Seawater Eel Intestine Research Articles

Molecular mechanisms underlying guanylin-induced transcellular Cl− secretion into the intestinal lumen of seawater-acclimated eels

Guanylin (GN) stimulates Cl− secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl− transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl− secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3– from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl− and Na+, which was blocked by serosal DNDS. These results suggest that Cl− is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+–HCO3– cotransporter (NBC) but not by Na+-K+-2Cl− cotransporter 1 (NKCC1), and Cl− is secreted by unknown DPC-sensitive Cl− channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42− transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl− secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl− secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine.

Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2β and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCβ genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2β was MI > PI, and that of NCCβ was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na(+)-K(+)-Cl(-) transport system governed by NKCC2β, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl(-) channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.

Effects of eel neuropeptide Y on ion transport across the seawater eel intestine.

A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (Isc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the Isc were concentration-dependent with a threshold concentration of 3 x 10(-9) M and a maximal effect at 3 x 10(-7) M. Similar concentration-response curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an alpha 2-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3 x 10(-8) M for eNPY, 3 x 10(-8) M for AD), but not at high dosage (10(-6) M eNPY, 10(-7) M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na(+)-K(+)-Cl- cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na(+)-K(+)-Cl- cotransporter.

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.

Alteration of sensitivity to various regulators in the intestine of the eel following seawater acclimation

Effects of serotonin (5-HT), methacholine (MCh; a muscarinic acetylcholine receptor agonist), eel atrial natriuretic peptide (eANP) and adrenalin (AD) on the transepithelial potential difference (PD), short-circuit current (Isc) and tissue resistance ( Rt) across the intestine were compared between seawater (SW) and freshwater (FW) eels. The dose-response curves and the median effective concentrations (ec 50) of 5-HT, MCh and eANP were not significantly different between SW and FW eels. However, the SW eel intestine was three times more sensitive to AD than FW eels. Cortisol treatment enhanced the AD sensitivity and bumetanide-sensitive Isc, an indicator of Na +-K +-Cl − cotransport activity. These results suggest that cortisol may synthesize AD receptor and Na +-K +-Cl − cotransport system in the intestine following SW acclimation of eels.

Somatostatin-related peptides isolated from the eel gut: effects on ion and water absorption across the intestine of the seawater eel.

Four somatostatin-related peptides were isolated from eel guts. Two of them were the same as eel SS-25II (eSS-25II) and eel SS-25I (eSS-25I) isolated from European eel pancreas. The remaining two peptides were C-terminal tetradecapeptides (eSS-14II and eSS-14I) of eSS-25II and eSS-25I, respectively. These four peptides all enhanced the serosa-negative transepithelial potential difference and short-circuit current across the seawater eel intestine after pretreatment with isobutylmethylxanthine, serotonin (5-HT) and methacholine, an agonist of acetylcholine (ACh). Among these peptides, eSS-25II was the most potent enhancer, followed by eSS-25I and eSS-14II. Since the large peptide (eSS-25II) acts at a lower concentration than the small somatostatin (eSS-14II), the 11 N-terminal amino acid residues seem to potentiate somatostatin action in the eel intestine. In contrast, eSS-14II was more potent than mammalian SS-14, indicating that the three amino acid residues (Tyr18, Gly21, Pro22) in the C-terminal portion also contribute to the potency of somatostatin. Endogenous somatostatin (eSS-25II) activated net Na+, Cl- and water fluxes across the seawater eel intestine. This stimulatory action was not inhibited by tetrodotoxin or yohimbine, an adrenergic antagonist, indicating that eSS-25II does not act through neuronal firing or through catecholamine release. Thus, eel somatostatins may act directly on the enterocytes, but on a distinct receptor from that for adrenaline, to antagonize the inhibition of NaCl and water absorption by 5-HT and ACh in the seawater eel intestine.

