Seawater Eels Research Articles

Further studies on the absence of hypocalcemic effects of eel calcitonin in the eel, Anguilla japonica.

Within 8 hr after an intraarterial injection of synthetic eel calcitonin (500 mU/100 g) into either freshwater- or seawater-adapted eels, no effect was observed on plasma Na, Ca, or Mg concentrations. In freshwater eels, a single injection of eel calcitonin (100 mU/100 g) into the caudal vein was without effect on ion concentrations of the urine or on blood pressure. There was no significant difference in plasma ion concentrations between freshwater eels infused with anticalcitonin rabbit serum and those infused with normal rabbit serum, although Ca and Mg concentrations decreased significantly after 3 days in both cases. There was also no difference in the circulating calcitonin levels between eels in fresh water and those in seawater. When the corpuscles of Stannius were removed from freshwater or seawater eels, plasma Ca concentration increased significantly after 2 to 4 weeks, whereas there was no change in the plasma calcitonin levels. Hypophysectomy of freshwater eels resulted in a significant decrease in plasma ion concentrations including Ca, but was without effect on plasma calcitonin levels. These observations indicate that calcitonin may play no significant role in maintenance of plasma Ca concentrations in the eel.

Branchial ATPases and ionic transport in the eel Anguila japonica—II. Ca2+-ATPase

Abstract 1. 1. Calcium-activated ATPase activity was demonstrated in the gills of both seawater and freshwater Japanese eels, Anguila japonica. The specific enzyme activity was highest in the heavy microsomal fraction. 2. 2. The enzyme characteristics in the microsomal fraction of the gills from freshwater and seawater adapted fish were studied and they showed similarities in the following aspects: (a) the enzyme was activated by either Ca2+ or Mg2+, maximal activation occurred at divalent ion concentration of 3 mM, and Mg2+ was less effective than Ca2+ in activating the enzyme; (b) the K m of the enzyme for ATP was 0.2–0.3 mM; (c) maximal enzyme activity was noted at 25–35°C and at pH 8.1–8.3 and (d) mersalyl acid and EDTA (ethylenediamine tetra-acetic acid) inhibited the enzyme while ouabain had no effect. 3. 3. When freshwater fish were transferred to seawater, serum Ca2+ level rose for the first 4 days and then returned to control value. Gill Ca2+-ATPase activity started to increase on day 4 after transfer and maintained at higher level throughout sea water adaptation. 4. 4. Enzyme isolated from the gills of seawater fish showed higher V max and K m values. These changes in enzyme activity and kinetics were suggested to be related to the calcium regulation in the fish.

Chloride-dependent sodium and water transport in the seawater eel intestine

Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped seawater eel intestine, and the effects of removal of Ca2+ and replacement of Cl− with other anions on these two parameters were examined. Removal of Ca2+ from normal (NaCl) Ringer solution on both mucosal and serosal sides reduced the serosa-negative PD and the net water flux. Since SO 4 2− binds Ca2+ strongly, the effects of substitution of SO 4 2− for Cl− could be due to deficiency in both Cl− and Ca2+. Among five anions used in this study, CH3SO 4 − (with low affinity to Ca2+) seems to be the most suitable substitute for Cl−. When both mucosal and serosal Cl− were replaced with CH3SO 4 − , both the PD and the net water flux decreased to approximately zero. When mucosal Cl− was replaced progressively with other anions, the serosa-negative PD and the net water flux decreased in association with the decrease in Cl− concentration, and a linear regression was observed between the decrease in the net water flux and that in the PD. These results indicate that Na+ and water transport depend closely on Cl− transport.

A light microscopic study of the action of sodium orthovanadate on the gills of fresh-water and sea-water eels (Anguilla anguillaL.)

Gills of fresh-water and sea-water eels were perfused at a constant pressure with physiological Ringer containing 10−6M sodium orthovanadate and examined by light microscopy. The secondary gill filaments were markedly vasoconstricted in both freshwater and sea-water fish although the peripheral blood route around the secondary filaments was unaffected. The central venous space in the primary filament was largely unaffected. Significant constriction of both afferent and efferent arteries on the primary filament occurred. We conclude that orthovanadate vasoconstricts eel gills mainly at the level of the secondary filaments. The study also emphasizes that chloride cells are located on both the primary and secondary filaments of fresh-water gills but solely on the primary filaments of sea-water gills.

