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.     

Ionic exchanges in isolated and open-circuited toad skin

Summary Net influxes of Na and Cl and effluxes of K and H (J _Na,J _Cl,J _K andJ _H) and volume flowJ _v across isolated open-circuited toad skins were measured using rotating chambers and a small volume of external solution, the ion fluxes being determined by chemical analysis of the external solution, in the range of 0.2 to 5.0mm external Na concentration. In this concentration range, with skin potential varying with (Na) _e ,J _Na is a linear function of the Na electrochemical potential difference across the skin, as expected on irreversible thermodynamic grounds. TheL _Na coefficient calculated asJ _Na/ _Na is equal to 5.5×10^–12 mole² joule^–1 cm^–2 min^–1, which is similar to values obtained for the same species in the short-circuited state and in higher ranges of (Na) _e . A positive correlation is observed betweenJ _Na andJ _K whenJ _Na varied with (Na) _e and also whenJ _Na varies in randomly selected skins. Antidiuretic hormone stimulatesJ _Na,J _K andJ _v in the range of 0.2 to 5.0mm (Na) _e and lowers the Na concentration in the equivalent solution absorbed by the skin (calculated asJ _Na/J _v ). Substitution of external Cl by SO₄ has no effect onJ _Na,J _K andJ _H and also in the skin potential in the range of (Na) _e studied. Substitution of external Na by K abolishesJ _Cl and reverses the skin polarity, the external solution now being positive to the internal one. Na removal from the external solution also reducesJ _K almost to zero.J _H is significantly reduced in this condition; however, a basal secretion still persists with (Na) _e equal to zero. The results of these experiments can be tentatively interpreted in terms of electrical coupling between ion fluxes, since only the procedures that result in alterations of skin potential are followed by changes in the rates of ion transport. The existence of other coupling mechanisms cannot be ruled out.     

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.     

Transient potassium fluxes in toad skin

Summary Experiments were carried out in the isolated short-circuited skin of the toadBufo marinus ictericus.⁴²K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 ±0.36 nmol cm^–2 hr^–1 (n=7) and efflux 3.62±0.38 nmol cm^– hr^–1 (n=7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of⁴²K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10^–4 m) to the outer solution, a sharp decline is observed in the rate of⁴²K discharge into the bathing solution,J ₂₁ ^K , which falls from 3.62±0.38 nmol cm^–2 hr^–1 to 2.02±0.04 nmol cm^–2 hr^–1 (n=7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline inJ ₂₁ ^K which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase inJ ₂₁ ^K and a linear relationship was observed betweenJ ₂₁ ^K and short-circuit current in the range of 0 to 115mm external Na concentration. The decline inJ ₂₁ ^K induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of⁴²K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of⁴²K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10^–3 m) to the inner bathing solution induces a transient rise in the rate of⁴²K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior ofJ ₂₁ ^K upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 11 Na–K pump.     

Sulfate transport in rabbit ileum: Characterization of the serosal border anion exchange process

Summary The preceding paper [30] shows that transepithelial ileal SO₄ transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO₄ was present at 0.22mm. In standard Cl, HCO₃ Ringer's,J _ms ^SO4 was 81.3±5.3 (1se) andJ _ms ^SO4 was 2.5±0.2 nmol cm^–2 hr^–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO₄ transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO₄ fluxes. In contrast to its effect on SO₄ transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO₃ and PO₄ with gluconate reducedJ _ms ^SO4 by 70% and increasedJ _ms ^SO4 by 400%. A small but significantJ _net ^SO4 remained.J _ms ^SO4 could be increased by addition to the serosal side of Cl, Br, I, NO₃ or SO₄. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ _ms ^SO4 in the presence of Cl was considerably higher than in the presence of SO₄ (73.1 and 42.2 nmol. cm^–2 hr^–1, respectively;p<0.001). TheK _1/2 value for Cl was 7.4mm, 10-fold higher than that for SO₄ (0.7mm). Omitting HCO₃ and PO₄ had no measurable effects on SO₄ fluxes.This study shows that (i) SO₄ crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO₄ may exchange for Cl, Br, I, NO₃ and SO₄ itself, but probably not for HCO₃ or PO₄; (iv) kinetics of the exchange system suggest there is a greater affinity for SO₄ than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport.     

Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

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.     

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.     

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.     

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.     

Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport

Summary Activation of protein kinase C has been shown to cause both stimulation and inhibition of transport processes in the brush-border membrane and renal tubule. This study was designed to examine the dose-response nature and time-dependent effect of 4 -phorbol-12-myristate-13-acetate (PMA) on the rates of bicarbonate absorption (J _HCO3) and fluid absorption (J _v) in the proximal convoluted tubule (PCT) of rat kidney. Bicarbonate flux was determined by total CO₂ changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of PMA (10^–10 10^–5 M) within 10 min caused a significant increase ofJ _HCO3 andJ _v. A peaked curve of the dose response was observed with maximal effect at 10^–8 M PMA on both bicarbonate and fluid reabsorption, which could be blocked completely by amiloride (10^–3 m) and EIPA (10^–5 M). On the other hand, with an increase of perfusion time beyond 15 min, PMA (10^–8 and 10^–6 M) could inhibitJ _HCO3 andJ _v. Amiloride (10^–3 M) or EIPA (10^–5 M) significantly inhibitsJ _HCO3 andJ _v, while there is no additive effect of PMA and amiloride or EIPA on PCT transport. An inactive phorbol-ester, 4-phorbol, that does not activate protein kinase C, had no effects onJ _HCO3 andJ _v. Capillary perfusion of PMA (10^–8 M) significantly stimulate bothJ _HCO3 andJ _v; however, PMA did not affect glucose transport from either the luminal side or basolateral side of the PCT. These results indicate that activation of endogenous protein kinase C by PMA could either stimulate or inhibit both bicarbonate and fluid reabsorption in the PCT dependent on time and dose, and these effects are through the modulation of Na⁺/H^– exchange mechanism.     

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.     

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.     

Function and regulation of the avian caecal bulb: influence of dietary NaCl and aldosterone on water and electrolyte fluxes in the hen (Gallus domesticus) perfused in vivo

Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na⁺ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg^–1 body mass) were also measured.Evidence was found for primary active net absorption of Na⁺, inducing parallel Na-linked absorption of water and Cl^– and secretion of K⁺. Around 20–35% of total Cl^– absorption and K⁺ secretion were independent of Na⁺ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10^–3 (G _Cl) and 1.1×10^–3 (G _K) S·cm^–2.Acetate (40mM) stimulated Na⁺ fluxes (8.5–9.9 Eq·cm^–2·h^–1) and Na-linked water fluxes (27–44 l·cm^–2·h^–1). Increased coupling ratios (2.9–4.6 l·Eq^–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na⁺ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J _Na (6.8–11.6);J _K (–3.2––4.3);J _Cl (4.3–5.6 Eq·cm serosal area^–2·h^–1);J _v (28–43 l·cm^–2·h^–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ _Na (10.8–14.0 Eq·cm^–2·h^–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ _K orJ _Cl.Abbreviations ECPD, EPD Electrochemical or electrical potential difference - G _Cl ,G _K ionic conductances (Cl^–, K⁺) - J _v ,J _ion net volume or ion flux rate, mucosa-serosa positive;P _d (Cl) diffusive permeability coefficient (of Cl^–) - SEDM standard error of difference between means     

Cell sodium activity and sodium pump function in frog skin

Summary Cell Na activity,a _Na ^c , was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO₃ Ringer on the apical surface. Under control conditionsa _Na ^c averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda _Na ^c to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina _Na ^c and cell current,I _c . On restoring Na after several minutes of exposure to apical Na-free solutionI _c rose rapidly to a stable value whilea _Na ^c increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa _Na ^c indicates that the pump Na flux is linearly related toa _Na ^c in the range 2–12mm. These results indicate thata _Na ^c plays an important role in relating apical Na entry to basolateral active Na flux.     

Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts

Ion and acid–base balance were examined in the freshwater-adapted mummichog (Fundulus heteroclitus) using a series of treatments designed to perturb the coupling mechanisms. Unidirectional Cl⁻ uptake (J^Cl_in) was extremely low whereas J^Na_in was substantial (three- to sixfold higher); comparable differences occurred in unidirectional efflux rates (J^Cl_out, J^Na_out). J^Cl_in was refractory to all treatments, suggesting that Cl⁻/base exchange was unimportant or absent. Indeed, no base excretion or modulation of ion fluxes occurred for acid–base balance for up to 8 h after NaHCO₃⁻ loading (injections of 1000 or 3000 nequiv.·g⁻¹). Acute environmental low pH (4.5) and amiloride (10⁻⁴ M) treatments caused concurrent inhibition of J^Na_in and net H⁺ excretion (J^H+_net), indicating the presence of Na⁺/H⁺ exchange. J^Na_in was elevated and J^H+_net restored during recovery from both treatments, but this exchange did not appear to be dynamically adjusted for acid–base homeostasis. High external ammonia exposure (1 mmol·l⁻¹) initially blocked ammonia excretion (J^Amm_net) but had no effect on J^Na_in, whereas high pH (9.4) reduced both J^Amm_net and J^Na_in. Inhibition of J^Na_in by the low pH and amiloride treatments had no effect on J^Amm_net. These results indicate that ammonia excretion is entirely diffusive and independent of both Na⁺uptake and the protons that are transported via the Na⁺/H⁺ coupling. In addition, ureagenesis served as a compensatory mechanism during high external ammonia exposure, as a marked elevation in urea excretion partially replaced the inhibited J^Amm_net. In all treatments, changes in the Na⁺–Cl⁻ net flux differential were consistent with changes in J^H+_net measured by traditional water titration techniques, indicating that the former can be used as an estimate of the acid–base status of the fish. Overall, the results demonstrate that the freshwater-adapted F. heteroclitus does not conform to the ion/acid–base relationships described in the standard model based on commonly studied species such as trout, goldfish, and catfish.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

Transients in toad skin: Short circuit current and ionic fluxes related to inner sodium substitution by monovalent cations

Summary When the Na electrochemical potential difference across the skin (_Na) is altered by perturbing the transmembrane electrical potential difference or the external Na concentration, effects on transport and associated oxygen consumption can be described by the formalism of linear nonequilibrium thermodynamics (Vieira, Caplan & Essig, 1972,J. Gen. Physiol. 59:77; Danisi & Lacaz-Vieira, 1974,J. Gen. Physiol. 64:372; Procópio and Lacaz-Vieira, 1977,J. Membrane Biol. 35:219). We now show that with modifications of _Na by substitution of Li or choline for Na in the inner bathing solution, this formalism is no longer applicable. Inner Na by K substitution ((Na×K) _i ) causes profound alterations in short-circuit current (SCC),J _Na ⁱⁿ , K efflux (J _K ^eff ) and PD. SCC drops transiently after (Na×K) _i in Cl and in SO₄ media, increasing subsequently. In Cl medium, following the initial transient, there is a late decline in SCC toward a steady state. The rate of SCC decline in Cl medium is more pronounced than that observed in SO₄ medium. (Na×K) _i causes a transient increase inJ _Na ⁱⁿ with a peak synchronous to the minimum in SCC, both in Cl and in SO₄ media. This was interpreted as due to depolarization of the inner membrane. In SO₄ medium, following the peak observed after (Na×K) _i J _Na ⁱⁿ drops, to increase again toward a steady state in which SCC andJ _Na ⁱⁿ are not statistically different, resembling the control condition before (Na×K) _i . In Cl medium, however, theJ _Na ⁱⁿ steady state is approximately 100% higher than SCC. This difference is due to an important K efflux (J _K ^eff ), which builds up progressively after the substitution. The apparent K permeability [J _K ^eff /(K _i )] is of comparable magnitude in Cl and in SO₄ media before (Na×K) _i , the apparent K permeability increases one order of magnitude as compared to the control condition before the ionic substitution. In Cl medium, the high levels ofJ _Na ⁱⁿ and ofJ _K ^eff observed in the steady state after (Na×K) _i were interpreted as being a consequence of cell swelling. SCC and PD follow very different temporal patterns after (Na×K) _i which are characterized by transients in SCC and a simple fall in PD. Reasons for these differences are discussed.