilos exhibited significantly greater pyruvate kinase and lactate dehydrogenase activities than those of Boleophthalmus boddaerti ( P <0.01). Under the control normoxic condition, the branchial lactate content of the former mudskipper was also significantly greater ( P < 0.01) than that of the latter. After hypoxic exposure, lactate and alanine accumulated in the gills of P. chrysospilos but not B. boddaerti . Succinate and propionate were not detected in the gills of these two mudskippers under the control and hypoxic conditions.     

Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

The physiology of hyper-salinity tolerance in teleost fish: a review

Hyper-saline habitats (waters with salinity >35 ppt) are among the harshest aquatic environments. Relatively few species of teleost fish can tolerate salinities much above 50 ppt, because of the challenges to osmoregulation, but those that do, usually estuarine, euryhaline species, show a strong ability to osmoregulate in salinities well over 100 ppt. Typically, plasma Na⁺ and Cl⁻ concentrations rise slowly or not at all up to about 65 ppt. At higher salinities ion levels do rise, but the increase is small relative to the magnitude of increase in concentrations of the surrounding water. A number of adjustments are responsible for such strong osmoregulation. Reduced branchial water permeability is indicated by the observation that with the exposure to hyper-salinities drinking rates rise more slowly than the branchial osmotic gradient. Lower water permeability limits osmotic water loss and greatly reduces the salt load incurred in replacing it. Still, increased gut Na⁺/K⁺-ATPase (NAK) activity is necessary to absorb the larger gut salt load and increased HCO₃ ⁻ secretion is required to precipitate Ca²⁺ and some Mg²⁺ in the imbibed water to facilitate water absorption. All Na⁺ and Cl⁻ taken up must be excreted and increased branchial salt excreting capacity is indicated by elevated mitochondrion-rich cell density and size, gill NAK activity and expression of chloride channels. Excretion of Na⁺ and Cl⁻ occurs against a larger gradient than in seawater and calculation of the equilibrium potential for Na⁺ across the gill epithelium indicates that the trans-epithelial potential required for excretion of Na⁺ climbs with salinity up to about 65 ppt before leveling off due to the increasing plasma Na⁺ levels. During acute transition to SW or mildly hyper-saline waters, some species have shown the ability to upregulate branchial NAK activity rapidly and this may play an important role in limiting disturbances at higher salinities. It does not appear that the opercular epithelium, which in SW acts in a way that is functionally similar to the gills, continues to do so in hyper-saline waters. Little is know about the hormones involved in acclimation to hyper-salinity, but the few studies available suggest a role for cortisol, but not growth hormone and insulin-like growth factor. Despite the increased transport capacity evident in both the gill and gut in hyper-saline waters there is no clear trend toward increased metabolic rate. These studies provide a general outline of the mechanisms of osmoregulation in these species, but significant questions still remain.     

Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na⁺,K⁺-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na⁺,K⁺-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.     

Circadian and circannual rhythms of LH, FSH, testosterone (T), prolactin, cortisol, T3 and T4 in plasma of mature, male white-tailed deer

Circadian and circannual rhythm of plasma LH, FSH, testosterone (T), prolactin, cortisol, triiodothyronine (T3) and thyroxine (T4) were investigated in two mature male white-tailed deer. No circadian rhythms were detected. Seasonal levels of LH and FSH were reached in September and October; troughs occur in May and June. Maximal T values were detected in November and December (the time of the rut); minimal levels occur between February and July. Prolactin peaked in May and June; minimal levels were detected between October and February. T3 exhibited two maxima; the first in the May-June period, the second in the September-October period. T4 showed no recognizable circannual rhythm. Cortisol levels were found to be much higher during cold months (December-April) than during the rest of the year. The least variable circadian levels were that of FSH and prolactin, with LH, T4, T3, cortisol and testosterone following in descending order. Cannulation stress might have some effect on the levels of testosterone, LH and cortisol. Correlation between LH and testosterone levels were detected mainly during sexually active periods.     