Angiotensin and water intake in the Japanese eel, Anguilla japonica

Intraarterial injection of Asn 1-Val 5 angiotensin II amide (AII) into water-replete eels in fresh water elicited drinking within a few minutes after the injection. The reliable drinking was induced at 50 ng/100 g and the amount of water intake increased in a dose-dependent manner. Semipurified eel angiotensins were as potent as AII for induction of drinking. In dehydrated eels in seawater, AII accelerated the drinking rate. AII was still effective in freshwater eels after destruction of various forebrain and midbrain regions and even in the decerebrated eels in which the telencephalon, diencephalon, and a part of mesencephalon were removed. However, vagotomized eels failed to respond to AII by drinking. These results seem to indicate that AII acts on the central nervous system at a level lower than the diencephalon in the eel.

Effect of pimozide on the cytology of the eel pituitary. II. MSH-secreting cells.

Pimozide, a specific blocker of dopaminergic receptors, was injected for 4 or 9 days in freshwater (FW) eels or eels acclimated to sea water (SW) for 10 or 30 days. The daily dose was 100 or 200 microgram/100 g. Melanophore index values increase in FW and in 1 month-SW injected eels. All the treated fish react by a total or subtotal degranulation of the lead-hematoxylin positive cells in the pars intermedia. These cells were previously identified as alpha-MSH-secreting cells. The MSH cell nuclear area is significantly increased, nucleoli are larger and the endoplasmic reticulum more developed. The intensity of the response is similar in FW and SW eels, but it does not increase with the higher dose. The rapid release of pituitary alpha-MSH is also visualized by immunofluorescence and immunoenzymologic techniques. No effect on the second cell type of the pars intermedia (PAS-positive cell) is detected. The amount of neurosecretory material is often reduced in the neurohypophysis. These results suggest that the hypothalamic inhibitory control of MSH release and synthesis is mediated through dopaminergic fibers in the eel, but other factors cannot be ignored in this regulation.

Neurohypophysial hormonal control of kidney function in the Furopean eel (Anguilla anguilla L.) adapted to sea-water or fresh water.

Low doses of arginine-vasotocin (AVT), isotocin and oxytocin (1 pg-1 ng/kg body weight) were antidiuretic in eels adapted to fresh water but not in those adapted to sea-water. High doses (more than 10 ng/kg) were always diuretic. No effects on tubular water reabsorption were observed and the glomerular filtration rate (GFR) was proportional to the maximum reabsorptive rate for glucose (Tm(glucose)) in eels adapted to sea-water. Increases in urinary flow appeared therefore to result from glomerular recruitment. Infusion of AVT or isotocin at low rates reduced the GFR and urinary flow of freshwater eels to the levels found in seawater eels. Vasopressin (lysine or arginine) had no direct effect on kidney function in freshwater eels but blocked both the diuretic and antidiuretic actions of the other hormones. When infused into seawater eels it was diuretic. This effect could have been due to blockade of the actions of endogenous AVT and/or isotocin.

Prolactin, hypercalcemia and corpuscles of Stannius in seawater eels.

In intact eels in sea water (SW), ovine prolactin (PRL) treatment induces hypercalcemia, but its mechanism of action, which is discussed, remains to be defined. Corpuscles of Stannius (CSt) are modified simultaneously: two cell categories then become evident. The first cell type (type 1) predominates; it has an oval shape and large granules, it shows a nuclear and nucleolar hypertrophy and a mitotic activity, and appears greatly stimulated by PRL; it may elaborate a hypocalcemic factor (hypocalcin) which would compensate for the PRL-induced hypercalcemia. A similar effect, although slightly less intense, is detected in hypophysectomized-PRL treated eels in SW. A second cell type (type 2), is more elongated, smaller in size, and has an oval nucleus and fine granules. Scarcely less active in SW, it is significantly stimulated by PRL despite an increased blood sodium and potassium level. This experiment does not help to clarify its function.

