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In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.

In fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K+ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K+ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K+ATPase was dose-dependent in both environmental adaptations. Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K+ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions.

The ability of eel intestinal epithelial cells to recover from an acute acid load was analysed using the fluorescent dye 2',7'-bis-carboxy-ethyl-5,6-carboxyfluorescein (BCECF) and cell suspensions. Under these experimental conditions (bicarbonate-free solutions) the resting pHi in cells prepared from sea-water (7.52 +/- 0.031) and fresh-water (7.50 +/- 0.094) adapted animals proved to be similar. The recovery rate (following an acid load) increases by increasing the Na ion concentration in the extracellular medium. This pHi recovery is competitively inhibited by the specific inhibitor dimethylamiloride (DMA) with a low Ki in sea- (1.2 microM) as well as in fresh-water (1.3 microM) adapted animals, indicating the presence of a specific Na/H exchange activity in these cells. Using basolateral membrane vesicles it could be demonstrated that this activity is located on the basolateral side of the enterocyte membrane. The kinetic parameters (Kapp and Jmax) of this exchanger are similar in fresh-water and sea-water adapted animals suggesting that no salinity adaptation occurs, thus excluding the involvement of the antiporter in the osmoregulatory processes. These results are in agreement with the presence in the plasma membrane of the eel enterocytes of a Na/H-1 (housekeeper) form of the antiporter.

Transport of L-ascorbate by intestinal brush-border membrane vesicles of European eel Anguilla anguilla was stimulated by a transmembrane Na gradient (out > in) but not by a similarly directed gradient of K. Under short-circuited membrane potential conditions, a kinetic analysis of L-ascorbate influx indicated the presence of a single Na-dependent carrier process (Kapp = 0.75 +/- 0.07 mM and Jmax = 0.33 +/- 0.03 nmol.mg protein-1.min-1) and a nonsaturable transfer component with an apparent diffusional permeability (P) of 0.27 +/- 0.02 microliter.mg protein-1.min-1. D-Isoascorbate was a competitive inhibitor of L-ascorbate influx exhibiting a Ki of 8.21 +/- 0.63 mM. The electrogenic nature of +Na-L-ascorbate cotransport was confirmed by a stimulatory effect of an inside-negative membrane potential on vitamin uptake. Hill analysis of L-ascorbate influx over a wide range of external Na concentrations suggested a 2 Na-to-1 L-ascorbate binding ratio. Results indicate that the vitamin L-ascorbate is transported across fish intestinal brush-border membranes by an electrogenic Na-dependent carrier process in conjunction with more than one sodium ion.

Brush-border membrane vesicles (BBMV) were prepared from European eel (Anguilla anguilla) intestinal epithelium by a magnesium-ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) precipitation technique. Amino acid transport by these purified vesicle preparations was investigated using either radiolabeled substrates or the voltage-sensitive fluorescent dye 3,3'-diethylthiadicarbocyanine iodide [DiSC2(5)]. All amino acids tested exhibited carrier-mediated, Na+-dependent and Na+-independent transfer processes plus diffusion. The only exceptions were glutamic acid and proline, which displayed Na+ dependency and diffusion but did not appear to be transported by Na+-independent agencies. Carrier-mediated transport kinetic constants (Kapp and Jmax) for several amino acids are reported. Cis-inhibition experiments suggested the presence of at least four distinct Na+-dependent transport systems in eel intestinal BBMV: 1) an anionic transport process for glutamic and aspartic acids; 2) a cationic mechanism for lysine and arginine; 3) a relatively specific neutral amino acid carrier for proline and alpha-(methylamino)isobutyric acid; and 4) a nonspecific neutral amino acid system for most other substrates of this group. This scheme for carnivorous fish intestine most closely approximates that reported for mammalian gut with minor dissimilarities that may relate to metabolic differences or specific dietary requirements of the two vertebrate groups.

Studies the mechanism of bicarbonate translocation across the luminal membrane of the eel kidney tubular cells. Monitored uptake of H14CO3- in isolated brush-border membrane vesicles; Indicated presence of a transmembrane for the accumulation of H14CO3- in the vesicular space; Suggested presence of an anion exchanger in the membrane vesicles.