High rates of intestinal bicarbonate secretion in seawater tilapia (Oreochromis mossambicus).

Osmoregulation in fish is a complex process that requires the orchestrated cooperation of many tissues. In fish facing hyperosmotic environments, the intestinal absorption of some monovalent ions and the secretion of bicarbonate are key processes to favor water absorption. In the present study, we showed that bicarbonate levels in the intestinal fluid are several fold higher in seawater than in freshwater acclimated tilapia (Oreochromis mossambicus). In addition, we analyzed gene expression of the main molecular mechanisms involved in HCO3- movements i.e. slc26a6, slc26a3, slc4a4 and v-type H-ATPase sub C in the intestine of tilapia acclimated to both seawater and freshwater. Our results show an anterior/posterior functional regionalization of the intestine in tilapia in terms of expression patterns, which is affected by environmental salinity mostly in the anterior and mid intestine. Analysis of bicarbonate secretion using pH-Stat in tissues mounted in Ussing chambers reveals high rates of bicarbonate secretion in tilapia acclimated to seawater from anterior intestine to rectum ranging between ~900 and ~1700nmolHCO3-cm-2h-1. However, a relationship between the expression of slc26a6, slc26a3, slc4a4 and the rate of bicarbonate secretion seems to be compromised in the rectum. In this region, the low expression of the bicarbonate transporters could not explain the high bicarbonate secretion rates here described. However, we postulate that the elevated v-type H-ATPase mRNA expression in the rectum could be involved in this process.

Effect of CdCl2 on Regulatory Volume Decrease (RVD) in Mytilus galloprovincialis digestive cells.

This study investigated the role of cadmium, a widespread heavy metal in the aquatic environment, on cell volume regulation of digestive cells isolated from the digestive gland of Mytilus galloprovincialis. These cells when exposed to a rapid change (from 1100 to 800 mOsm/kg) of the bathing solution osmolality swelled but thereafter underwent a Regulatory Volume Decrease (RVD), tending to recover the original size. This homeostatic response is altered by cadmium, as suggested by experiments performed both on isolated cells pre-incubated with cadmium (10(-5)M) and on cells isolated from animals exposed to sub-lethal concentrations of the metal (40 µg/l for 21 days). It is suggested that cytoskeleton and Na(+)/K(+)ATPase are the possible targets of cadmium which impairment is responsible of the altered homeostatic response.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus aurata L.).

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 µmol l(-1)), PI3K (LY-294002, 75 µmol l(-1)) or MEK (U-012610, 10 µmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla.

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla.

The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10(-4) mol x l(-1)) or aspirin (10(-4) mol x l(-1)) in different experimental conditions. Both the antiinflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO3- in the incubation bath. The effects of prostaglandin E2 (PGE2) on acid secretion rate (J(H)) and on alkaline secretion rate (J(OH)) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE2 (10(-8)-10(-5) mol x l(-1)) increased JH in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10(-4) mol x l(-1)), PGE2 stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO3- or Na+ and by serosal ouabain (10(-4) mol x l(-1)). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO3- transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na+ transport.

Effects of acetylcholine, serotonin and noradrenalin on ion transport in the middle and posterior part of Anguilla anguilla intestine.

The effects of acetylcholine analogues, serotonin and catecholamines on ion transport were studied in both the middle and the posterior intestine of Anguilla anguilla, mounted in an Ussing chamber, with the aim of understanding whether these regulators affect different mechanisms in the different tracts. In the middle intestine, acetylcholine analogues and serotonin decreased the serosa negative transepithelial potential and short-circuit current without altering the transepithelial resistance; catecholamines reversed the inhibitory effects of both regulators. Similar opposite effects were produced by both the acetylcholine analogues and noradrenalin in the posterior intestine. However, the lowering of the short-circuit current elicited by serotonin was paralleled by the decrease of the transepithelial resistance, whilst noradrenalin had the opposite effects on both parameters. These observations, together with the results of experiments performed by measuring the dilution potential in the control condition and in the presence of either serotonin or serotonin plus noradrenalin, led us to hypothesize that serotonin increases the anion conductance of the paracellular pathway while noradrenalin decreases it. In both the middle and posterior intestine, these regulators probably affect transcellular transport mechanisms by acting on the Na-K-Cl transporter; both acetycholine and serotonin decrease its activity while noradrenalin increases it.