Effects of L-phenylalanine on alkaline phosphatase activity and ion and water absorption in the eel intestine

1. Specific inhibition of intestinal alkaline phosphatase by L-phenylalanine was shown in eels adapted to both seawater and freshwater. 2. L-phenylalanine-sensitive alkaline phosphatase was demonstrated histochemically at the luminal surface of the sea water eel intestine. 3. Net absorption of water, Na + and Cl − by the everted intestine was reduced significantly by adding L-phenylalanine to both internal and external Ringer incubation medium. 4. No appreciable change in transepithelial potential difference was observed by adding 30 mM L-phenylalanine to NaCl, sulfate or choline Ringer solutions.

Properties and nature of (Na + + Mg 2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na + + Mg 2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na + + K + + Mg 2+)-dependent ATPase in both cases. (Na + + Mg 2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI 2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea. It is concluded that the (Na + + Mg 2+)-dependent ATPase activity of gills arises from the presence of a (Na + + K + + Mg 2+)-dependent ATPase.

Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport.

Ouabain circulating in blood inhibits Na-K-ATPase in the gills of seawater eels at a concentration similar to that necessary for inhibition in vitro. By contrast, a much higher concentration is required when ouabain is applied to the exterior of the gill. Inhibition by external ouabain occurs only when the drug gains access to the circulation of the fish, as evidenced by simultaneous inhibition of Na-K-ATPase in the kidney. These results suggest that the Na-K-ATPase of gill chloride cells faces inward, lining intracytoplasmic tubular channels continuous with the extracellular fluid. Inhibition of gill Na-K-ATPase by ouabain in intact salt water eels results in almost complete inhibition of the efflux of both Na+ and Cl-. The efflux is tritiated water was much less reduced, to 60% of normal. Since chloride is actively transported outward across the gill of seawater teleosts, it is suggested that active chloride transport is coupled to Na-K-ATPase. A neutral sodium chloride carrier is postulated that is energized by the movement of sodium from extracellular fluid down its electrochemical gradient into the chloride cell.

Influence of acclimatization temperature and salinity on [formula omitted] adenosine triphosphatase and fatty acid composition in the gills of the eel, ( Anguilla anguilla)

1. 1. Microsomal Na/K-ATPase from the gills of natural freshwater eels had an Arrhenius plot discontinuity at 20°C. The corresponding enzyme from natural seawater eels had a discontinuity at 12°C. Fatty acids from total lipids of the gills of freshwater and seawater eels were rich in (n-6) and (n-3) fatty acids respectively. 2. 2. Natural freshwater eels adapted to seawater for 3 months without a temperature change had an unchanged Arrhenius plot discontinuity and an unchanged fatty acid composition. The situation was unchanged after a further 3 months in sea water at low temperature. 3. 3. Natural seawater eels adapted to a higher temperature in sea water for 3 months yielded an Arrhenius plot discontinuity at 20°C and their gills fatty acids were now more saturated. These changes were reversed by re-adapting the eels for a further 3 months in sea water at a lower temperature. 4. 4. It is concluded that temperature and not salinity determines the degree of unsaturation of gill lipids. The position of discontinuity in the Arrhenius plot of a biomembrane enzyme can be correlated with the degree of unsaturation of membrane lipids under natural conditions reflecting the fluidity of the biomembrane. The (n-3) and not the (n-6) polyunsaturated fatty acids yield fluid biomembranes at low temperatures.

Eel esophagus as an osmoregulatory organ.

Ion and water permeabilities were measured in the isolated esophagus of the eel (Anguilla anguilla and A. japonica), and compared with those in the stomach and the intestine. The freshwater eel esophagus was impermeable both to Na+ and Cl- ions and to water, whereas permeabilities to the ions increased selectively after seawater adaptation. The ion permeabilities of both the freshwater and the seawater eel stomach were lower than in the seawater eel esophagus, although water permeability was greater than in the esophagus. Sea water enclosed in the lumen was diluted three times more efficiently in the seawater eel esophagus than in the stomach. The intestinal permeabilities were greater than those of the esophagus and the stomach, and increased after seawater adaptation. In the eel, ingested sea water seems to be diluted mainly in the esophagus by passive diffusion of the ions into the blood without addition of water. After further but less important dilution in the stomach with salt removal and with water addition, the water is absorbed by the intestine, following active absorption of the ions. Thus the eel in sea water is able to replace water lost osmotically by drinking hypertonic sea water.

