Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl- -induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH4Cl-loaded); and alkalotic turtles (NaHCO3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl- -addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl- reabsorption, the mucosal and serosal addition of Cl- to Na+-free, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl- reabsorption and cAMP-dependent electrogenic alkali secretion (positive Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.     

Electrogenic, K+-dependent chloride transport in locust hindgut

Potassium chloride is the major salt recycled in most insect secretory systems. Ion and water reabsorption occur in the rectum by active transport of Cl- and largely passive movement of K+. Both these processes are stimulated several fold by a neuropeptide hormone acting via cyclic AMP (cAMP). This Cl- transport process was investigated by using intracellular ion-sensitive microelectrodes, radiotracer flux measurements, voltage clamping, ion substitutions and inhibitors. the mucosal entry step for Cl- is energy-requiring and highly-selective, and is stimulated directly by cAMP and luminal K+. Under some experimental conditions, measured electrochemical potentials for cations across the mucosal membrane are too small to drive C;- entry by NaCl or KCl cotransport mechanisms; moreover, net 36Cl- flux is independent of the apical Na+ potential. Similarly no evidence for a HCO3- -Cl- exchange was obtained. We conclude that Cl- transport in locust gut is different from mechanisms currently proposed for vertebrate tissues.     

Electrical properties and fluxes of Na+ and Cl- across lizard intestine

Electrical parameters and unidirectional Na+ and Cl- fluxes were determined in vitro across the duodenum, ileum and colon of lizard (Gallotia galloti). Electrical potential difference (PD) and short circuit current (Isc) were low in the three segments studied, whilst tissue conductance (Gt) was high. A net active transport of Na+ and Cl- was observed in the three segments. Net Na+ absorption was higher across duodenum and ileum than across the colon, while net Cl- absorption was similar in duodenum, ileum and colon. Ouabain virtually abolished Isc, PD and net Na+ and Cl- fluxes in all the segments. Amiloride abolished net Cl- flux in duodenum, ileum and colon, whereas net Na+ flux was abolished in colon but decreased in duodenum and ileum. PD and Isc were not affected by the presence of the diuretic.     

Aldosterone and chloride conductance of amphibian skin

Chloride influx (JCl) across the skin of toads maintained in dilute MgCl2 or Na2SO4 was determined after overnight incubation with(out) aldosterone, and related to mitochondria-rich cell (MRC) density of the preparations. Adaptation to MgCl2 vs. Na2SO4 was reflected by higher plasma aldosterone in the former group (17 vs. 3 nmol/l, respectively) while JCl was lower, even after overnight incubation (172 vs. 318 pmol cm-2 s-1). Incubation with aldosterone induced a more pronounced increase in JCl in the case of Na2SO4- vs. MgCl2-adapted toads (delta JCl: 242 vs. 25 pmol cm-2 s-1, respectively), which could be related to difference in MRC density between these two groups (1078 vs. 615 cells/mm2, respectively). On the other hand, the in vitro effect of aldosterone on Na+ transport (assessed by Isc) was equally pronounced in both groups, and thus independent of MRC density. These data suggest that aldosterone, rather than being involved in MRC proliferation, stimulates Cl- conductance by influencing the functional state of MRC.     

