Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

Passive sodium movements across the opercular epithelium: The paracellular shunt pathway and ionic conductance

Summary The unidirectional Na⁺, Cl^–, and urea fluxes across isolated opercular epithelia from seawater-adaptedFundulus heteroclitus were measured under different experimental conditions. The mean Na⁺, Cl^–, and urea permeabilities were 9.30×10^–6 cm·sec^–1, 1.24×10^–6 cm·sec^–1, and 5.05×10^–7 cm·sec^–1, respectively. The responses of the unidirectional Na⁺ fluxes and the Cl^– influx (mucosa to serosa) to voltage clamping were characteristic of passively moving ions traversing only one rate-limiting barrier. The Na⁺ conductance varied linearly with, and comprised a mean 54% of, the total tissue ionic conductance. The Cl^– influx and the urea fluxes were independent of the tissue conductance. Triaminopyrimidine (TAP) reduced the Na⁺ fluxes and tissue conductance over 70%, while having no effect on the Cl^– influx or urea fluxes. Mucosal Na⁺ substitution reduced the Na⁺ permeability 60% and the tissue conductance 76%, but had no effect on the Cl^– influx or the urea fluxes. Both the Na⁺ and Cl^– influxes were unaffected by respective serosal substitutions, indicating the lack of any Na⁺/Na⁺ and Cl^–/Cl^– exchange diffusion.The results suggest that the unidirectional Na⁺ fluxes are simple passive fluxes proceeding extracellularly (i.e., movement through a cation-selective paracellular shunt). This pathway is dependent on mucosal (external) Na⁺, independent of serosal (internal) Na⁺, and may be distinct from the transepithelial Cl^– and urea pathways.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

Summary The Ehrlich tumor cell possesses and anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl^–+Na⁺ and/or K⁺) is Cl^–-selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na⁺ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na⁺, ouabain-insensitive K⁺ and DIDS-insensitive Cl^– transport (KCl cotransport) are low and equivalent to that found in 150mm Na⁺ medium containing furosemide. Increasing the [Na⁺] results in parallel increases in K⁺ and Cl^– transport. The maximum rate of each (18 to 20 meq/(kg dry wt)·min) is reached at about 20mm Na⁺ and is maintained up to 55mm. Thus, over the range 1 to 55mm Na⁺ the stoichiometry of KCl cotransport is 11. In contrast to K⁺ and Cl^–, furosemide-sensitive Na⁺ transport is undetectable until the [Na⁺] exceeds 50mm. From 50 to 150mm Na⁺, it progressively rises to 7 meq/(kg dry wt)·min, while K⁺ and Cl^– transport decrease to 9 and 16 meq/(kg dry wt)·min, respectively. Thus, at 150mm Na⁺ the stoichiometric relationship between Cl^–, Na⁺ and K⁺ is 211. These results are consistent with the proposal that the Cl^–-dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na⁺].     

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

Summary 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 P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- 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.     

Effects of ouabain on chloride movements across the seawater eel intestine

Summary 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.     

Steady states and the effects of ouabain in theNecturus gallbladder epithelium: A model analysis

Summary A simple numerical model for theNecturus gallbladder epithelium is presented. K⁺, Na⁺ and Cl^– cross the mucosal and serosal membranes as well as the junctions by means of electrodiffusion; furthermore the mucosal membrane contains a neutral entry mechanism for NaCl and the serosal membrane contains an active pump for K⁺ and Na⁺. The values which have been used for the model are taken from the literature. The model can only attain steady states if the resistance of the serosal membrane is lower than 1000 cm². Values reported in the literature for the resistance of this membrane vary from about 3000 to about 100 cm². We shall argue, however, that the higher estimates are in error because they are derived from a model of the tissue in which each membrane and the junction are modeled by a resistor; this procedure is invalid because the resistance of the lateral intercellular space relative to the resistance of the tight junctions is neglected and consequently the resistance of the serosal membrane is overestimated by a factor of about four. Apart from predicting a realistic steady state at normal external concentrations the model can predict quantitatively several experimental results obtained from the living epithelium. We have focused on the experiments which test the permeabilities of the serosal membrane and the properties of the pump:i) Replacement of serosal Cl^– by an impermeant ion.ii) Replacement of serosal K⁺ by Na⁺.iii) Inhibiting the (Na⁺, K⁺)-pump. The best correspondence between model and experiments is obtained when the pump is assumed to be electrogenic (or rheogenic) with a ratio of coupling between Na⁺ and K⁺ of 32. In this case both model and direct experiments (also presented in this paper) show an initial abrupt depolarization of 6 to 7 mV. The model also shows that it cannot be concluded fromi andii that the Cl^– permeability of the serosal membrane is low. The model explains, even with high passive Cl^– permeabilities, why the intracellular Cl^– concentration is relatively unaffected by paracellular currents, a fact which in other epithelia has been taken as an implication of a low Cl^– permeability of the serosal membranes.     

