Na and Cl transport and short-circuit current in rabbit ileum

Summary Na and Cl fluxes and short-circuit current (I _sc) in rabbit ileum have been studied as a function of ionic concentrations in HCO₃-free solutions. Both net Na flux (J _net ^Na ) andI _sc show similar saturation functions of [Na] at fixed [Cl]. They show no significant difference between zero and 112mm Na but at 140mm NaI _sc is significantly greater than theJ _net ^Na . Net Cl transport, secretion, is observed only at 140mm Na and is approximately equivalent to the difference between theI _sc andJ _net ^Na . The transcellular mucosa-to-serosa Na fluxes measured at 140 and 70mm Na do not differ significantly from the correspondingI _sc. The net Cl flux varies with [Cl] at fixed [Na] whileI _sc is virtually not affected by [Cl]. These results suggest that the absorptive Na transport process is electrogenic and responsible for theI _sc and that the secretory fluxes of Na and Cl are coupled, require high [Na], vary with [Cl], and do not contribute toI _sc. K-free solution abolishes theI _sc after a prolonged lag. Finally, the effect of a low resistance shunt pathway on active Na absorption is examined with a four-compartment model.Deceased (October 16, 1974).     

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.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana Pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.Reprinted from The Journal of Membrane Biology, Vol. 54, No. 3, pages 191–202. Our apologies for deleting the author's names on the original version.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

Ion transport by rabbit colon

Summary Descending rabbit colon, stripped ofmuscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO₃ secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit currentI _sc is determined solely by the rate of active Na transport and that the net movements of Cl and HCO₃ are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO₃ secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxesJ _sm ⁱ of Na, K and Cl suggest thatJ _sm ^Na ,J _sm ^K and one-third ofJ _sm ^Cl may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such thatP _KP _NaP _Cl=1.00.070.11. Electrolyte transport byin vitro rabbit colon closely resembles that reported fromin vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.     

Transepithelial transport in cell culture: Stoichiometry of Na/phlorizin binding and Na/d-glucose cotransport. A two-step,two-sodium model of binding and translocation

Summary The renal cell line LLC-PK₁ cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I _sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm^–2hr^–1, andI _sc, 0.50±0.50(8) eq cm^–2hr^–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI _sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI _sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation.     

Active sulfate absorption in rabbit ileum: Dependence on sodium and chloride and effects of agents that alter chloride transport

Summary Unidirectional fluxes of³⁵SO₄ across and into rabbit ileal epithelium were measured under short-circuit conditions, mostly at a medium SO₄ concentration of 2.4mm. Unidirectional mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) were 0.456 and 0.067 moles hr^–1 cm^–2, respectively.J _ms was 2.7 times higher in distal ileum than in mid-jejunum. Ouabain abolished net SO₄ transport (J _net) by reducingJ _ms. Epinephrine, a stimulus of Cl absorption, had no effect on SO₄ fluxes. Theophylline, a stimulus of Cl secretion, reducedJ _ms without affectingJ _sm, causing a 33% reduction inJ _net. Other secretory stimuli (8-Br-cAMP, heat-stable enterotoxin, Ca-ionophore A23187) had similar effects. Replacement of all Cl with gluconate markedly reducedJ _net through both a decrease inJ _ms and an increase inJ _sm. The anion-exchange inhibitor, 4-acetoamido-4-isothiocyano-2,2-sulfonic acid stilbene (SITS), when added to the serosal side, reducedJ _ms by 94%, nearly abolishingJ _net. SITS also decreasedJ _sm by 75%. Mucosal SITS (50 m) was ineffective. 4,4-diisothiocyano-2,2-sulfonic acid stilbene (DIDS) had effects similar to SITS but was less potent. Measurements of initial rates of epithelial uptake from the luminal side (J _me) revealed the following: (1)J _me is a saturable function of medium concentration with aV _max of 0.94 moles hr^–1 cm^–2 and aK _1/2 of 1.3mm; (2) replacing all Na with choline abolishedJ _me; (3) replacing all Cl with gluconate increasedJ _me by 40%; (4) serosal SITS had no effect onJ _me; and (5) stimuli of Cl secretion had no effect onJ _me or increased it slightly. Determination of cell SO₄ with³⁵SO₄ indicated that, at steady-state, the average mucosal concentration is 1.1 mmoles per liter cell water, less than half the medium concentration. Cell SO₄ was increased to 3.0mm by adding SITS to the serosal side. Despite net transport rates greater than 1.4 Eq hr^–1 cm^–2, neither addition of SO₄ to the SO₄-free medium nor addition of SITS to SO₄-containing medium altered short-circuit current. The results suggest that (1) ileal SO₄ absorption consists of Na-coupled influx (symport) across the brush border and Cl-coupled efflux (antiport) across the basolateral membrane; (2) the overall process is electrically neutral; (3) the medium-to-cell Cl concentration difference may provide part of the driving force for net SO₄ absorption; and (4) since agents affecting Cl fluxes (both absorptive and secretory) have little effect on SO₄ fluxes, the mechanisms for their transcellular transports are under separate regulation.     