New evidence for active sodium transport from fluid-filled rat lungs

The hypothesis that fluid reabsorption from the air spaces is mediated at least in part by active transport of Na+ was investigated in six sets of experiments conducted in isolated fluid-filled rat lungs. Fluid reabsorption was monitored by following the changes in the air space concentration of labeled albumin. We found that incorporation of bicarbonate rather than a nonvolatile buffer (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) in the air space solution more than doubled the rate of fluid reabsorption. Addition of 10(-4) M amiloride to the air space solution reduced the rate of fluid reabsorption over a 2-h experiment from 1.2 +/- 0.1 to 0.7 +/- 0.1 ml and decreased reabsorption of both labeled and unlabeled Na+ from the air spaces. To show that Na+ could be reabsorbed from the air spaces even if the concentrations of Na+ in the perfusate increased above those in the air space, mannitol (150 mM) was added to the perfusate and air space solutions and the concentrations of Na+ and Cl- were reduced to 90 and 60 mM, respectively. Mannitol diffuses across the pulmonary epithelium very slowly, and it osmotically restrained the movement of water out of the air spaces. Na+ concentrations in the perfusate increased by 10 +/- 2 mM, but concentrations in the air space remained unchanged. Despite an increasingly unfavorable concentration gradient for Na+, 0.2 mmol Na+ and 0.6 ml water were reabsorbed from the air spaces in 2 h. Ouabain (10(-4) M) did not appear to slow fluid reabsorption in the presence of mannitol, but it reduced K+ secretion into the air spaces and increased K+ appearance in the perfusate in a manner consistent with inhibition of Na+-K+-adenosinetriphosphatase at the basolateral surface of the epithelial cells. Fluid reabsorption was not altered when the lungs were exposed to a hypotonic solution (185 mM), but secretion of K+ into the air spaces was accelerated and K+ was lost from the perfusate. These experiments are consistent with active Na+ transport from the air spaces.     

Anion transport in the colon

The principal anions transported by colonic epithelium are Cl-, HCO3- and organic anions (OA-), particularly acetate, butyrate and pyruvate, these last being formed by microbial degradation of carbohydrate. In the normal absorptive rat colon, Cl- is transported from lumen to plasma both by the transcellular and paracellular pathways. The transcellular route appears to depend on amiloride-insensitive coupling of Na+-Cl- at the mucosal (apical) membrane, the Na+ electrochemical gradient energizing Cl- uptake. Intraluminal [HCO3-] rises as Cl- as absorbed, and a mucosal Cl- -HCO3- exchange carrier has been postulated. In some species (and in distal colon of the rat when sodium-depleted), the putative Na+-Cl- carrier is absent so that Cl- absorption then depends largely on the paracellular electrochemical gradient. Absorption of OA- is independent of the transepithelial p.d., is associated with HCO3- secretion and is considerably reduced by acetazolamide. In the absence of Cl-, OA- supports Na+ absorption but does not depend on it continuing unchanged when the latter is blocked. Colonic epithelium can become secretory and an example of this state is congenital chloridorrhoea in which an elevated transepithelial p.d. is associated with excessive Cl- secretion. Here, it appears that the Na+-Cl- and Cl- -HCO3- carriers are lost and Cl- conductance of the mucosal membrane substantially increased. The transepithelial uphill movements of Cl- or HCO3- in the absorptive and secretory colon appear to depend on coupling to other ionic flows, and there seems to be no need to postulate active transport of these ions.     

Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion.     

Interaction of heptanol and pressure on sodium and chloride transport by toad skin

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.     

Urates and allantoic regulation in embryonic Japanese quail, Coturnix coturnix japonica

During embryonic development, allantoic fluid represents the shifting balance between renal excretion and reabsorption by chorioallantoic membranes. Allantoic contents of Na+, K+, Cl-, urate, pH, and water were followed over days 10-15 of the 16 day incubation. Water volume remained near 0.9 ml until day 13, then declined very rapidly. The pH declined more steadily, from 8 to 5.5. Contents of Na+ and Cl- fell regularly to final values 80-88% below day 10. The K+ content changed differently and nearly doubled by day 13 but returned to day 10 values at the end. Urate content rose until day 13, then fell suddenly to low levels. This was due to the abrupt precipitation of most urate into masses not sampled by our method, so that after day 13, urate was underestimated (probably by 90-96%). Ion binding by urates was low (about 3% of Na+ and Cl-, 10% of K+) and appeared to be nonspecific. The underestimate of urate contents means, however, that in late incubation about one third of allantoic Na+ and 65-70% of K+ and Cl- are bound to precipitated urate and do not appear in balance sheets of allantoic ions. These precipitated ions account for the significant amounts of Na+ and K+ that remain in the allantoic remnant, left in the eggshell after hatching, but whose presence is not predicted by analysis of allantoic fluid.     

