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.

Na+/H+ exchange and osmotic shrinkage in isolated trout hepatocytes

The ability of rainbow trout liver cells to regulate their intracellular pH (pHi) was studied using two methods on hepatocytes isolated by collagenase digestion: (i) by monitoring pHi with the fluorescent dye BCECF-AM, and (ii) by measuring the amiloride-sensitive uptake of 22Na, which represents Na+/H+ exchange. In low-Na+ medium (¾16mmoll-1), Na+ uptake was reduced by approximately 70% in the presence of amiloride derivatives (DMA or MPA, 10(-4)moll-1). Changing separately either the extracellular pH (pHe) or the intracellular pH (pHi, clamped by treating the cells with nigericin in the presence of 140mmoll-1 K+) between 6 and 8 induced an increase in the rate of Na+ uptake when pHe was raised or when pHi was reduced. When transferred to hypertonic medium, hepatocytes shrank to nearly 72% of their initial volume, and thereafter a slow and partial regulatory volume increase phase was observed, with an increase in the amiloride-sensitive rate of Na+ uptake and an increase in intracellular pH. As DIDS-sensitive Cl- uptake was concomitantly enhanced, it is suggested that hypertonic stress activates Na+/H+ and Cl-/HCO3- exchange.

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).

Ion flux changes induced by voltage clamping or by amphotericin B in the isolated urinary bladder of the trout.

1. When bathed with Ringer solutions on both sides in Ussing chambers, isolated urinary bladders of trout carry out an electrically silent transport of Na+ and Cl- ions from mucosa to serosa. In the present experiments, an electrical gradient was set up across the epithelium mounted under these conditions either by using a voltage-clamp technique or by submitting the mucosal side to the polyene antibiotic Amphotericin B. 2. With stable potential differences between -40 and +40 mV applied to the tissue, both unidirectional fluxes (Jms and Jsm) for Na+ and for Cl- varied as a function of the electrical gradient. This indicates that a sizeable fraction of these ionic exchanges is due to passive diffusion across a low-resistance pathway. 3. Amphotericin B (40 micrograms/ml.) applied to the mucosal side induced an immediate and large (up to 50 mV, serosa positive) potential difference and a sustained diminution in tissue resistance. These effects were strictly Na+-dependent and were reduced in the presence of ouabain. Therefore they are due to increased passive entry of Na+ along its electrochemical gradient across the cellular apical membrane. 4. The effect of Amphotericin B was enhanced when Cl- was replaced by an impermeant anion such as gluconate. Therefore the shunting effect of Cl- on the induced potential observed in the control conditions appears to be due to an increase in Cl- permeability across an anion pathway. Flux data indicate that this pathway is transcellular.

The mechanism of coupled transport of sodium and chloride in isolated urinary bladder of the trout.

1. Transepithelial fluxes of Na and Cl were measured in dissected trout bladders mounted in Ussing chambers. The mucosa-to-serosa fluxes appeared to comprise a linear and a saturating (Km = 8 mM) component as a function of the luminal concentration of the ion. The over-all permeability for Cl was higher than for Na. Removal of either ion from the mucosal side induced the disappearance of the net flux of the co-ion even when the substituting ions were not impermeant. This indicated a complete and specific mutual dependence between the Na and Cl transports. A large part (30-50%) of Cl exchanges was ascribed to an exchange-diffusion process. 2. In order to localize the site of ion copuling, the apical uptakes of Na and Cl were measured by short-term exposure (2 min) to radioisotopes. The apical fluxes were similar to the transepithelial fluxes and therefore appeared to be the limiting factor for ion transfer. Ionic replacements on the mucosal side showed that the coupling between Na and Cl and Cl/Cl exchange occurs at this step.

Failure of 2,4,6-triaminopyrimidine (TAP) to block sodium pathways in a "leaky" epithelium: the urinary bladder of the trout.

The organic cation 2, 4, 6 - Triaminopyrimidine (TAP) used to block sodium permeability across leaky epithelia is ineffective on isolated urinary bladder of the trout. This observation suggests that this membrane does not present cation-selective paracellular pathways.