Ion transport by rabbit colon: II. Unidirectional sodium influx and the effects of amphotericin B and amiloride

Summary The unidirectional influx of Na from the mucosal solution into the epithelium ofin vitro descending rabbit colon (J _me ^Na ) determined under short-circuit conditions, is comprised of two components: one represents entry of Na into transporting epithelial cells and is abolished by amiloride which also abolishes Na absorption (J _net ^Na ). The other represents diffusional Na entry into paracellular pathways traversing the epithelium. In all instances, exposure of the mucosal surface to amphotericin B increased tissue conductance andJ _me ^Na and elicited K secretion. Tissues showing a spontaneousI _sc of approximately 4 eq/cm²hr did not respond to amphotericin B with increasedI _sc andJ _net ^Na . However, in tissues characterized by a lowerI _sc under control conditions, amphotericin B increasedI _sc andJ _net ^Na to approximately 4eq/cm²hr. These findings suggest that amphotericin increasesJ _net ^Na and elicits K secretion by disrupting the normal permselectivity of the mucosal membrane. Under these conditions the extrusion of Na from cell-to-serosal solution becomes the rate limiting step in transepithelial Na transport. Finally, a close correlation betweenJ _me ^Na andJ _net ^Na was observed when the rate of Na absorption varied either spontaneously or experimentally with amiloride, suggesting that the backflux of Na from cell-to-mucosal solution is undetectably small.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

Summary The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na⁺-enriched cells. Subsequent incubation in K⁺-containing NaCl medium results in the reaccumulation of K⁺, Cl^–, water and the extrusion of Na⁺. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (K _M for K ₀ ⁺ =3.5mm;J _max=30.1 mEq/kg dry min), which in these experiments was coupled 1K⁺/1 Na⁺. The second is the Cl^–-dependent (Na⁺+K⁺) cotransport system (K _M for K ₀ ⁺ =6.8mm;J _max=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K⁺1Na⁺2Cl^–, the exchange of K _i ⁺ for K ₀ ⁺ . The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Sulfate influx across the rabbit ileal brush border membrane: Sodium and proton dependence,and substrate specificities

Summary In intact ileal mucosa, uptake of SO₄ across the brush border membrane requires the presence of Na and is saturable, withK1/2=1.3mm at 140mm Na (P.L. Smith, S.A. Orellana & M. Field, 1981.J. Membrane Biol. 63:199–206). The present study examines the substrate specificities and transport stoichiometry of the Na-dependent SO₄ uptake process. The effects of variations in medium anion and cation composition on lumen-to-epithelium influx of SO₄ (J _me ^SO4 ) were determined under short-circuit conditions.J _me ^SO4 is inhibited by thiosulfate, but not by phosphate, methylsulfate, vanadate or taurocholate. Cl is weakly inhibitory. Uptake of SO₄ is poorly supported by Li, and is unaffected by K, indicating a specific dependence on Na. At low SO₄ concentration (0.22mm),J _me ^SO4 is a hyperbolic function of medium Na concentration; the corresponding Hill plot is linear with a slope of 1.0, suggesting a transport stoichiometry of 1 Na: 1 SO₄. At high SO₄ concentration (6.7mm), the Na-dependent SO₄ velocity curve is sigmoidal and yields a Hill plot which is again linear but has a slope of 1.56, suggesting transport of more than 1 Na per SO₄. SO₄ uptake in presence of Na exhibits a dependence on medium pH. At 0.22mm SO₄ and 140mm Na,J _me ^SO4 was doubled by lowering pH from 7.4 to 6.8. However, at 6.7mm SO₄ and 140mm Na, changing pH had no effect onJ _me ^SO4 over the range 6.8 to 8.5. The pH dependence ofJ _me ^SO4 at 6.7mm SO₄ was restored when medium Na was lowered to 3mm, suggesting that pH sensitivity is a function of the concentration of preformed NaSO ₄ ^– ion pair. The results suggest that SO₄ influx across the ileal brush border occurs by electroneutral Na⁺/NaSO ₄ ^– or Na⁺/H⁺/SO ₄ ^2– cotransport, the former being favored by high concentrations of Na and SO₄.     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Kinetics of sodiumd-glucose cotransport in bovine intestinal brush border vesicles

