Toxicity of fatty acid 18:5n3 from Gymnodinium cf. mikimotoi: II. Intracellular pH and K+ uptake in isolated trout hepatocytes.

Effects of octadecapentaenoic acid 18:5n3 and other related polyunsaturated fatty acids present in gymnodinium cf. mikimotoi were tested in isolated trout hepatocytes. These exotoxins decreased intracellular pH followed by a slow recovery to initial value and alkalinization of acidic compartments, suggesting an inhibition of vacuolar H(+)-ATPases. Moreover, addition of 18:5n3 to the extracellular medium induced a decrease of K+ uptake into hepatocytes as a result of Na,K-ATPase inhibition. However, high concentrations (10(-5)-10(-3) M) are necessary to induce these effects.

Toxicity of fatty acid 18:5n3 from Gymnodinium cf. mikimotoi: I. Morphological and biochemical aspects on Dicentrarchus labrax gills and intestine.

We present evidence for the toxic effects of fatty acid 18:5n3 (octadecapentaenoic acid) in the gills and intestine of the sea bass Dicentrarchus labrax. Light microscopic observation of gills showed strong mucus production and alteration of ionocytes. The Mg- and Na,K-ATPase activities were inhibited, with IC50 values of 10(-3) and 1.6 x 10(-4) M, respectively. Results are discussed in relation to osmoregulation.

Effect of caulerpenyne, a toxin extracted from Caulerpa taxifolia on mechanisms regulating intracellular pH in sea urchin eggs and sea bream hepatocytes.

The proliferation of the green marine alga Caulerpa taxifolia in the Mediterranean led us to investigate the toxic effects on marine organisms of caulerpenyne (Cyn), the major secondary metabolite synthesized by the alga. This study was performed on sea urchin eggs (Paracentrotus lividus) and isolated hepatocytes from the sea bream (Sparus aurata), in which accumulation of the toxins by metabolic processes may be of significance. Cyn provoked an acidification of seawater containing both unfertilized and fertilized eggs, as revealed by a titrable efflux of protons. The pHi in unfertilized eggs continuously increased in the presence of Cyn, whereas there was a biphasic response in both fertilized eggs and isolated hepatocytes, with a decrease of the pHi followed by recovery to the initial value. Cyn inhibited the accumulation of 14C-methylamine in acidic granules present in the cortical zone of sea urchin eggs. Dicyclocarbodiimide (DCCD), a well-known H(+)-ATPase inhibitor, provoked a similar inhibition. Both molecules increased pH in the acidic compartments of isolated bream hepatocytes. These results suggest that Cyn inhibits intracellular sequestration of protons and thus liberates protons into the cell cytoplasm from which they leak toward the extracellular medium.

Potassium uptake and water content in hepatocytes isolated from rat livers preserved in Euro-Collins and UW solutions and after transplantation.

Isolated hepatocytes from the rat were used to assess the maintenance of liver cell function in relation to the composition of the preservation medium. After separation by collagenase, they were incubated in Krebs-Ringer-Bicarbonate medium (KRB), Euro-Collins (EC), or University of Wisconsin (UW) solutions. Potassium influx, cell volume, and transaminase release were measured in cells freshly separated from control livers or from livers preserved in vitro up to 12 h in these media or having undergone orthotopic liver transplantation (OLT). While ion exchange levels were retained in all media, cells shrank significantly in UW but were able to restore their volume after 3 h of liver preservation. With regard to in vivo conditions, UW appears to be the best medium to prevent edema and to maintain more stable potassium exchange and enzyme production. These results are of value for liver transplantation in humans.

Effects of hyposmotic shock on taurine transport in isolated trout hepatocytes.