Taurine transport by rabbit kidney brush-border membranes: Coupling to sodium,chloride, and the membrane potential

Summary Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na⁺-d-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na⁺ gradient (K⁺ diffusion potential) and could be completely inhibited by other -amino acids. It displayed a specific anion requirement (Cl^–Br^–SCN^–>I^–>NO ₃ ^– ). At chemical Na⁺ equilibrium, Cl^– gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl^– gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl^– as well as Br^–. The Cl^– stoichiometry was found to be one, whereas taurine transport, in the presence of Cl^–, was sigmoidally related to the Na⁺ concentration, resulting in a coupling ratio of 2 to 3 Na⁺: 1 taurine. Upon Cl^– replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na⁺ concentration (up to 150mm). These results suggest a 2 to 3 Na⁺:1 Cl^–:1 taurine cotransport mechanism driven mainly by the Na⁺ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl^– appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.     

Effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout,Salmo gairdneri

Summary Daily intramuscular injection of cortisol (4 mg kg^–1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm² of filament epithelium. Concomitantly, whole body influxes of sodium (Na⁺) and chloride (Cl^–) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na⁺ or Cl^– influx. A significant correlation was observed between both Na⁺ and Cl^– influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na⁺ and Cl^– transport capacity. We conclude that chronic cortisol treatment enhances whole body Na⁺ and Cl^– influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.     

On the interrelations of the Na+-and Cl?-influxes in the pulmonate freshwater snailLymnaea stagnalis

Summary In the freshwater snailLymnaea stagnalis the influxes of Na⁺ and Cl^– were studied at different external concentrations of these ions. The characteristies of the Na⁺- and Cl^–-influxes are similar with respect to saturation kinetics,K _m (0.1 mM) and activation by low-salt adaptation. In short-term experiments the Na⁺- and Cl^–-influxes are independent. Because of the counter-ions (H⁺ and HCO ₃ ^– ) involved, this indicates a potential acid-base regulatory capacity. Low-salt adaptation, due to either Na⁺-or Cl^–-depletion, activates both the Na⁺- and the Cl^–-influx. It is suggested that under both conditions the number of active integumental pumps, involved in Na⁺- as well as in Cl^–-uptake, is increased.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

Effect of K+ channels in the apical plasma membrane on epithelial secretion based on secondary active Cl− transport

Summary Models of epithelial salt secretion, involving secondary active transport of Cl^– [9], locate the K⁺ conductance of the plasma membrane exclusively in the basolateral membrane, although there is considerable experimental evidence to show that many secretory epithelia do have a significant apical K⁺ conductance. We have used an equivalent circuit model to examine the effect of an apical K⁺ conductance on the composition and flow rate of the fluid secreted by an epithelium in which secretion is driven by the secondary active transport of Cl^–. The parameters of the model were chosen to be similar to those measured in the dog tracheal mucosa when stimulated with adrenaline to secrete. We find that placing a K⁺ conductance in the apical membrane can actually enhance secretion provided that proportion of the total cell K⁺ conductance in the apical membrane is not greater than about 60%, the enabling effect on secretion being maximal when the proportion is around 10–20%. We also find that even when the entire cell K⁺ conductance is located in the apical membrane, the secreted fluid remains relatively Na⁺ rich. Analysis of the sensitivity of model behavior to the choice of values for the parameters shows that the effects of an apical K⁺ conductance are enhanced by increasing the ratio of the paracellular resistance to the transcellular resistance.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na⁺ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl^– conductance and we show that the sizes of both conductances vary with the Cl^– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl^– concentration rises from 5 to 150 mmol/liter, the size of the inward Na⁺ current declines, the decline being half-maximal when the Cl^– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl^– concentration increases, the inward Cl^– current remains at a constant low level until the Cl^– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl^– in the pipette solution was replaced by other anions indicate that the Na⁺ current is suppressed by intracellular Br^-, Cl^– and NO ₃ ^- but not by intracellular I^-, glutamate or gluconate. Our studies also show that the Cl^– conductance allows passage of Cl^– and Br^- equally well, I-less well, and NO ₃ ^- , glutamate and gluconate poorly, if at all. The findings with NO ₃ ^- are of particular interest because they show that suppression of the Na⁺ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl^– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na⁺ and Cl^– conductances by the cytosolic Cl^– concentration. Since the cytosolic Cl^– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na⁺ and Cl^– conductances may provide a mechanism by which ductal Na⁺ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements.     