Catecholamine receptor in the seawater eel intestine

Dose-response relationships of catecholamines on seawater eel intestinal ion transport were obtained; the potency order being (-)adrenalin>(-)noradrenalin=clonidine>(±)noradrenalin (racemic form of noradrenalin)>(-)phenylephrine>dopamine>(±)isoproterenol, indicating that α2 are more potent than α1- or β-agonists. In addition, the effects of adrenalin were completely blocked by yohimbine (α2-antagonists) but not by prazosin (α1-antagonists) or propranolol (β-antagonist). These results indicate the existence of an α2-receptor in the seawater eel intestine. Adrenalin may activate the α2-receptor physiologically, since adrenalin is the most potent stimulant and is the predominant catecholamine in American eel plasma (Hathaway and Epple 1989). Presumably ion and water absorption across the seawater eel intestine will be maintained by adrenalin. From the structure and the action of various agents used in the present study, structure-activity relationships of catecholamines are considered: hydroxyl groups on the benzene ring (catechol) seem to be essential for the α2-action in the seawater eel intestine and the presence of OH and CH3 on β-carbon and amide, respectively, seems to potentiate the α2-action.

Noradrenalin antagonizes effects of serotonin and acetylcholine in the seawater eel intestine

After inhibiting ion and water transport with 10-6 mol·l-1 serotonin and 10-6 mol·l-1 methacholine, a muscarinic agonist of acetylcholine, 10-5 mol·l-1 (±)noradrenaline restored the serosa-negative transepithelial potential difference and short-circuit current in a step-like manner, accompanied by an increase in water absorption across the seawater eel intestine. Such recovery by noradrenalin was not obtained after pretreatment with 10-7 mol·l-1 eel atrial natriuretic peptide. This means that the inhibitory mechanisms of serotonin and acetylcholine are different from those of atrial natriuretic peptide. Similarly, 10-7 mol·l-1 clonidine and guanabenz (α2-agonists) also reversed the inhibitory action of serotonin and methacholine, but 10-7 mol·l-1 phenylephrine (α1-agonists) and 10-7 mol·l-1 isoproterenol (β-agonist) did not antagonize serotonin and methacholine actions. Further, the enhancement by 10-5 mol·l-1 noradrenalin was blocked by 10-4 mol·l-1 yohimbine (α2-agonists) and 10-4 mol·l-1 prazosin (α1-agonists), but not by 10-4 mol·l-1 propranolol (β-antagonist). Although relatively high dosage is required to obtain a significant effect, and discrimination between α1- and α2- is not successful in the present study, these results suggest that noradrenalin acts on an α-type receptor. The α-type receptor may exist on the enterocytes, since the effects of noradrenalin are observed even in the presence of 10-6 mol·l-1 tetrodotoxin. Interestingly, the tissue resistance also increased in parallel with increase in the short-circuit current after treatment with noradrenalin in the posterior part of the seawater eel intestine.

Effects of eel atrial natriuretic peptide on NaCl and water transport across the intestine of the seawater eel.

Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaCl and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N-terminally truncated eel atrial natriuretic peptide (5-27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was required to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-terminal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent inhibitor, with 100% inhibition at 10(-7) M; 50% inhibition was obtained at 10(-2) M in acetylcholine, and 30% inhibition in serotonin (10(-5) M) and histamine (10(-3) M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetrodotoxin, and were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate. Based on these results, structure-activity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.

Mechanism of Cl- transport in eel intestinal brush-border membrane vesicles.

Cl- transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. 36Cl- uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K+ diffusion potential obtained by imposing a K+ concentration gradient (Cout greater than Cin) in the presence of valinomycin, (c) an inwardly directed H+ ion concentration gradient. The membrane-potential-driven Cl- transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropyl-amino)-benzoic acid. Arachidonic acid also inhibited Cl- uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na+ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl- influx was the same in the presence of inwardly directed gradients of Li+, Na+ or K+, arguing against the presence of Na(+)-Cl-, as well as K(+)-Cl- cotransport. The absence of a significant contribution of the Na(+)-K(+)-2Cl- cotransport mechanism to the Cl- uptake in seawater eel intestinal BBMV was indicated from the observations that Cl- uptake was not stimulated by the simultaneous presence of inwardly directed Na+ and K+ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na+ and K+. Furthermore, no evidence for the dependence of Cl- uptake on the Na+ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K+ and valinomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate Transport Systems in the Intestine of the Seawater EEL

