Modes of operation and variable stoichiometry of the furosemide- sensitive Na and K fluxes in human red cells

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

The Linkage of Light-stimulated Cl Influx to K and Na Influxes in Hydrodictyon africanum

There are reciprocal stimulations of Cl influx by K and Na,and of K and Na influx by Cl, in the light in Hydrodictyon africanum.The component of the K influx which stimulates, and is stimulatedby, Cl, is independent of the ouabainsensitive mechanism forK influx also found in H. africanum. The concentration dependenceof the cation effects on Cl influx and on the Cl-stimulatedportion of their own influxes are similar. The stimulation withK saturates at about 0.3 mM K; that with Na saturates at about2 mM Na. The Cl-dependent portions of the K and Na influxeshave similar responses to changes in photo-synthetic metabolism(far-red illumination, CDMU, and CCCP) as does the light-stimulatedCl influx. This suggests that Cl influx, and the Cl-stimulatedportions of K and Na influxes are both dependent on photosystem2 of photosynthesis, and are less sensitive to the uncouplerCCCP than is ¹⁴CO₂ fixation or the K-Na pump. It would thusappear that the Cl-dependent portions of the K and Na influxesin the light are linked to the cation-stimulated portion ofthe Cl influx. There is no very great change in the electricalcomponent of the inwardly directed passive driving force oncations under conditions in which Cl is being pumped comparedwith those under which it is not. It is not clear whether suchincrease in this driving force as do occur could account quantitativelyfor the increase in the cation influxes associated with Cl transport,or whether chemical coupling must be invoked. In addition tothe Cl-stimulated portions of the cation influxes, there arealso light-stimulated portions of K and Na influx which areindependent of Cl, not associated with the cation regulatingmechanism, and which seem to have a similar linkage to photosynthesisas does the Cl-K-Na pump. Since the light-stimulated portionof the K efflux appears to be similar to this portion of theK influx, these Cl-independent light-stimulated portions ofK and Na influxes are tentatively related to light-induced changesin cation permeability.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

The effect of hypertonic urea solution on Na+ and K+ transport through the smooth muscle membrane.

The aim of this work was to examine the effect of a hypertonic solution (Krebs solution + 290 mM urea) on K+ and Na+ transport. The experiments were carried out on the guinea-pig taenia coli preparations using the method of Na-24 and K-24 loading and washout. The efflux curves were analysed by means of the digital computer technique. The following parameters were determined: efflux rate constant k2, influx rate constant k1, intracellular ion concentration C1 ion flux M and permeability P. Any significant difference between PNa/PK ratio in hypertonic urea and isotonic Krebs solutions was found.     

Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.     

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes

A detailed study of hypertonically stimulated Na-K-2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to better understand the 1 K(+):1 Cl(-) stoichiometry of transport that was previously observed. In this study, we derived the velocity equations for K(+) influx under both rapid equilibrium assumptions and combined equilibrium and steady-state assumptions and demonstrate that the behavior of the equations and curves in Lineweaver-Burke plots are consistent with a model where Cl(-) binds first, followed by Na(+), a second Cl(-), and then K(+). We further demonstrate that stimulation of K(+) movement by K(+) on the trans side is an intrinsic property of a carrier that transports multiple substrates. We also demonstrate that K(+) movement through NKCC1 is strictly dependent upon the presence of external Na(+), even though only a fraction of Na(+) is in fact transported. Finally, we propose that the larger transport of K(+), as compared with Na(+), is a result of the return of partially unloaded carriers, which masks the net 1Na(+):1K(+):2Cl(-) stoichiometry of NKCC1. These data have profound implications for the physiology of Na-K-2Cl cotransport, since transport of K-Cl in some conditions seems to be uncoupled from the transport of Na-Cl.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Passive potassium transport in LK sheep red cells. Modification by N- ethyl maleimide

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5- min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3- substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site.     

Sodium-coupled sugar and amino acid transport in an acidic microenvironment

1. Nutrient transport mechanisms of lobster hepatopancreatic epithelial brush border membrane vesicles (BBMV) are strongly influenced by the acidic nature of the tubular lumen. 2. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport Ki, without affecting JM. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. Increased H+ concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport JM. 5. At pH 4.0 leucine was transported as a cation by two dissimilar carrier systems: a Na-independent process shared by polar amino acids, and an electroneutral Na-2Cl-dependent mechanism shared with non-polar amino acids. 6. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chloride and sodium influx: a coupled uptake mechanism in the squid giant axon