Ontogeny of osmoregulatory patterns in the South American shrimp Macrobrachium amazonicum: Loss of hypo-regulation in a land-locked population indicates phylogenetic separation from estuarine ancestors

Ontogenetic changes in osmoregulation were compared between two geographically separate populations of a South American shrimp, Macrobrachium amazonicum, originating from the Amazon delta (A) and the Pantanal (P), respectively. Population A lives in coastal rivers and estuaries in northern Brazil, whereas population P may be considered as land-locked, spending its entire life cycle in inland freshwater (FW) habitats in southwestern Brazil. All life-history stages of population A tolerated brackish and seawater (SW) conditions, being hyper-osmoregulators at salinities < 17, iso-osmotic at ca. 17, and hypo-regulators at higher concentrations. The capabilities to survive and osmoregulate in FW were in this population expressed already at hatching (zoea I), but not any longer in the subsequent larval stages (II-IX), which showed complete mortality during an experimental 24 h exposure to fully limnic conditions. FW tolerance re-appeared only in the juvenile and adult life-history stages. Similarly, the ability to hyper-regulate at salinities 1-5 was strong in the zoea I, weaker in the subsequent larval stages, and increasing again after metamorphosis. The function of hypo-regulation in concentrated media including SW was present throughout ontogeny, particularly in late larval and early juvenile stages. These ontogenetic patterns of osmoregulation and FW tolerance are congruent with a diadromous life cycle, which includes larval release in FW and a subsequent downstream transport of the salt-dependent early larvae towards estuarine or coastal marine waters, where development to metamorphosis is possible. The FW-tolerant juveniles can later migrate upstream, recruiting to riverine populations. In the land-locked population P, all life-history stages tolerated FW and brackish conditions up to salinity 25, but mortality was high in SW (100% in adults). All postembryonic stages of this population were hyper-osmoregulators at salinities < 17, with a strong osmoregulatory capacity in FW. Unlike in population A, all stages were osmoconformers at higher salinities, lacking the function of hypo-regulation. In summary, our results show in two hydrologically and genetically isolated shrimp populations close relationships between differential patterns of ontogenetic change in osmoregulatory functions, salinity tolerance, and the ecology of successive life-history stages. In all postembryonic stages of the hololimnetic Pantanal population, the acquisition of an increased ability to hyper-osmoregulate in FW and, in particular, the complete loss of the ability to hypo-osmoregulate at high salt concentrations represent striking differences to the diadromous population from the Amazon estuary. These differences reflect different life styles and reproductive strategies, suggesting an at least incipient phylogenetic separation.     

Alkaline environmental pH has no effect on ammonia excretion in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti

Experiments were performed to evaluate the effects of alkaline environmental pH on urea and ammonia excretion rates and on tissue urea, ammonia, and free amino acid concentrations in two mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti. Periophthalomodon schlosseri is known to be capable of actively excreting ammonia. The rate of ammonia excretion in B. boddaerti exposed to 50% seawater (brackish water, BW) at pH 9 decreased significantly during the first 2 d of exposure when compared with that of specimens exposed to pH 7 or 8. This suggested that B. boddaerti was dependent on NH(3) diffusion for ammonia excretion, as in most fishes. It was incapable of detoxifying the accumulating endogenous ammonia to urea but could store and tolerate high concentrations of ammonia in the muscle, liver, and plasma. It did not undergo reductions in proteolysis and/or amino acid catabolism in alkaline water, probably because the buildup of endogenous ammonia was essential for the recovery of the normal rate of ammonia excretion by the third day of exposure to a pH 9 medium. Unlike B. boddaerti, P. schlosseri did not accumulate ammonia in the body at an alkaline pH (i.e., pH 9) because it was capable of actively excreting ammonia. Periophthalmodon schlosseri did not undergo partial amino acid catabolism (no accumulation of alanine) either, although there might be a slight reduction in amino acid catabolism in general. The significant decrease in blood pCO(2) in B. boddaerti at pH 9 might lead to respiratory alkalosis in the blood. In contrast, P. schlosseri was able to maintain its blood pH in BW at pH 9 despite a decrease in pCO(2) in the blood. With 8 mM NH(4)Cl in BW at pH 7, both mudskippers could actively excrete ammonia, although not to the same extent. Only P. schlosseri could sustain ammonia excretion against 8 mM NH(4)Cl in BW at pH 8. In BW containing 8 mM NH(4)Cl at pH 9, both mudskippers died within a short period of time. Boleophthalmus boddaerti consistently died faster than did P. schlosseri. This indicates that the body surfaces of these mudskippers were permeable to NH(3), but the skin of P. schlosseri might be less permeable to NH(3) than that of B. boddaerti. Both mudskippers excreted acid (H(+)) to alter the pH of the alkaline external medium. Such a capability, together with modifications in gill morphology and morphometry as in P. schlosseri, might be essential to the development of an effective mechanism for the active excretion of NH+4.     