Energetics of coupled Na+ and Cl- entry into epithelial cells of bullfrog small intestine.

Na+, K+ and Cl- concentrations (cij) and activities (aij), and mucosal membrane potentials (Em) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25 degrees C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl- and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl- concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. Em was measured with conventional open tip microelectrodes, aiCl with solid-state Cl-selective silver microelectrodes and aiNa and aiK with Na+ and K+-selective liquid ion-exchanger microelectrodes. The average Em recorded was -34mV. ciNa, ciK and ciCl were 51, 105 and 52 mM. The corresponding values for aiNa, aiK and aiCl were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is 'bound' or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl-. aiCl significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl- is implicated in intracellular Cl- accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl- electrochemical potential differences (deltamuNa and deltamuCl). deltamuNa (-7000 J . mol-1; cell minus mucosal medium) was energetically more than sufficient to account for deltamuCl (1000--2000 J . mol-1).     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Na+, Li+ and Cl− transport by brush border membranes from rabbit jejunum

Na+, Li+, K+, Rb+, Br-, Cl- and SO4(2-) transport were studied in brush border membrane vesicles isolated from rabbit jejunum. Li+ uptakes were measured by flameless atomic absorption spectroscopy, and all others were measured using isotopic flux and liquid scintillation counting. All uptakes were performed with a rapid filtration procedure. A method is presented for separating various components of ion uptake: 1) passive diffusion, 2) mediated transport and 3) binding. It was concluded that a Na+/H+ exchange mechanism exists in the jejunal brush border. The exchanger was inhibited with 300 microM amiloride or harmaline. The kinetic parameters for sodium transport by this mechanism depend on the pH of the intravesicular solution. The application of a pH gradient (pHin = 5.5, pHout = 7.5) causes an increase in Jmax (50 to 125 pmol/mg protein . sec) with no change in Kt (congruent to 4.5 nM). Competition experiments show that other monovalent cations, e.g. Li+ and NH4+, share the Na+/H+ exchanger. This was confirmed with direct measurements of Li+ uptakes. Saturable uptake mechanisms were also observed for K+, Rb+ and SO4(2-), but not for Br-. The Jmax for K+ and Rb+ are similar to the Jmax for Na+, suggesting that they may share a transporter. The SO4(2-) system appears to be a Na+/SO4(2-) cotransport system. There does not appear to be either a Cl-/OH- transport mechanism of the type observed in ileum or a specific Na+/Cl- symporter.     

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane delta pH and not to membrane potential difference. In brush-border membrane vesicles, an Na+ (Li+)-H+ exchange was confirmed. At physiological Na+ concentrations, 40-70% of Na+-H+ exchange was mediated by the electroneutral Na+-H+ antiporter; the remainder consisted of Na+ and H+ movements through parallel conductive pathways. Both modes of Na+-H+ exchange were saturable, with half-maximal rates at about 13 and 24 mM Na+, respectively. Besides a Na+ gradient, a K+ gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H+ and K+ in brush-border membranes. Experiments with Cl- or SO2-4 gradients failed to demonstrate measurable Cl--OH- or SO2-4-OH- exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl- conductance was found. In basolateral membrane vesicles, neither Na+(Li+)-H+ exchange nor Na+ or K+ conductances were found. However, in the presence of valinomycin-induced K+ diffusion potential, H+ conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid. A Cl- conductance of the membranes was also found, but antiporter-mediated electroneutral Cl--OH- or SO2-4-OH- exchange could not be detected by the dye method. The restriction of the electroneutral Na+-H+ exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Independence of apical membrane Na+ and Cl- entry in Necturus gallbladder epithelium

Transepithelial fluid transport (Jv) and intracellular Na+ and Cl- activities (aNai, aCli) were measured in isolated Necturus gallbladders to establish the contribution of different proposed apical membrane entry mechanisms to transepithelial salt transport. In 10 mM HCO3- Ringer's, Jv was 13.5 +/- 1.1 microliter X cm-2 X h-1, and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10(-3)M) or SITS (0.5 X 10(-3)M) to the mucosal bathing solution. Bumetanide (10(-5)M) was ineffective. Bilateral Na+ removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis of the following results, all obtained during mucosal bathing solution changes: during Na+ removal, aNai fell 4.3 times faster than aCli; during Cl- removal, aCli fell 7.5 times faster than aNai; amiloride (10(-3) M) reduced aNai at a rate of 2.4 +/- 0.3 mM/min, whereas aCli was not changed; bumetanide (10(-5) M) had no significant effects on Jv or aCli. The hypothesis of Na-K-Cl cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Ba2+ addition) did not alter aNai or aCli. The average rate of NaCl entry under normal transporting conditions, estimated from Jv, assuming that the transported fluid is an isosmotic NaCl solution, was 22.5 nmol X cm-2 X min-1. Upon sudden cessation of NaCl entry, assuming no cell volume changes, aNai and aCli should fall at an average rate of 4.8 mM/min. To compare this rate with the rates of Na+ and Cl- entry by ion exchange, the Na+ or Cl- concentration in the mucosal bathing solution was reduced rapidly to levels such that electroneutral cation or anion exchange, respectively, should cease. The rate of Na+ or Cl- entry before this maneuver was estimated from the initial rate of fall of the respective intracellular ionic activity upon the mucosal solution substitution. aNai and aCli decreased at initial rates of 3.7 +/- 0.4 and 5.9 +/- 0.8 mM/min, respectively. The rate of fall of aNai upon reduction of external [Na] was not affected by amiloride (10(-3) M), and the rate of fall of aCli upon reduction of external [Cl] was unchanged by SITS (0.5 X 10(-3) M), which indicates that net cation or anion exchange was, in fact, abolished by the changes in Na+ and Cl- gradients, respectively. I conclude that double exchange (Na+/H+ and Cl-/HCO-3) is the predominant or sole mechanism of apical membrane NaCl entry in this epithelium.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Intracellular gradients of ion activities in the epithelial cells of theNecturus gallbladder recorded with ion-selective microelectrodes

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.     

Prostaglandin-mediated inhibition of Na+/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine

Prostaglandins stimulate repair of the ischemia-injured intestinal barrier in the porcine ileum through a mechanism involving cAMP-dependent Cl- secretion and inhibition of electroneutral Na+/H+ exchanger (NHE) activity. In the present study, we focused on the role of individual NHE isoforms in the recovery of barrier function. Ischemia-injured porcine ileal mucosa was mounted on Ussing chambers. Short-circuit current (I(sc)), transepithelial electrical resistance (TER), and isotopic fluxes of 22Na were measured in response to PGE2 and selective inhibitors of epithelial NHE isoforms. Immunoassays were used to assess the expression of NHE isoforms. Forty-five minutes of intestinal ischemia resulted in a 45% reduction in TER (P < 0.01). Near-complete restitution occurred within 60 min. Inhibition of NHE2 with HOE-694 (25 microM) added to the mucosal surface of the injured ileum stimulated significant elevations in TER, independent of changes in I(sc) and histological evidence of restitution. Pharmacological inhibition of NHE3 or NHE1 with mucosal S-3226 (20 microM) or serosal cariporide (25 microM), respectively, had no effect. Ischemia-injured tissues treated with mucosal S-3226 or HOE-694 exhibited equivalent reductions in mucosal-to-serosal fluxes of 22Na+ (by approximately 35%) compared with nontreated ischemia-injured control tissues (P < 0.05). Intestinal ischemia resulted in increased expression of the cytoplasmic NHE regulatory factor EBP50 in NHE2 but not in NHE3 immunoprecipitates. Selective inhibition of NHE2, and not NHE3, induces recovery of barrier function in the ischemia-injured intestine.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.