Dopamine, prolactin control, and osmoregulation in eels

In freshwater eels, administration of l-Dopa or 2-Br-α-ergocryptine induced a granule storage in pituitary prolactin cells. These cells appear less active, as shown by a significant decrease in nuclear diameter and atrophy of the endoplasmic reticulum; blood sodium level and skin mucus content (assayed by N-acetyl-neuraminic acid determination in the skin), both under prolactin control in freshwater, are reduced. These two drugs act through a dopaminergic mechanism in mammals and some fish. Conversely, pituitary transplantation or 6-hydroxy-dopamine treatment stimulates prolactin cells which show an intense degranulation. In seawater eels, no clear effect was detected after a similar treatment with l-Dopa; prolactin cells remained sparsely granulated, blood sodium and skin mucus content were not affected. It appears, therefore, that both the release and the synthesis of prolactin have already decreased after 1 mo in seawater and that dopamine has no further effect. In eels, prolactin secretion is regulated by external and blood osmolality; pituitary activity is also controlled by prolactin circulating levels through a direct short feedback. Hypothalamus control, from the nucleus lateralis tuberis (NLT), seems to involve a dopaminergic mechanism. Recent evidence has shown that dopamine may be the prolactin-inhibiting factor (PIF) in the rat; this hypothesis may apply to freshwater eels, but in seawater, the regulation of prolactin synthesis and release may well be different or more complex. The role of a prolactin releasing factor (PRF) simultaneously modulating these processes must be taken into consideration.

Activities and localization of succinic dehydrogenase and Na +/K +-activated adenosine triphosphatase in the gills of fresh water and sea water eels ( Anguilla anguilla)

1. 1. Gill tissue from eels was mechanically disrupted and subjected to density gradient centrifugation to yield an enriched population of chloride cells and an enriched population of respiratory epithelial cells. 2. 2. Chloride cells isolated in this way appeared morphologically intact in the light microscope but electron microscopic examination showed that the plasma membrane was damaged. 3. 3. The specific activity of Na +/K +-ATPase in sea water fish was more than thirty times greater in the chloride cell population than in the respiratory epithelial cell population. 4. 4. Cells released from gill tissue (prior to density gradient centrifugation) catalysed the reduction of iodonitrotetrazolium violet in the presence of succinate; the specific activity of succinate dehydrogenase measured in this way was 2·5 times greater in cells from sea water fish than in those from fresh water fish. 5. 5. Density gradient separation of cells reacted with succinate and iodonitrotetrazolium showed that the enhanced succinic dehydrogenase activity of sea water gills was localized in the chloride cells.

Kidney function of the American eel Anguilla rostrata.

Urine formation in the eel, acclimated to freshwater (FW) and seawater (SW) was studied. SW eels maintained higher plasma and urine osmotic and ionic concentrations than FW eels. Polyethylene-1,2-glycol-14C (PEG-14C) clearance was 29% greater than methoxy-inulin-3H clearance. PEG-14C clearance was considered to be the glomerular filtration rate (GFR). The GFR of SW and FW eels did not differ. Urine flow rate in SW eels was one-third of that in FW eels. The primary urinary solutes in both groups were Na and Cl. Apparently, tubular fluid secretion occurred in FW and, to a lesser degree, in SW eels. With the assumption that water reabsorption was secondary to Na reabsorption in the kidney, the clearance data were used to evaluate all possible explanations for what appeared to be fluid secretion. The data were inconsistent with the possibility that GFR had been underestimated due to glomerular sieving of PEG or active or passive reabsorption of the marker, but consistent with tubular fluid secretion. Furosemide caused diuresis in both groups of eels apparently by inhibition of Na reabsorption in the distal tubule, but it had no effect on the apparent tubular fluid secretion. Tubular ion secretion could not be conclusively implicated as a driving force for fluid secretion. However, the possibility of K, Ca, or Mg secretion in a proximal segment followed by reabsorption in a more distal part of the nephron was not ruled out.