Chloride-sulphate exchange chemically measured in human erythrocyte ghosts.

The purpose of this paper is to provide a clear description of a new chemical method to measure and analyze the net anion exchange (Cl-/SO4--) in resealed human erythrocyte ghosts. With this technique, we measured the influx of an inorganic divalent anion (SO4--) against a monovalent anion (Cl-) as a function of temperature. We found an apparent activation enthalpy Ea of 21-24 Kcal/mol. Besides the Cl-/SO4-- influx pH - dependence, measured under condition of electric anion equilibrium, shows a bell shape with a maximum around pH 6.3. In addition we accounted for the extracellular pH changes during the exchange of chloride for sulphate across the erythrocyte ghosts when bicarbonate is present in the medium. The pH drops sharply, then increases for the remaining experimental time course.

Bicarbonate absorption in eel intestine: evidence for the presence of membrane-bound carbonic anhydrase on the brush border membranes of the enterocyte.

Bicarbonate absorptive fluxes through the isolated intestine of the European eel (Anguilla anguilla) were evaluated by the pH-stat method under short-circuited conditions. It was found that bicarbonate absorptive flux was dependent on the luminal Na+ and was inhibited by luminal 4-acetamido-4' stilbene-2-2' disulfonic acid (SITS; 2.5 x 10(-4) M) and luminal acetazolamide (10(-4) M), while luminal amiloride (1 mM) was without effect. Furthermore, by using brush border membrane vesicles (BBMV) isolated from eel intestine, the existence of two carbonic anhydrase (CA) isoforms, one tightly associated to the brush border membrane (BBM) and the other soluble in the cytosol, was demonstrated. The membrane-bound CA differs from the cytoplasmic isoform in that 1) it is relatively resistant to treatment with 0.045% lauryl sulfate sodium salt (SDS); 2) it is less inhibitable by ethoxzolamide and sulfanilamide; and 3) its Kmapp is significantly lower than that of the cytoplasmic isoform. These results suggest that a BBM-bound CA isozyme would play an important role in bicarbonate absorption from the lumen, facilitating the HCO3- transfer through the luminal membrane of the eel enterocyte most likely via a Na+ (HCO3-) or (OH-) cotransport system.

Different effects of cGMP and cAMP in the intestine of the European eel, Anguilla anguilla.

The regulation of salt absorption in the sea water eel intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E1 on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increases the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying C1- transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine Basoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.

Requirement of HCO3- for Cl(-)-absorption in seawater-adapted eel intestine.

The role of HCO3-/CO2 buffer in Cl- absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl- absorption, expressed as short-circuit current (Isc), was measured in either 20 mM HCO3-/1% CO2 Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO3-/CO2 with HEPES/O2 was without effect on Isc and transepithelial voltage (Vt), whereas bilateral removal of HCO3-/CO2 reduced Isc and Vt by 50%, indicating that the presence of HCO3-/CO2 buffer at one side of the epithelium is sufficient to keep Cl- absorption at the maximum rate. We examined in further detail the individual components of the HCO3-/CO2 system that stimulates Cl- absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO2 to the luminal or serosal solution (final pH = 7.6 in the chamber) had no effect on Isc and Vt, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO3-/1% CO2 Ringer or 20 mM HCO3- alone. Stimulation of Isc induced by unilateral (luminal or serosal) HCO3-/CO2 was inhibited by luminal or serosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (0.25 mM) or by serosal Na+ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of Isc induced by luminal addition of HCO3-/CO2, whereas it was without effect when HCO3-/CO2 was added serosally or bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory effect of anthranilate derivatives on HCO3-/Cl- exchange in red blood cells of human, pigeon and trout.