Adenylate cyclase activity in fish gills in relation to salt adaptation

The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [alpha-32p]-ATP into [alpha-32p]-cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted-mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones (glucagon, VIP) and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE1. These results are interpreted in relation to the general process of osmoregulation.     

Branchial Na,K-ATPase and osmoregulation in the purple shore crab,Hemigrapsus nudus (Dana)

The purple shore crab, Hemigrapsus nudus, controls its hemolymph osmolality over a wide range of external salinities: it is a strong hyperosmoregulator in 25%, 50% and 75% sea water (SW) and is isosmotic in 100% SW. The role of branchial sodium + potassium-activated, magnesium-requiring adenosine triphosphatase (NA, K-ATPase) in osmoregulation was investigated by assaying enzyme-specific activity (SEA) in gills from crabs acclimated for 14 d in the four sea water media. Assay conditions were characterized for optimal ESA with crude homogenates of gills; ion and cofactor requirements were found to be similar to those of other crustacean Na, K-ATPases. Branchial ESA was highest in crabs acclimated for 2 weeks in 50% SW and was significantly correlated with the osmotic gradient across the body wall in 50%, 75% and 100% SW. Gills 6, 7 and 8 had the highest ESA in all media and possessed approximately 70% of the total branchial Na, K-ATPase activity, but all gills showed significant, approximately twofold increases of ESA in 50% SW compared with values in 100% SW. The time courses of increased branchial Na, K-ATPase ESA and decreased hemolymph osmotic pressure in crabs transferred from 100% SW to 50% SW are consistent with both increased in vivo activity of existing enzyme in the short term and a longer-term synthesis of new enzyme by the gills which is measured by our in vitro assay.     

Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia, Oreochromis mossambicus, is capable of surviving a wide range of salinities and temperatures. The present study was undertaken to investigate the influence of environmental salinity and temperature on osmoregulatory ability, organic osmolytes and plasma hormone profiles in the tilapia. Fish were acclimated to fresh water (FW), seawater (SW) or double-strength seawater (200% SW) at 20, 28 or 35 degrees C for 7 days. Plasma osmolality increased significantly as environmental salinity and temperature increased. Marked increases in gill Na(+), K(+)-ATPase activity were observed at all temperatures in the fish acclimated to 200% SW. By contrast, Na(+), K(+)-ATPase activity was not affected by temperature at any salinity. Plasma glucose levels increased significantly with the increase in salinity and temperature. Significant correlations were observed between plasma glucose and osmolality. In brain and kidney, content of myo-inositol increased in parallel with plasma osmolality. In muscle and liver, there were similar increases in glycine and taurine, respectively. Glucose content in liver decreased significantly in the fish in 200% SW. Plasma prolactin levels decreased significantly after acclimation to SW or 200% SW. Plasma levels of cortisol and growth hormone were highly variable, and no consistent effect of salinity or temperature was observed. Although there was no significant difference among fish acclimated to different salinity at 20 degrees C, plasma IGF-I levels at 28 degrees C increased significantly with the increase in salinity. Highest levels of IGF-I were observed in SW fish at 35 degrees C. These results indicate that alterations in gill Na(+), K(+)-ATPase activity and glucose metabolism, the accumulation of organic osmolytes in some organs as well as plasma profiles of osmoregulatory hormones are sensitive to salinity and temperature acclimation in tilapia.     