Summary Brush border membrane vesicles (BBMV) purified from steer jejunum were used to study the kinetics of sodiumd-glucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport (J _gluc) was measured over a wide range of glucose concentrations ([S]=0.01–20mm), curvature of the Woolf-Augustinsson-Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rateJ _max=5.8–8.8 nmol/mg·min) saturable system. Further studies indicated that changes incis [Na] altered theK _t , but not theJ _max, suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first.Trans sodium inhibitedJ _gluc hyperbolically. KCl-valinomycin diffusion potentials, inner membrane face positive, loweredJ _gluc, while potentials of the opposite polarity raiseJ _gluc. At low glucose concentrations ([S]<0.05mm), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by sodium activation curves, which were hyperbolic when [S]=0.5mm, but were sigmoidal when [S]=.0.01mm. Simultaneous fluxes of²²Na and [³H]glucose at 1mm glucose and 30mm NaCl yielded a cotransport-dependent flux ratio of 21 sodium/glucose, suggestive of 11 (Na/glucose) high capacity, low affinity system and a 31 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Na-dependent saturable systems. Extravesicular (cis) Na changed theK _t , but not theJ _max of the major system.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

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.     

Mechanism of Cl secretion in canine trachea: Changes in intracellular chloride activity with secretion

Summary Cl-sensitive microelectrodes were employed to investigate the mechanism of Cl secretion by canine tracheal epithelium. In control tissues with a mean calculated short-circuit current (I _sc) of 18.1 A/cm², the intracellular Cl activity (a _Cl ⁱ ) was 47.2mm. This value is 30.1mm (or 27.0 mV) above the electrochemical equilibrium for Cl across the apical membrane. Epinephrine, which stimulates Cl secretion, increased the calculatedI _sc to 160 A/cm² and decreaseda _Cl ⁱ to 32.2mm, a value only 11.2mm (or 10.9 mV) above equilibrium for the apical membrane. These results indicate a secretagogue induced decrease in the impedance to Cl exit from the cell via the apical membrane. From these and prior measurements we calculate that epinephrine-induced Cl efflux from the cell can occur by simple diffusion across the apical membrane. Further implications of these calculations are also discussed.     

The steady-state relationship between sodium and chloride transmembrane electrochemical potential differences inNecturus gallbladder

Summary Intracellular C1, K and Na activities (a _Cl ⁱ ,a _k ⁱ anda _Na ⁱ ) and transmucosal membrane potential (E _m) in epithelial cells ofNecturus gallbladder were measured at different external Na concentrations ([Na]_o), with liquid ion-exchanger and conventional microelectrodes. Bladders were mounted in a divided chamber at 23°C between identical HCO₃-free Ringer solutions containing 5mm K. The pH was 7.2. Tris was substituted for Na. Measurements were made under steady-state conditions as determined by the constancy of the transepithelial potential difference. Both,a _Cl ⁱ anda _Na ⁱ increased in a saturable fashion with [Na]_o.E _m did not change significantly. Average values (±sem) under normal conditions ([Na]_o=100mm) fora _Cl ⁱ ,a _Na ⁱ andE _m were 16.8±0.8mm (n=9), 9.7±0.6mm (n=10) and –52.6±0.6 mV (n=26), respectively. In Na-free mediaa _Cl ⁱ declined to its equilibrium value.a _K ⁱ (96±2mm;n=7) did not change when [Na]_o was varied between 100 and 10mm but decreased to 80±3mm (n=4) in Na-free media.Transmembrane electrochemical potential differences, , for Cl and Na were calculated at four different [Na]_o levels. A highly significant linear relation between and was found, indicating that Cl and Na transport are energetically linked. The results support the view that the energy necessary for intracellular Cl accumulation is derived from the simultaneous dissipation of the chemical potential gradient of Na across the apical membrane and that the coupled entry mechanism is electroneutral.     

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

Summary We evaluated the effects of vawrying aqueous Cl^– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl^– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl^– concentrations and/or PGO on the relation between holding voltageV _H -mV) and open-time probability (P _o).Reducingcis Cl^– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P _v) with a half-maximal reduction inP _o atcis Cl^–170mM. Channel activity was sustained by equimolar replacement ofcis Cl^– with F^–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl^– concentration andP ₀ at 10mm Cl^–. Reducingcis Cl^– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl^– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl^– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl^– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl^– channels.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .