Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.     

Apical Na+ permeability of frog skin during serosal Cl− replacement

Summary Gluconate substitution for serosal Cl^– reduces the transepithelial short-circuit current (I _sc) and depolarizes shortcircuited frog skins. These effects could result either from inhibition of basolateral K⁺ conductance, or from two actions to inhibit both apical Na⁺ permeability (P _Na ^ap ) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na⁺ concentration (C _Na ^c ) andP _Na ^ap have been monitored by measuring the currentvoltage relationship for apical Na⁺ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates ofP _Na ^ap ) and CNa/c were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl^– replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition ofI _sc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations.P _Na ^ap was either unchanged or increased, and CNa/c either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl^– replacement inhibitsP _Na ^ap and Na, K-pump activity. Gluconate substutition likely reduces cell volume, triggering inhibition of the basolateral K⁺ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C Macknight (J. Membrane Biol. 83:119–137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na⁺ entry. The volume-conductance coupling serves to conserve volume by reducing K⁺ solute loss. Its molecular basis remains to be identified.     

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.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled alpha, beta, and gamma subunits of epithelial Na(+) channel (ENaC) showed that, although most of the short-circuit current (I (sc)) was amiloride sensitive (AS-I (sc)), there was also an amiloride-insensitive component (NS-I (sc)) due to Cl(-) secretion (Morris and Schafer, J Gen Physiol 120:71-85, 2002). In the present studies, we observed a progressive increase in NS-I (sc) and a corresponding decrease in AS-I (sc) during experiments. There was a significant negative correlation between AS-I (sc) and NS-I (sc) both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I (sc) could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca(2+)-activated Cl(-) channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I (sc) by 40-60%, with a proportional decrease in NS-I (sc). Addition of 100 muM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I (sc) that was followed by a sustained increase of 50-60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl(-) secretion and the proportional fall in ENaC-mediated Na(+) absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl(-) secretion and Na(+) absorption.     

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.     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: II. Na+ and Cl− effect of inward movements of Cl−

Summary At low concentration (1mm) of Cl^– in the outer solution, the influx of chloride through the isolated skin (J ₁₃ ^Cl ) of the South American frogLeptodactylus ocellatus (L.) seems to be carried by two mechanisms: (i) a passive one that exhibits the characteristics of an exchange diffusion process, and (ii) an active penetration. Studies of the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl ) indicate, that the presence of a high (107mm) concentration of Cl^– in the outer solution activates the translocation of this ion through the cells. Studies of the unidirectional flux of Cl^– across the outer barrier (J ₁₂ ^Cl ) indicate that Na⁺ out stimulates the penetration of Cl^– at this level. Cl^– out, in turn, stimulates, theJ ₁₂ ^Na , but this effect is only detected at low concentrations of Na⁺ out.     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: III. Na+ and Cl− effect on electrical phenomena

Summary During their flux through the skin of the frogLeptodactylus ocellatus, Na⁺ and Cl^– interact with each other. This interaction gives rise to electrical phenomena which are studied in the present paper. The skin is mounted in Na₂SO₄ Ringer's with 115 mM Na⁺ on the inside, and a variety of outer solutions,. The osmolarity of all solutions is kept constant at 237.8 mosmol by adding sucrose. When the main anion used on the outside is SO ₄ ⁼ the electrical potential difference () rises steadily with the concentration of sodium (Na⁺)_o up to 87 mV, which is reached at about 20mm. Thereafter remains constant. When the main anion is Cl^– it is observed that rises steadily with (NaCl)_o with a slope similar to the curve obtained with SO ₄ ⁼ (37 mV per decade), but with a lower intercept attributed to an inward Cl pumping which is characteristic of this frog species. At 2–9 mM (NaCl)_o a Cl-specific channel is activated. Further increases of (NaCl)_o produce a decrease of . The specificity of the activation of this site by monovalent cations and its use by monovalent anions is also studied.     

Properties and distribution of Mg2+-HCO−3-ATPase in brush border membranes isolated from rat small intestine

• 1.1. The small intestine was cut into seven segments and properties and distribution of brush border Mg²⁺-HCO⁻₃-ATPase activity in each segment were examined.
• 2.2. The optimal Mg²⁺ concentration was 1.0 mM.
• 3.3. The optimal HCO⁻₃ concentration was 100 mM in the first (duodenal), 50 mM in the 3rd and 40 mM in the 5th segment, respectively.
• 4.4. The optimal pH value was about 9.0.
• 5.5. l-phenylalanine (above 1 mM) and SCN⁻ (above 50 mM) significantly inhibited both Mg²⁺- and Mg²⁺-HCO⁻₃-ATPase activity.
• 6.6. The enzyme activity was found to be highest in the duodenal segment and then gradually decreased in consecutive segments as well as β-glycerophosphatase, Na⁺-K⁺-ATPase and supernatant carbonic anhydrase.
• 7.7. The functional significance of this ATPase and the relationship with carbonic anhydrase was discussed.