The effect of acute or chronic administration of prolactin on renal function in feral chickens

The role of the hormone prolactin in avian osmoregulation has not been clearly defined. The increases in plasma prolactin concentrations which have been demonstrated in previous studies in response to osmotic stress are strongly suggestive of a role for prolactin in avian osmoregulation. The present study investigated the effects of either acute or chronic administration of ovine prolactin on plasma electrolytes and renal function in the feral chicken. The effect of acute administration of prolactin depended on the dose of prolactin used. All plasma concentrations of prolactin achieved by the infusions were greater than reported values for endogenous prolactin. Acute infusion of prolactin at the lower dose increased the fractional excretion of sodium and chloride significantly, whereas the higher dose of prolactin had no effect. However, chronic administration of ovine prolactin had no significant effects on plasma electrolytes and renal function. It is possible that the role of prolactin in avian osmoregulation is a combination of its effects on the kidneys and also on extrarenal tissues such as the intestine and nasal salt glands (where present). Accepted: 22 August 1997     

Triiodothyronine is needed for the acclimatization of brown trout (Salmo trutta) or rainbow trout (Oncorhynchus mykiss) to seawater]

Brown and rainbow trout, held in freshwater at 13 +/- 1 degrees, were injected, every 3 days, with iopanoic acid (IOP: 5 mg/100 g body wt), an inhibitor of deiodination of thyroxine (T4) to triiodothyronine (T3). One group of IOP-treated rainbow trout was immersed in T3 (20 micrograms/l water). In IOP trout, plasma T3 fell to very low levels by day 7, while changes in T4 levels were less marked. In IOP + T3 trout plasma T3 increased fivefold, plasma T4 being unchanged. No mortality occurred and plasma osmolarity (OP) was not altered by any treatment. After direct transfer to seawater (30/1000), IOP trout were unable to acclimate to salinity: all died within 2 or 3 days, while the survival at day 3 was 100% in control brown trout and 45 and 74% in control and IOP + T3 rainbow trout respectively. OP increased more in IOP and less in IOP + T3 than in controls. There was a significant inverse correlation between T3, but not T4, plasma level, at the time of transfer and the OP 1 day later. In conclusion, although T3 does not play a significant role in osmoregulation in freshwater, T3 and therefore the deiodination of T4 into T3, were required for the development of hypo-osmoregulatory capacity involved in acclimation of trout to seawater.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Body fluid volume regulation in elasmobranch fish

This review addresses an often overlooked aspect of elasmobranch osmoregulation, i.e., control of body fluid volume. More specifically the review addresses the impact of changes in blood volume in elasmobranchs exposed to different environmental salinities. Measurement of blood volume in the European lesser-spotted dogfish, Scyliorhinus canicula, following acute and chronic exposure to 80% and 120% seawater (SW) is reported. In 80%, 100% and 120% SW-adapted S. canicula, blood volume was 6.3+/-0.2, 5.6+/-0.2 and 4.6+/-0.2 mL 100 g(-1) body mass, respectively. Blood volume was significantly higher and lower in 80% and 120% SW-acclimated animals compared to 100% SW controls. Comparisons are made between these results and previously published data. The role of drinking and volume regulation in elasmobranchs is discussed. For the first time measured water reabsorption rates and solute flux rates across the elasmobranch intestinal epithelia are presented. Water reabsorption rates did not differ between 100% SW-adapted bamboo shark, Chiloscyllium plagiosum, and fish acutely transferred to 140% SW. For the most part net solute flux rates and direction for both the 100% and 140% SW groups were the same with the exception of a net efflux of chloride and potassium in the 140% group and influx of these ions in the 100% adapted group. The significance of the intestine as part of the overall elasmobranch osmoregulatory strategy is discussed as is the role of the kidneys, rectal gland and gills in the regulation of body fluid volume in this class of vertebrates.     