Isolated trout hepatocytes exposed to hypotonic medium undergo a regulatory volume decrease (RVD) that occurs via two separate routes, K(+)-Cl- cotransport and amino acid release, the ion efflux accounting for 70% of the total osmolyte loss. Taurine, glutamine and glutamic acid are the most important and represent 73% of the total amino acid content (53 mmol (l cell water)-1). The osmolarity-sensitive release of amino acids was studied using [3H]taurine. Kinetic studies indicated two components for taurine influx: a linear Na(+)-independent transport and a saturable Na(+)-dependent system with a Michaelis-Menten constant (Km) of 122 microM and a maximum velocity (Vmax) of 31.2 pmol (mg protein)-1 min-1. This second way of uptake was also chloride dependent and indicated an apparent coupling ratio Na+:Cl-:taurine of 2:1:1. The latter component and the taurine efflux were stimulated during RVD, leading to intracellular amino acid loss. Taurine efflux activation during volume recovery was transient and also dependent on the presence of both Na+ and Cl- in the extracellular medium. Furthermore, taurine release and RVD were slowed down when Ca2+ was omitted from the medium. These results suggested two distinct and complementary mechanisms for volume regulation in trout hepatocytes during hypotonic conditions.

Activation by N-ethylmaleimide of a Cl--dependent K+ flux in isolated trout hepatocytes.

Isolated trout hepatocytes when swollen in hypotonic medium undergo a regulatory volume decrease (RVD), which occurs via KCl loss. The system shows characteristics similar to those of the transporter described in red cells. This led us to investigate, in trout hepatocytes, the effect of another signal known to activate this flux in red cells, i.e. treatment with the sulphhydryl-group reagent N-ethylmaleimide (NEM). NEM treatment resulted in a striking increase in ouabain-resistant K+ uptake measured by an isotope pulse uptake technique. The time course of the response to NEM was similar to that obtained with a hypotonic shock, indicating that the effect of NEM was immediate and transient. The NEM-stimulated K+ influx demonstrated the same anion sensitivity as the volume-induced K+ influx, i.e. a specific requirement for Br- or Cl-. Efflux experiments showed that NEM treatment produced a stimulation of both K+ and Cl- effluxes leading to a substantial net loss (10%) of cellular KCl, as confirmed by analysis of ionic contents. This KCl loss is consistent with the rapid cell shrinkage observed after addition of NEM. The Cl--dependent K+ influx was found to be independent of external Na+; in addition, NEM had no effect on Na+ content, indicating that Na+ is not implicated in this process. The effect of loop diuretics was tested on the NEM-stimulated K+ influx. As observed for the volume-induced K+ flux, a high concentration of furosemide (10(-3) mol l-1) is required for full inhibition of this flux; no effect was obtained with bumetanide (10(-4) mol l-1). Consequently, NEM appears to activate a KCl cotransport similar to the one induced in hypotonically swollen cells. Finally, the combination of the two treatments, NEM and hypotonic shock, was found to increase the K+ fluxes even further, suggesting additivity of the two stimuli by mutual positive interaction.

Characteristics of ouabain binding to isolated trout hepatocytes.

Na+, K+ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10(-4) M produced maximal inhibition (95%) of K+ uptake and enhanced intracellular Na+ accumulation, showing that active fluxes account for a very large proportion of Na+ and K+ exchanges. Inhibition of the Na-K pump by ouabain was significant at low concentrations (10(-8) M). When external K+ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC50) of K+ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [3H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K+ concentration in the medium, the other linear and independent of external K+. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5 x 10(5) per cell) and independent of the external K+ concentration (7, 0.5 or 0 mM), while the dissociation constant (KD) decreased from 4.2 microM to 7.3 nM when K+ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min-1), as estimated from ouabain-sensitive K+ flux, in comparison to those described in other cell types of higher vertebrates. At each external K+ concentration studied, the inhibition of K+ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.

Inhibitory effect of neurohypophysial peptides on cyclic AMP accumulation by isolated hepatocytes of the trout.