ABSTRACT Utilizing a pH-stat method, the rates of mucosal and serosal alkalinization were measured separately in the seawater eel intestine. These two rates were dependent on contralateral HCO3− concentration and were inhibited by contralateral application of DIDS, an inhibitor of HCO3− transport, indicating that the mucosal and serosal alkalinization are due to HCO3− secretion and absorption, respectively. The mucosal alkalinization was enhanced after inhibiting Na+/K+/Cl− cotransport by treatment with bumetanide, furosemide or Ba2+, with a latent period of more than 10 min, suggesting that HCO3− absorption from mucosa to serosa depends on Na+/K+/Cl− cotransport. The serosal alkalinization caused by HCO3− absorption was completely abolished after mucosal application of bumetanide. After pretreatment with bumetanide, mucosal omission of Cl− halved the enhanced rate of mucosal alkalinization, and Na+ omission had no effect on it; this indicates that the exit of HCO3− into the lumen depends on luminal Cl−, i.e. on the existence of the usual C1−/HCO3− exchange on the brushborder membrane. When serosal Na+ was removed under the same conditions, mucosal alkalinization was reduced, indicating that HCO3− entry from the serosal fluid depends on Na+. Serosal omission of Cl− did not reduce mucosal alkalinization. In addition, serosal alkalinization was enhanced by serosal removal of Na+ but not of Cl−. These results suggest that there is a Na+/HCO3− cotransport on the basolateral membrane. A possible model for HCO3− transport systems in the seawater eel intestine is proposed, and a possible role for these transport systems is discussed in relation to Na+, Cl− and water transport.

Effects of Bicarbonate on Salt and Water Transport Across the Intestine of the Seawater EEL

ABSTRACT To elucidate whether the Na+/K+/Cl− cotransport system depends on HCO3− in the seawater eel intestine, the effects of HCO3− on the transepithelial potential difference (PD) and on net water and ion fluxes were examined. When HCO3− buffer was replaced with phosphate buffer, the serosa-negative PD and net Na+, Cl− and water fluxes from mucosa to serosa were inhibited, indicating that the Na+/K+/Cl− cotransport system is inhibited in phosphate-buffered solutions. Similar inhibitory effects were also observed in solutions buffered with Hepes, Tris or Tes, indicating that the inhibitory effects are not specific for the phosphate buffer but are caused by omission of the HCO3− buffer system. Although the HCO3− buffer system consists of HCO3− and CO2, higher CO2 pressure with constant HCO3− concentration did not enhance, but inhibited, the PD and the net water flux: this indicates that the inhibition observed after removal of the HCO3− buffer system is due to omission of HCO3− rather than CO2. The inhibition of PD and the net water flux was greater after removal of HCO3− from the serosal side than from the mucosal side. Similarly, the inhibitory effects of 4, ′ -diisothio-cyanostilbene-2,2′-disulphonic acid (DIDS), an inhibitor of HCO3− transport, were more pronounced on the serosal side than on the mucosal side. Mucosal Ba2+ also inhibited PD and the short-circuit current (Isc) and enhanced the tissue resistance (Rt), presumably through partially blocking the apical K+ channels. However, these effects of Ba2+ were completely abolished after pretreatment with serosal DIDS, suggesting that Ba2+ and DIDS evoked the same effect. These results are combined and a possible role for HCO3− in the Na+/K+/Cl− co transport system is discussed.

Amino Acid Metabolism and Water Transport Across the Seawater Eel Intestine

ABSTRACT To elucidate how intracellular L-alanine enhances water transport across the seawater eel intestine, effects of various metabolic inhibitors were examined. The L-alanine-induced water flux was inhibited by amino-oxyacetate, an inhibitor of aminotransferase. After blocking the synthesis of pyruvate from L-alanine with this drug, water transport was stimulated with pyruvate, whose effects were inhibited by oxythiamine, an inhibitor of pyruvate dehydrogenase. 2,4-Dinitro-phenol (DNP) also inhibited the effects of L-alanine. Furthermore, L-alanine enhanced ouabain-sensitive O2 consumption in this tissue, and the enhancement in O2 consumption preceded that in the transepithelial potential difference (PD) and the net water flux. These results indicate that L-alanine is metabolized through the citric acid cycle to produce ATP, and that a metabolic product stimulates ion and water transport. L-Glutamine also seems to be metabolized just like L-alanine because: L-glutamine acted from inside the enterocyte; DNP inhibited the effects of L-glutamine; neither of the effects of L-glutamine and L-alanine were additive but they were mutually complementary; L-glutamine also enhanced ouabain-sensitive O2 consumption; and the increment in O2 consumption preceded that in the PD and the net water flux. The effects of L-glutamine on the PD and the net water flux depended on glutamine concentration and the concentration-response curve was of the Michaelis-Menten type, indicating that the rate of L-glutamine uptake into the enterocyte limits the overall rate of L-glutamine metabolism. A regulatory role of amino acids for ion and water transport is discussed.