Effect of Ambient Salinity,Food-deprivation and Prolactin on the Thyroidal Response to TSH,and in vitro Hepatic T4 to T3 Conversion in Yearling Coho Salmon,Oncorhynchus kisutch

This study examines the effect of food deprivation, increased ambient salinity and prolactin administration on the thyroidal response to ovine TSH, and in vitro hepatic monodeiodination of T4 to T3 in coho salmon, Oncorhynchus kisutch. Fed fish and fish food-deprived for 18 days showed similar significant increases in plasma T4 9 and 24 h after a single injection of TSH. Plasma T3 levels were also elevated in both fed and food-deprived fish 9 h after the TSH injection but plasma T3 levels in the food-deprived fish were markedly lower than in the fed salmon. The increase in T4 and T3 evident in freshwater-acclimated fish after TSH administration was not found in salmon acclimated to 65% sea water. Prolactin, given alone (either as a single injection or a series of three daily injections) had no effect on plasma T4 or T3 levels. When given together with TSH prolactin prolonged the T4 and T3 elevating effect of TSH. Food-deprived salmon had lower in vitro hepatic T4 to T3 conversion rates than fed groups but T4 to T3 conversion did not appear to be affected by increased ambient salinity, or by prolactin and/or TSH administration.     

Plasma osmolality and oxygen consumption of perch Perca fluviatilis in response to different salinities and temperatures

The present study determined the blood plasma osmolality and oxygen consumption of the perch Perca fluviatilis at different salinities (0, 10 and 15) and temperatures (5, 10 and 20° C). Blood plasma osmolality increased with salinity at all temperatures. Standard metabolic rate (SMR) increased with salinity at 10 and 20° C. Maximum metabolic rate (MMR) and aerobic scope was lowest at salinity of 15 at 5° C, yet at 20° C, they were lowest at a salinity of 0. A cost of osmoregulation (SMR at a salinity of 0 and 15 compared with SMR at a salinity of 10) could only be detected at a salinity of 15 at 20° C, where it was 28%. The results show that P. fluviatilis have capacity to osmoregulate in hyper‐osmotic environments. This contradicts previous studies and indicates intraspecific variability in osmoregulatory capabilities among P. fluviatilis populations or habitat origins. An apparent cost of osmoregulation (28%) at a salinity of 15 at 20° C indicates that the cost of osmoregulation in P. fluviatilis increases with temperature under hyperosmotic conditions and a power analysis showed that the cost of osmoregulation could be lower than 12·5% under other environmental conditions. The effect of salinity on MMR is possibly due to a reduction in gill permeability, initiated to reduce osmotic stress. An interaction between salinity and temperature on aerobic scope shows that high salinity habitats are energetically beneficial during warm periods (summer), whereas low salinity habitats are energetically beneficial during cold periods (winter). It is suggested, therefore, that the seasonal migrations of P. fluviatilis between brackish and fresh water is to select an environment that is optimal for metabolism and aerobic scope.     

Adaptive diversity in congeneric coastal crabs: Ontogenetic patterns of osmoregulation match life-history strategies in Armases spp (Decapoda, Sesarmidae)

Ontogeny of osmoregulation and salinity tolerance were investigated throughout the larval development of two congeneric species of sesarmid crab, Armases ricordi (H. Milne Edwards) and A. roberti (H. Milne Edwards), and compared with previous observations from two further congeners, A. miersii (Rathbun) and A. angustipes (Dana). In the semiterrestrial coastal species A. ricordi, the zoeal stages were only at moderately reduced salinities (17-25.5‰) capable of hyper-osmoregulation, being osmoconformers at higher concentrations. The megalopa was the first ontogenetic stage of this species, which exhibited significant hyper-osmoregulation at further reduced salinities (≥ 5‰), as well as a moderately developed function of hypo-regulation at high concentrations (32-44‰). The riverine species A. roberti showed similar overall patterns in the ontogeny of osmoregulation, however, also some striking differences. In particular, its first zoeal stage showed already at hatching a strong capability of hyper-osmoregulation in salinities down to 5‰. Interestingly, this early expressed function became significantly weaker in the subsequent zoeal stages, where survival and capabilities of hyper-osmoregulation were observed only at salinities down to 10‰. The function of hyper-regulation in strongly dilute media re-appeared later, in the megalopa stage, which tolerated even an exposure to freshwater (0.2‰). Differential species- and stage-specific patterns of osmoregulation were compared with contrasting life styles, reproductive behaviours, and life-history strategies. In A. ricordi, the larvae are released into coastal marine waters, where salinities are high, and thus, no strong hyper-osmoregulation is needed throughout the zoeal phase. The megalopa stage of this species, by contrast, may invade brackish mangrove habitats, where osmoregulatory capabilities are required. Strong hyper-osmoregulation occurring in both the initial and final larval stages (but not in the intermediate zoeal stages) of A. roberti correspond to patterns of ontogenetic migration in this species, including hatching in freshwater, larval downstream transport, later zoeal development in estuarine waters, and final re-immigration of megalopae and juvenile crabs into limnic habitats, where the conspecific adults live. Similar developmental changes in the ecology and physiology of early life-history stages seem to occur also in A. angustipes. A. miersii differs from all other species, showing an early expression and a gradual subsequent increase of the function of hyper-osmoregulation. This ontogenetic pattern corresponds with an unusual reproductive biology of this species, which breeds in supratidal (i.e. land-locked) rock pools, where variations in salinity are high and unpredictable. Matching patterns in the ontogeny of osmoregulation and life-history strategies indicate a crucial adaptive role of osmoregulation for invasions of (by origin marine) crabs into brackish, limnic and terrestrial environments.     