Cyclic AMP levels were measured in freshly isolated hepatocytes of the rainbow trout. Compared with basal values, the average levels were increased up to 60 times in a dose-dependent manner either by mammalian glucagon (concentration range 1 nmol-1 mumol/l; dose giving half maximum response (EC50) 0.18 mumol/l) or by forskolin (concentration range 0.1-100 mumol/l; EC50 about 10 mumol/l). These stimulatory effects were partially inhibited by fish or mammalian neurohypophysial hormones used at relatively high concentrations (1-5 mumol/l). It is suggested that these results are evidence for the presence of V1-type receptors in fish hepatocytes. Together with previous results obtained with gills on the hormonal inhibition of adenylate cyclase activity, they suggest that teleost fish may possess only V1-type receptors (or two V1-related types), while the V2 receptors have evolved (or have become functional) in higher vertebrates.

Effects of hyposmotic shock on ion fluxes in isolated trout hepatocytes.

Isolated trout hepatocytes exposed to hypotonic Hank's medium (isotonicity x 0.70) swelled to 1.17 times the control volume after 3 min; by 15 min the cell volume had returned to normal. The ouabain-insensitive K+ uptake increased, indicating an immediate rise in K+ membrane permeability. As indicated by analysis of cellular contents, the regulatory volume decrease (RVD) was ensured by a release of intracellular K+. Na+ was not implicated in this mechanism. This potassium permeability induced by hypotonic shock was transient (maximum at 6 min), insensitive to blocking agents of voltage- and Ca2+-dependent K+ channels, and chloride-dependent. This result, together with a time-course of Cl- uptake similar to that of K+, suggests a K+/Cl- cotransport mechanism. This cotransport is inhibited by high furosemide concentrations (10(-3) mol l-1) but not by bumetanide (10(-4) mol l-1) or piretanide (10(-4) mol l-1).

Interaction of new 19 nor progesterone derivatives with progestagen, mineralocorticoid and glucocorticoid cytosolic receptors.

Structure-activity relationships were studied in vitro on a number of natural and artificial steroids in order to assess their progestagen specificity. These substances included a new compound derived from 19 nor progesterone, TX066 or nomegestrol acetate, and two synthetic intermediates, TX045 and TX071, all prepared by Laboratoires Theramex. The experiments involved binding competition on the cytosolic receptors prepared from target organs against specific radiolabelled ligands. Relative affinities were determined in rats against progesterone (P), aldosterone (A) and dexamethasone (DM), by displacement of: (3H)-ORG 2058 from the progestagen receptor of uterus, (3H)-A from Type I (mineralocorticoid) receptor of the kidneys and (3H)-DM from glucocorticoid receptors of the kidney (Type II) and of the liver. The various modifications introduced in the progesterone molecule led to sequences in competition potency which were either parallel or opposite in the progesterone compared to the 19 nor progesterone series. The main practical conclusion is that TX066 which is intended for use as a progestagen presents very little mineralo- and glucocorticoid activities at the receptor level, while its affinity for the progestin receptor is nearly as good as that of progesterone. The two derivatives TX045 and TX071 displayed very little or no progestagen affinity while their mineralo- and glucocorticoid potencies were between those of 19NP and TX066.

In vivo and in vitro studies on the release of cortisol from interrenal tissue in trout. I. Effects of ACTH and prostaglandins.

The secretion of cortisol from the interrenal tissue in trout Salmo gairdnerii was studied in vivo and by an in vitro superfusion method in relation to the effect of adrenocorticotropic hormone (ACTH), prostaglandins (PGE1, PGF2 alpha) and prostaglandin inhibitors. The experiments were performed between April and July. In control, uninjected animals, circulating cortisol was 206 +/- 24 ng/ml. At 24 h following the intraperitoneal injection of indomethacin, dexamethasone, or both, the levels were reduced to 84 +/- 10, 43 +/- 20 and 63 +/- 11 ng/ml respectively. In control animals receiving solvent (ethanol), the value was 190 +/- 30. With longer term duration (72 h) the levels of plasma cortisol following injection of indomethacin, dexamethasone and both drugs together were 120 +/- 29, 120 +/- 10 and 37 +/- 8 respectively. On the contrary, no significant change was produced by prostaglandins. In vitro release of cortisol from superfused head kidneys (which contain the interrenal tissue of teleosts) decreased gradually with time and reached a minimum after 50 min. Addition of ACTH or of PGE1 to the medium induced an immediate, dose dependent increase in cortisol output which reached a maximum after 15-30 min. On the contrary, PGE2 alpha or indomethacin did not modify cortisol release. These results show that cortisol production in trout, which is controlled by ACTH as in other vertebrates, is also under the influence of other hormonal substances acting on the adenyl-cyclase system.