All ten of anthranilate derivatives were found to be inhibitors of HCO3-/Cl- exchange in human, trout and pigeon red blood cells. The individual l50 values covered a range of 2-3 orders of magnitude for human, trout and pigeon RBCs. The results obtained confirm that the anion transporting region of the band 3 protein in human, trout and pigeon red blood cells are highly preserved even though the RBCs belong to animals living in entirely different habitats. However, in spite of the general similarities, significant differences between human trout and pigeon red blood cells could be observed. They could be attributed to differences of the hydrophobic character of the various probes.

Transport properties of the basolateral membrane of the oxyntic cells in frog fundic gastric mucosa.

The conductive properties of the basolateral membrane of oxyntic cells (OC) of frog fundic gastric mucosa were investigated by utilizing the microelectrode technique. By examining the response of the basolateral cell membrane potential difference, Vcs, to sudden ion concentration changes in the serosal bath it was concluded that the basolateral membrane of OC has a high Ba2+-sensitive K+-conductance, and no Cl- -conductance both in resting (cimetidine) and in stimulated (histamine) state. The response of Vcs to serosal Cl- -removal, consisting in a slight hyperpolarization (anomalous Nernst response), could not be explained by possible permeability changes to K+ and Na+ since the potential response to Cl- was essentially preserved by blocking K+-permeability with Ba2+ and replacing all Na+ by choline. Conversely, hyperpolarization of Vcs after Cl- -free perfusion was abolished by exposure to HCO3- -free solution, indicating that HCO3- -ions are required at the serosal bath for Cl- to get his effect. It was investigated wether the effect of Cl- was due to an electrogenic Na+ (HCO3-)n/Cl- exchange mechanism on the basolateral membrane. Experiments showed that the potential response to HCO3- -removal and to Na+-removal, consisting in a depolarization of Vcs, was similar both in presence and in absence of Cl-. Furosemide (0.5 mmol/l) had no effect on steady Vcs and Vt. The electrophysiological analysis of the data led to excluding the involvement of Na-Cl, Na-2Cl and NaK-2Cl cotransports, and to including the existence of an electrogenic Na+(HCO3-)n/Cl- exchange process, while suggests the presence of an electroneutral Cl-/HCO3- exchange mechanism to explain Cl- -transport across the basolateral membrane of OC.

[Inhibition of anion transport in human erythrocyte ghosts under diverse experimental conditions]. / Inibizione del trasporto anionico in ghosts di eritrociti umani in diverse condizioni sperimentali.

The anion and cation permeability of the red blood cells can be modified by a large variety of chemical agents. The interaction of many of these agents with the membrane seem to involve a reaction with positively charged groups. A series of unrelated amphiphilic compounds which should not react with amino-groups nevertheless, produce reciprocal effects on anion and cation permeability. An important exception to the pattern of reciprocal anion-cation effects is found with the disulfonic stilbene derivate, SITS, developped by Maddy as a covalent bonding agent for amino groups of the cell surface. As derivative we used H2DIDS. This agent is just effective as other in reducing anion permeability, but has no effect on cation permeability. The unique specificity of H2DIDS has been attributed to its inability to penetrate into membrane and reacts with a small sites superficially located on the outer face of the membrane. We studied the inhibition of 35SCN efflux by mean H2DIDS in the human red cell ghosts as a function of concentration, incubation time, pH in the medium. We found that the best concentration to obtain the maximum of inhibition is about 50 microM H2DIDS, increasing H2DIDS concentration does not produce further inhibition. Incubation time must be around one hour, increasing the incubation time, there isn't any variation in the inhibition. H2DIDS acts better in a basic range then in an acid one.