Intestinal water transport and chloride pump in relation to sea-water adaptation of the eel, Anguilla japonica

Abstract 1. The electrical potential difference (PD) and net water flux across the everted eel intestinal sac were estimated with a same preparation. 2. The water flux was closely correlated with serosa-negativity of the PD in sea-water eels but not in fresh-water eels. Similar situations were found in the conger eel (marine) and goldfish (fresh water). 3. When Cl− concentration of bathing solution was diminished, the high rate of water flux in sea-water eels decreased to a low level of fresh-water eels. 4. Osmotic permeability was six times higher in sea-water eels than in fresh-water eels. 5. The mechanism of intestinal water transport is discussed in relation to the development of Cl− pump during sea-water adaptation of the eel.

Some factors regulating water intake by the eel, Anguilla japonica.

1. Internal as well as external factors affecting water ingestion in the eel were analysed using oesophagus-cannulated eels. 2. Acute withdrawal of the blood induced an immediate drinking response in the freshwater eel, whereas infusion of a large amount of hypertonic saline interrupted the copious drinking observed in the seawater eel. 3. The freshwater eel responded to slow infusion of hypertonic NaCl solution by constant drinking. 4. Inhibition of drinking was observed in the seawater eel by distension of the stomach or intestine with isotonic mannitol solution. 5. The freshwater eel started drinking immediately after transfer to sea water, and stopped drinking immediately after return to fresh water. 6. Application of various salt solutions revealed that chloride ions are responsible for the induction of drinking in sea water. 7. Stimulation of drinking by chloride ions and inhibition by fresh water may be an anticipatory drinking behaviour, which facilitates adaptation of eels to both sea water and fresh water.

Studies on the production and metabolic clearance rates of cortisol in the European eel. Anguilla anguilla (L.)

Aspects of cortisol metabolism have been studied in the eel, Anguilla anguilla L., adapted to fresh water and sea water environments. The effects of hypophysectomy and removal of the corpuscles of Stannius on cortisol dynamics were assessed. High specific activity tritiated cortisol reached a steady state in plasma 7 h after beginning a constant intravenous infusion. Plasma cortisol concentrations were determined in hourly blood specimens during the steady state situation. Plasma cortisol concentration, its metabolic clearance and production rates were obtained for the various experimental groups. Plasma electrolytes were also determined. Similar cortisol concentrations occur in fresh water and sea water eels. Sea water adapted eels had higher metabolic clearance and production rates than fresh water animals. After hypophysectomy both fresh water and sea water adapted eels had reduced plasma cortisol concentrations and cortisol production rates, although the metabolic clearance rates were significantly elevated. These alterations in cortisol dynamics following hypophysectomy were returned to normal by infusion of mammalian ACTH in a group of sea water adapted eels. In fresh water eels removal of the corpuscles of Stannius produced almost twofold increases in the metabolic clearance and production rates of cortisol without there being any change in plasma cortisol concentration. Removal of the corpuscles of Stannius from sea water adapted eels induced a slight decrement in plasma cortisol concentration.

The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (Anguilla anguilla) adapted to fresh water and sea water

1. Gill tissue from eels adapted to fresh water or to sea water was disrupted in 0.32m-sucrose containing 0.1% (w/v) sodium deoxycholate and the subcellular distribution of (Na(+)+K(+))-dependent adenosine triphosphatase was determined. 2. About 70% of the recovered enzyme was in a fraction sedimenting between 225000g(av.)-min and 6000000g(av.)-min; the specific activities of enzymes from tissues of freshwater and seawater eels were 16 and 51 mumol of phosphate/h per mg of protein respectively. 3. The enzymes from gills of freshwater and seawater eels were indistinguishable on the basis of a number of parameters. These included phosphorylation by [gamma-(32)P]ATP, the binding of [(3)H]ouabain, the extent to which bound [(3)H]ouabain was displaced by increasing concentrations of KCl and pH optima. 4. Electrophoresis on polyacrylamide gels in sodium dodecyl sulphate showed that enzyme preparations from both sources had an identical number of protein components. 5. The higher specific activity of (Na(+)+K(+))-dependent adenosine triphosphatase from tissue of seawater eels was accompanied by increased amounts of two protein components. One of these proteins retained (32)P after treatment of the enzyme with [gamma-(32)P]ATP and had mol.wt. 97000; the other component was a glycoprotein with mol.wt. approx. 46000. 6. The results are discussed in terms of the nature of the transepithelial NaCl pumps in the gills of freshwater and seawater fish.