Growth hormone and prolactin actions on osmoregulation and energy metabolism of gilthead sea bream (Sparus auratus)

The gilthead sea bream (Sparus auratus) is an euryhaline fish where prolactin (PRL) and growth hormone (GH) play a role in the adaptation to different environmental salinities. To find out the role of these pituitary hormones in osmoregulation and energy metabolism, fish were implanted with slow release implants of ovine GH (oGH, 5 microg g(-1) body mass) or ovine prolactin (oPRL, 5 microg g(-1) body mass), and sampled 7 days after the start of the treatment. GH increased branchial Na(+),K(+)-ATPase activity and decreased sodium levels in line with its predicted hypoosmoregulatory action. GH had metabolic effects as indicated by lowered plasma protein and lactate levels, while glucose, triglycerides and plasma cortisol levels were not affected. Also, GH changed liver glucose and lipid metabolism, stimulated branchial and renal glucose metabolism and glycolytic activity, and enhanced glycogenolysis in brain. PRL induced hypernatremia. Furthermore, this hormone decreased liver lipid oxidation potential, and increased glucose availability in kidney and brain. Both hormones have opposite osmoregulatory effects and different metabolic effects. These metabolic changes may support a role for both hormones in the control of energy metabolism in fish that could be related to the metabolic changes occurring during osmotic acclimation.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.     

Growth hormone stimulates the peripheral conversion of thyroxine into triiodothyronine by increasing the liver 5'-monodeiodinase activity in the fasted and normal fed chicken

Normal fed and 2 days fasted Warren chickens were injected intravenously with 100 micrograms of ovine growth hormone (GH) and ovine prolactin and plasma concentrations of thyroid hormones were assayed prior and up to 2 h after injection. Fasting alone decreases T3, but increases T4. An injection of GH resulted in increases of plasma T3 concentrations in two fasting experiments by 40% (after 3/4 h) and 104% (after 1 h). In normal fed animals no increase is observed in the first experiment, whereas a 35% increase occurs in the second one. An injection of 100 micrograms prolactin does not influence T3 in normal fed or fasting animals. Both GH and prolactin, however, may decrease plasma concentrations of T4. In a separate experiment 50 micrograms and 200 micrograms of GH raised the decreased T3 levels after fasting by 39% and 60% respectively 1 h after injection and by 24 and 61% respectively in normal fed chicken, whereas prolactin was ineffective in this regard. Using Hisex chickens, the influence of an injection of 100 micrograms GH on plasma concentrations of thyroid hormones could be confirmed. At the same time GH increases the liver 5'-monodeiodinase activity by 330% after 1 h and by 147% after 2 h. The peroxidase activity is not influenced in normal fed chickens, but GH decreases this activity in food deprived animals after 1 h and 2 h. It is concluded that ovine GH, but not prolactin, stimulates the peripheral conversion of T4 into T3 in both normal fed and food deprived chicken and that this effect is dose dependent.