     

Foliar and root absorption of Na+ and Cl− in maize and barley: Implications for salt tolerance screening and the use of saline sprinkler irrigation

Above-canopy sprinkler irrigation with saline water favours the absorption of salts by wetted leaves and this can cause a yield reduction additional to that which occurs in salt-affected soils. Outdoor pot experiments with both sprinkler and drip irrigation systems were conducted to determine foliar ion accumulation and performance of maize and barley plants exposed to four treatments: nonsaline control (C), salt applied only to the soil (S), salt applied only to the foliage (F) and salt applied to both the soil and to the foliage (F+S). The EC of the saline solution employed for maize in 1993 was 4.2 dS m^–1 (30 mM NaCl and 2.8 mM CaCl₂) and for barley in 1994, 9.6 dS m^–1 (47 mM NaCl and 23.5 mM CaCl₂). The soil surface of all pots was covered so that in the F treatment the soil was not salinized by the saline sprinkling and drip irrigation supplied nutrients in either fresh (treatments C and F) or saline water (treatments S and F+S).Saline sprinkling increased leaf sap Na⁺ concentrations much more than did soil salinity, especially in maize, even though the saline sprinkling was given only two or three times per week for 30 min, whereas the roots of plants grown in saline soil were continuously exposed to salinity. By contrast, leaf sap Cl^– concentrations were increased similarly by saline sprinkling and soil salinity in maize, and more by saline sprinkling than saline soil in barley. It is concluded that barley leaves, and to a greater extent maize leaves, lack the ability to selectively exclude Na⁺ when sprinkler irrigated with saline water. Moreover, maize leaves selectively absorbed Na⁺ over Cl^– whereas barley leaves showed no selectivity. When foliar and root absorption processes were operating together (F+S treatment) maize and barley leaves accumulated 11–14% less Na⁺ and Cl^– than the sum of individual absorption processes (treatment F plus treatment S) indicating a slight interaction between the absorption processes. Vegetative biomass at maturity and cumulative plant water use were significantly reduced by saline sprinkling. In maize, reductions in biomass and plant water use relative to the control were of similar magnitude for plants exposed only to saline sprinkling, or only to soil salinity; whereas in barley, saline sprinkling was more detrimental than was soil salinity. We suggest that crops that are salt tolerant because they possess root systems which efficiently restrict Na⁺ and Cl^– transport to the shoot, may not exhibit the same tolerance in sprinkler systems which wet the foliage with saline water. ei]T J Flowers     

Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Nature of the neutral Na+-Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl−/HCO 3 − double exchange,hydrochlorothiazide-sensitive Na+-Cl− symport and Na+-K+-2Cl− cotransport are all involved

Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 4-acetamido-4'-iso-thiocyanostilbene-2,2'-disulfonate (SITS) did not affect fluid absorption, 25 mM SCN- abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3 +/- 1.6 (n = 21) to 8.6 +/- 1.6 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN- or bumetanide. Cl- influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10(-4) M bumetanide, but was slowly inhibited by 10(-3) M HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10(-4) M bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl- influx eliminated. Intracellular Cl- accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10(-3) M HCTZ, reached a minimum within 15-30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent. The results obtained show that Na(+)-Cl- symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport. Thus, the three forms of neutral Na(+)-Cl(-)-coupled transport so far evidenced in epithelia, Na+/H+, Cl-/HCO3- double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na(+)-Cl- symport and bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport, are all present in the apical membrane of rabbit gallbladder.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

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.     

Stoichiometry and ion affinities of the Na−K−Cl cotransport system in the intestine of the winter flounder (Pseudopleuronectes americanus)

Summary Na–K–Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics withK _1/2 values of 5 and 4.5mm and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship withK _1/2 of 20mm and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (I _sc) were also determined. TheK _1/2 for Na was 7mm with a Hill coefficient of 0.9 and theK _1/2 for Cl was 46mm with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na1K2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 21. Therefore Na recycling from serosa to mucosa does not significantly contribute to theI _sc. Addition of serosal ouabain (100 m) inhibited Rb influx, indicating that Na–K–Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na–K-ATPase on the basolateral membrane and the apical Na–K–2Cl cotransporter.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.