Three-year field response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity: Trunk growth and leaf ion accumulation

Irrigated olive is rapidly increasing in arid and semiarid areas, many of which may be negatively affected by soil salinity. We evaluated changes in trunk growth and leaf Cl^–, Na⁺ and K⁺ concentrations in young Arbequina olives (Olea europaea L.) grown in a saline-sodic field over a three-year period. The trunk diameter was measured at the beginning and the end of the 1999 (70 trees), 2000 (59 trees) and 2001 (42 trees) growing periods. Leaves, sampled in August of each year, were analyzed for Cl^–, Na⁺ and K⁺ concentrations. Soil salinity (apparent electrical conductivity, ECa) of each monitored tree was measured 14 times during the 1999–2001 experimental period with an electromagnetic sensor and converted to root zone electrical conductivity of the soil saturation extract (ECe) based on ECa–ECe calibration curves. Salinity tolerance was determined using the Maas and Hoffman threshold–slope response model. Based on salinity thresholds (ECe_thr), the tolerance of olive in terms of trunk growth was high in 1999 (ECe_thr = 6.7 dS m^–1), but declined with age and time of exposure to salts by 30% in 2000 (ECe_thr = 4.7 dS m^–1) and by 55% in 2001 (ECe_thr = 3.0 dS m^–1). Based on the high absolute slopes obtained in all years (values between 16% and 23% dS^–1 m), olive was classified as very sensitive to ECe values above the threshold. Trunk growth thresholds based on leaf ion concentrations varied, depending on years, between 2.6 and 4.0 mg g^–1 (Cl_thr) and between 1.0 and 1.2 mg g^–1 (Na_thr), indicating that Arbequina olive was less sensitive to leaf Cl^– and much more sensitive to leaf Na⁺ than values reported as toxic in greenhouse studies. Leaf K⁺ slightly decreased with increasing salinity, whereas the K⁺/Na⁺ ratio sharply decreased with increasing salinity. We concluded that the initial salinity tolerance of olive was high, but declined sharply with time of exposure to salts and became quite sensitive due primarily to increasing toxic concentrations of Na⁺ in the leaves.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Intracellular and luminal ion concentrations in sea turtle salt glands determined by x-ray microanalysis

Summary The lachrymal salt glands of hatchlings of the green sea turtle (Chelonia mydas) secrete a hyperosmotic (up to 2000 mosmol·kg^–1) NaCl solution. X-ray microanalysis of frozen-hydrated glands showed that during secretion intracellular Na⁺ concentration in the principal cells increased from 13 to 34 mmol·l^–1 of cell water, whilst Cl^– and K⁺ concentrations remained unchanged at 81 mmol·l^–1 and 160–174 mmol·l^–1, respectively. The high Cl^– concentration and the change in Na⁺ concentration are consistent with the prevailing paradigm for secretion by the structurally and functionally similar elasmobranch rectal gland. Concentrations of Na⁺, Cl^– and K⁺ in the lumina of secretory tubules of secreting (Na⁺ 122, Cl^– 167, K⁺ 38 mmol·l^–1) and non-secreting (Na⁺ 114, Cl^–1 174, K⁺ 44 mmol·l^–1) glands were similar and the fluid was calculated to be approximately isosmotic with blood. In the central canals Na⁺ and Cl^– concentrations were similar but K⁺ concentration was lower (11–15 mmol·l^–1). It is concluded that either a high transepithelial NaCl gradient in secretory tubules and central canals is very rapidly dissipated during the short time between gland excision and freezing, or that ductal modification of an initial isosmotic secretion occurs.     

Cl−, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose_in/sucrose_out), while maximal acidification was observed with NaCl_in/KCl_out·K _in ⁺ was a poor substitute for Na _in ⁺ , and Cl _out ^– could be replaced by other anions with the following efficacy of acidification: Cl^–>Br^–>I^–>PO ₄ ^3– >gluconate>SO ₄ ^2– . Flux studies using³⁶Cl^– and²²Na⁺ showed that the vesicles had a permeability for Cl^– and Na⁺, and that ATP-dependent H⁺ pumping was accompanied by a net influx of Cl^– and a net efflux of Na⁺ provided that there was a Na⁺ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H⁺ was countered to about 45% by the Cl^– influx and to about 42% by the Na⁺ efflux. These studies demonstrated that both Cl^– and Na⁺ fluxes are necessary for optimal endocytic vesicle acidification.