The squid giant axon was internally dialyzed while the unidirectional fluxes of either Cl or Na were measured. The effects of varying the internal or external concentration of either Na or Cl were studied. Chloride influx was directly proportional to the external Na concentration whereas Cl efflux was unaffected by changes of the external Na concentration between 0 and 425 mM. Neither Cl influx nor efflux were affected by changes of internal Na concentration over the range of 8-158 mM. After ouabain and TTX treatment a portion of the remaining Na influx was directly dependent on the extracellular Cl concentration. Furthermore, when the internal Cl concentration was increased from 0 to 150 mM, the influxes of Cl and Na were decreased by 14 and 11 pmol/cm2.s, respectively. The influx of both ions could be substantially reduced when the axon was depleted of ATP. The influxes of both ions were inhibited by furosemide but unaffected by ouabain. It is concluded that the squid axolemma has an ATP-dependent coupled Na-Cl co-transport uptake mechanism.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mg _i ) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO₃ medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO₃) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations ?1 mm. The inhibitory effect of Q was manifested in Cl, but not in NO₃, whereas QD reduced K and Rb fluxes both in Cl and NO₃ media. The mean 50% inhibitory concentration (IC⁵⁰) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mm. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous. This work was supported by a grant from the National Institutes of Health (NIH DK5RO1 37,160).     

Anion/anion exchange in human neutrophils

Of the total one-way chloride fluxes (approximately 1.4 meq/liter cell water X min) in steady state human polymorphonuclear leukocytes bathed in 148 mM Cl media, approximately 70% behaves as self-exchange mediated by a nonselective anion carrier that is not inhibited by stilbene disulfonates. Five properties of this carrier-mediated exchange were investigated: substrate saturation is seen with respect to 36Cl influx as a function of the external Cl concentration [for normal-Cl cells, the apparent Km(Cl) is approximately 22 mM when Cl replaces para-amino- hippurate (PAH) and approximately 5 mM when Cl replaces glucuronate], and with respect to 36Cl efflux as a function of the concentration of internal Cl replacing PAH [apparent Km(Cl) congruent to 35 mM for cells bathed in 148 mM Cl]; there is trans stimulation of 36Cl influx by internal Cl (replacing PAH) with an apparent Km(Cl) congruent to 35 mM, and of 36Cl efflux by external Cl with an apparent Km(Cl) congruent to 22 mM (Cl replacing PAH) or approximately 5 mM (Cl replacing glucuronate); there is substrate competition between Cl and PAH, but the carrier appears devoid of affinity for glucuronate; influxes and effluxes mediated by the carrier are subject to competitive inhibition by extracellular alpha-cyano-4-hydroxycinnamate (CHC), with an apparent Ki congruent to 9 mM in Cl medium or approximately 1 mM in PAH medium (transport of the inhibitor itself is very slow); and internal Cl and external Cl or PAH undergo 1:1 countertransport, which is CHC sensitive. A simple equilibrium-competition model is proposed that accounts for all the extracellular ligand interactions presented for normal-Cl cells. Least-squares values of the carrier's true Michaelis constants for extracellular Cl, PAH, and CHC are 5.03 +/- 0.83, 50.3 +/- 14.9, and 0.29 +/- 0.09 mM, respectively.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Pleural surface fluorescence measurement of Na+ and Cl- transport across the air space-capillary barrier.

We developed a pleural surface fluorescence method to measure Na(+) and Cl(-) transport in perfused mouse lungs. The air space was filled with aqueous fluid containing membrane-impermeant fluorescent indicators of Cl(-) (lucigenin) or Na(+) (Sodium Green). After instillation of a Cl(-)-free solution into the air space, an increase in perfusate Cl(-) concentration from 0 to 30 mM produced a decrease in surface lucigenin fluorescence (6.5%/min) corresponding to Cl(-) influx of 1.0 mM/min. Cl(-) influx was increased to 2.1 +/- 0.3 mM/min by forskolin, and the increase was inhibited by glibenclamide. cAMP-stimulated Cl(-) influx was decreased by 57% in CFTR null mice. After instillation of a Na(+)-free solution into the air space, an increase in perfusate Na(+) concentration from 0 to 30 mM gave increased Sodium Green fluorescence (Na(+) influx of 1.2 mM/min), which increased approximately fivefold after cAMP agonists. Cl(-) and Na(+) transport were not affected in lungs from mice lacking aquaporins AQP1 or AQP5. Our results establish a pleural surface fluorescence method to measure unidirectional Cl(-) and Na(+) flux in intact lung and provide evidence for cAMP-stimulated transcellular Cl(-) and Na(+) transport.     

Lithium transport pathways in human red blood cells

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.