A new technique for measuring water transport across the seawater eel intestine.

A new technique was developed for measuring net water flux across the eel intestine in vitro. The new perfusion method was suitable for long duration experiments because of continuous oxygen supply to both the external and the perfusion medium. Net water flux was calculated directly from the difference between the rates of effluent and perfusate flow without measuring the concentration of marker substances. The calculated value of net flux appears to be reliable because: its direction is always from mucosa to serosa in both everted and non-everted intestine; ouabain diminishes it to zero; it is identical with the standard water flux obtained under zero perfusion; and it is identical with the value obtained by means of [14C]PEG following the previous perfusion method. The net water fluxes obtained by the new method were steadier than those obtained with the previous complicated perfusion method. In this new experimental system, the net water flux and the transepithelial potential difference (PD) decreased gradually with time, and were not restored by application of adrenergic agonists or cortisol. These parameters were stimulated by 5 mmol l-1 L-alanine, but not by D-glucose, L-valine or L-glycine, indicating a specific action of L-alanine.

Relationship between coupled Na+?K+?Cl? transport and water absorption across the seawater eel intestine

Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na+, K+, Cl− and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl− was removed from both sides of the intestine, net K+ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl− and water fluxes depend on K+ transport under the standard condition (Ando 1983), the interdependence of K+ and Cl− transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl− and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na+-free Ringer solutions, suggesting the existence of a Na+-dependent KCl transport system and linkage of water absorption to the coupled Na+−K+−Cl− transport. Since ouabain blocked the active Na+−K+−Cl− transport almost completely, the permeability coefficients for K+ and Na+ through the paracellular shunt pathway were estimated as PK=0.076 and PNa=0.058 cm/h, and PCl was calculated as 0.005 cm/h. Although Na+-independent K+ and Cltt- fluxes were observed again in the present study, these fluxes were not inhibited by CN−, ouabain or diuretics, and evoked even after blocking the Na+−K+−Cl− transport completely with ouabain. These results indicate that the Na+-independent K+ and Cl− fluxes are distinct from the active Na+−K+−Cl− transport and are not themselves active.

Potassium-dependent chloride and water transport across the seawater eel intestine

Simultaneous measurements of net ion and water fluxes and transepithelial potential difference (PD) were made in the stripped intestine of the seawater eel, and it was examined whether Cl- was driven following electrochemical gradient for Na+ across the brush border membrane of the epithelium or not. When mucosal Na+ was completely replaced with K+, while the serosa was being bathed with normal Ringer's solution, net Cl- and water fluxes were maintained as high as those in normal Ringer's solution. After serosal Na+ was completely replaced with choline+ while the mucosa was being bathed with Na+-free KCl Ringer's solution, 80% of the original Cl- and water fluxes still persisted, indicating significant Na+-independent Cl- and water transport. These results are against a hypothesis that Cl- is driven by electrochemical gradient of Na+ across the brush border membrane. The Na+-independent Cl- and water fluxes were a saturable function of mucosal K+ concentration, suggesting K+-dependent Cl- and water transport. A possible mechanism of Cl- transport is discussed in relation to K+ transport. On the other hand, a good correlation was observed between the net Cl- and water fluxes. This suggests that water transport depends on Cl- transport system; NaCl and/or KCl cotransport.

Effects of ouabain on chloride movements across the seawater eel intestine

Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K+ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na+−K+ pump by ouabain, K+-free or Na+-free Ringer solution increased the diffusion potential for Cl−. Inhibition of Cl− transport and increment in Cl− permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl− transport as well as Cl− permeability is dependent on Na+−K+ pump activity. A possible mechanism of dependence of Cl− transport on the Na+−K+ pump is discussed in relation to the increment in Cl− permeability.

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.