Nuclear binding of cortisol in intestinal mucosa and liver of a teleost fish (Salmo gairdnerii irideus).

Nuclear binding of corticosteroids in fish tissues was investigated in two target organs of the trout Salmo gairdnerii irideus: the liver and the intestinal mucosa. Incubation of intact nuclei with [3H]-cortisol or [3H]-dexamethasone failed to demonstrate high-affinity binding of these steroids to proteins. Exchange assay of [3H]-cortisol in high-salt nuclear extracts indicated an association constant Ka = 1.9 X 10(4) M-1 for intestinal mucosa and 2.1 X 10(4) M-1 for liver. In sea water-adapted trout, the association constant remained the same as in fresh water. These results extend previous observations obtained on the cytosol which showed that no high-affinity receptors could be disclosed in fish tissues using these two corticosteroids.

Uptake and binding of glucocorticoids in fish tissues.

Cytosols prepared from the liver and various tissues of goldfish (intact or hypophysectomized) and trout (intact) were incubated at 2 degrees C in the presence of tritiated cortisol or dexamethasone (3 x 10(-9) to 3 x 10(-6) mol/l) with or without a 1000-fold excess of unlabelled steroid. In contrast to mammals, the specifically bound component represented a very low fraction of the total bound steroid retained on DEAE cellulose filters and did not show saturation over a large range of concentrations. The subcellular distribution of [3H]dexamethasone was studied in trout liver after intra-vascular injection of the labelled steroid with and without an excess of unlabelled steroid. The amount of protein-bound steroid in the cytosol again corresponded to a small (4%) proportion of the free steroid. The large reduction in the uptake of tritiated dexamethasone, which was induced in both the cytosol and nuclei by competing unlabelled dexamethasone, was interpreted as evidence for mediated entry across cellular and nuclear membranes. These results indicate that high-affinity binding sites are absent, or present only in very small numbers in cytosol from teleost tissues. The entry of glucocorticoids into the nucleus may not require the hormone to be bound to high-affinity cytosolic receptors unless the binding, though quantitatively small, displays a high rate of turnover.

[Adaptation of trout to seawater. Effects on sodium plasma concentration, gill exchange and intestinal transport]. / Adaptation de la truite d'élevage à l'eau de mer. Effects sur les concentrations plasmatiques, les échanges branchiaux et le transport intestinal du sodium

10. Trout Salmo irideus are adapted to sea water (S W) within four weeks by submitting them to a stepwise increase in external salinity (successively: 1/3 SW, 1/2 SW, 3/4 SW, full SW). 20. In completely adapted individuals mean plasma electrolyte concentrations vary only slightly (by a few %) from one medium to another. Thus, the trout may be regarded as a euryhaline, eventually homeosmotic species. 30. With increasing outside salinity there is a progressive diminution in the overall gill permeability to ions which is suggested by saturation curves obtained for sodium fluxes (maximum at about 500 muEq/h. 100 g at 15 degrees C, for unshocked fish). 40. Disturbance of the animals provokes a striking elevation and desequilibrium in these exchanges and this in turn induces an abnormal rise in plasma concentrations and a subsequent failure to adapt to hypertonic media. 50. In vitro absorption of water and sodium by intestinal everted-sacs increases only after transfer to full sea water. Mucosal entry of ions into intestinal epithelial cells measured by the technique of Schultz et al. (1967), is diminished in sea water-adapted animal (by 42% in the case of sodium). 60. These results demonstrate that Salmo irideus possesses efficient osmoregulatory mechanisms which operate with minimal energy expenditure in hypertonic media.