Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

Fluxes and compartmentation of K+, Na+ and Cl−, and action of auxins in suspension-cultured Petroselinum cells

Transport of ⁸⁶Rb⁺/K⁺, ²²Na⁺, ³⁶Cl^?, and [³H]indole acetic acid (IAA) has been studied on suspension-cultured cells of the parsley, Petroselinum crispum (Mill) Nym. By compartmental analysis two intracellular compartments of K⁺, Na⁺, and Cl^? have been identified and ascribed to the cytoplasm and vacuole; half-times of exchange were around 200 s and 5 h, respectively. According to the Ussing-Teorell flux equation, active transport is required for the influx into the cytoplasm at the plasmalemma (K⁺, Cl^?) and the tonoplast (K⁺, Na⁺, Cl^?). The plasmalemma permeability pattern, P_K:P_Na:P_Cl=1.00:0.24:0.38, features an increased chloride permeability compared with cells from higher plant tissues. IAA uptake showed an exponential timecourse, was half-maximal after 10 min, and a linear function of the IAA concentration from 10^?9 to 10^?5 M. IAA and 2,4-dichlorophenoxy acetic acid reduce the apparent influx of K⁺, Na⁺, Cl^? during the initial 30 min after addition and subsequently accelerate both in- and efflux of these ions. We discuss that auxins could affect the ion fluxes in a complex way, e.g. by protonophorous activity and by control of the hypothetical proton pump.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

The ionic components of the current pulses generated by developing fucoid eggs.

Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na⁺, Mg²⁺, or SO₄^2?, are not needed for the first 20 min of pulsing. In fact, lowering external Na⁺ or Mg²⁺ (or K⁺) actually stimulates pulsing. Since tracer studies show that Ca²⁺ entry is speeded by Na⁺, Mg²⁺, or K⁺ reduction, these findings suggest that Ca²⁺ entry triggers pulsing. A sevenfold reduction in external Cl^? raises pulse amplitudes by 60%. Moreover, Cl^? is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl^? efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca²⁺ within the growing tip opens Cl^? channels. The resulting Cl^? efflux slightly depolarizes the membrane and thus drives a balancing amount of K⁺ out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca²⁺ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.     

Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll

Ionic mechanisms of salt stress perception were investigated by non‐invasive measurements of net H⁺, K⁺, Ca²⁺, Na⁺, and Cl^? fluxes from leaf mesophyll of broad bean (Vicia faba L.) plants using vibrating ion‐selective microelectrodes (the MIFE technique). Treatment with 90 m M NaCl led to a significant increase in the net K⁺ efflux and enhanced activity of the plasma membrane H⁺‐pump. Both these events were effectively prevented by high (10 m M ) Ca²⁺ concentrations in the bath. At the same time, no significant difference in the net Na⁺ flux has been found between low‐ and high‐calcium treatments. It is likely that plasma membrane K⁺ and H⁺ transporters, but not the VIC channels, play the key role in the amelioration of negative salt effects by Ca²⁺ in the bean mesophyll. Experiments with isotonic mannitol application showed that cell ionic responses to hyperosmotic treatment are highly stress‐specific. The most striking difference in response was shown by K⁺ fluxes, which varied from an increased net K⁺ efflux (NaCl treatment) to a net K⁺ influx (mannitol treatment). It is concluded that different ionic mechanisms are involved in the perception of the ‘ionic’ and ‘osmotic’ components of salt stress.     

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

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

Effect of temperature on ion content, ion fluxes and energy metabolism in Chara corallina

Abstract Effects of temperature on the ionic relations and energy metabolism of Chara corallina were investigated. Measurements were made of the ionic content, tracer ion fluxes, and photosynthetic and dark CO₂ fixation in isolated cells, and of O₂ exchange in photosynthesis and respiration in isolated shoot apices. The total intracellular concentration of K⁺, Na⁺ and Cl^? was the same in cells held for 5 days in non-growing medium at 15°C (the growth temperature) as in those held at 25°C or 5°C. The tracer influx in the light of all ions tested (Rb⁺, Na⁺, CH₃NH₃⁺, Cl^? and H₂PO₄^?) was lower at 5°C than at 15°C in experiments in which cells were subjected to 5°C for less than 24 h in toto. The influx at 25°C was greater than that at 15°C for H₂PO^?₄, there was no difference between the two temperatures for Na⁺, while the influx at 25°C was less than that at 15°C for Cl^?, Rb⁺ and CH₃NH₃⁺ For Cl^? and H₂PO^?₄ similar results were found in later experiments with cells grown at 20—23°C. Photosynthetic CO₂ fixation and O₂ evolution, and respiratory O₂ uptake, are greater at 25°C, and lower at 5°C, than they are at the growth temperature of 15°C. In longer-term pretreatments at the different temperatures, tracer Cl^? influx at 15°C and particularly at 25°C were lower than in short-term experiments, while the influx at 5°C was higher. It was concluded from these experiments, and from previous data on H⁺ free energy differences across the plasmalemma, that (1) the maintenance of internal ion concentrations involves a close balancing of influx and efflux of K⁺, Na⁺ and Cl^? at all experimental temperatures; (2) the regulation of the tracer fluxes of the ions is kinetic rather than thermodynamic and (3) that the tracer fluxes at low temperatures are not restricted by the rate at which respiration or photosynthesis can supply energy to them.     

Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor‐regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (E_m) measurements. Turgor recovery was inhibited by Gd³⁺, tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl‐induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na⁺ but not K⁺ and Cl^? in the incubation media. Na⁺ uptake was strongly decreased by amiloride and changes in net Na⁺ and H⁺ fluxes were oppositely directed. This suggests active uptake of Na⁺ in V. erythrospora mediated by an antiport Na⁺/H⁺ system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K⁺ efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.     

INHIBITION OF CARBON UPTAKE AND STIMULATION OF NITRATE UPTAKE AT LOW SALINITIES IN FUCUS DISTICHUS (PHAEOPHYCEAE)1

Inorganic carbon and nitrate uptake were examined in whole plants of Fucus distichus L. (Powell) incubated in dilutions of synthetic ocean water and media with different concentrations of Na⁺, K⁺ and Cl^?. Reduction in salinity from normal seawater (33 ppt) decreased carbon uptake rate but increased nitrate uptake rate by 50% each. Substitution of K⁺ for Na⁺ at constant ionic strength decreased nitrate uptake. Substitution of K⁺ or mannitol for Na⁺ decreased carbon uptake. Neither the uptake of nitrate or carbon was changed by substituting SO₄^2- for Cl^?. Ionophores, valinomycin and monensin, inhibited both nitrate uptake and carbon fixation from 20 to 70% of control rates. The stimulation of nitrate uptake at low salinity may be beneficial to plants in estuarine tidal environments in which nitrate is supplied by the fresh water source.     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

Effects of Inhibitors of Protein and Nucleic Acid Synthesis on the Development of Ion Uptake Mechanisms in Beetroot Slices (Beta vulgaris)

Mechanisms for the uptake of K⁺, Na⁺ and Cl^- develop sequentially in thin slices of beetroot tissue washed under aerobic conditions. Actinomycin D inhibited or prevented the development of K⁺, Na⁺ and Cl^- uptake mechanisms when added to freshly cut slices, but had no effect on net ion uptake when added after the development of the ion uptake mechanisms. The use of puromycin as a specific inhibitor of protein synthesis was unsatisfactory as it caused leakage of pigments and excessive loss of ions from the disks. Cycloheximide prevented the development of ion uptake mechanisms when added at the start of the experiment, but when added after the development of ion uptake mechanisms its inhibitory effect did not become apparent until after a certain time interval which varied from 3 hours for Cl^- to 25 hours for K⁺ uptake in the same experiment. p-Fluorophenylalanine caused an appreciable shortening of the time required for the development of Na⁺ and K⁺ uptake capabilities, while it completely prevented the development of a Cl^- uptake mechanism. p-Fluorophenylalanine-induced early uptake of Na⁺ and K⁺, however, was followed by periods of net leakage of these ions. It is suggested that the development of ion uptake mechanisms depends on the production of m-RNA, which appears to be relatively stable after its synthesis. The synthesis and decay characteristics of specific proteins required for the ion uptake mechanisms appear to differ for each ion species.     

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Cl− transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl^? transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl^? into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl^? uptake and medium osmolarity and (2) complete release of accumulated Cl^? by treatment with detergent. The rate of Cl^? uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl^? was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA⁺), uptake of Cl^? was minimal; transport was stimulated equally by the substitution of either K⁺ or Na⁺ for TMA⁺. Valinomycin in the presence of K⁺ enhanced further Cl^? uptake, while amiloride reduced Na⁺-stimulated Cl^? uptake towards the minimal level observed with TMA⁺. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na⁺ and K⁺; (2) the membrane permeability to the medium counterion determines the rate of Cl^? uptake; (3) Cl^? transport is not specifically coupled with either Na⁺ or K⁺; and, finally (4) Cl^? crosses the tracheal luminal membrane via an electrogenic transport mechanism.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na⁺/l-glutamate (l-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K⁺ and Cl^?. The presence of 100 mM K⁺ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K⁺ concentration and that in the absence of any sodium gradient, an outward K⁺ gradient was unable to influence the Na⁺/acidic amino acid transport system. It was also found that Cl^? could specifically activate the Na⁺-dependent l-glutamate (l-aspartate) uptake either in the presence or in the absence of K⁺. Also the effect of Cl^? was observed only in the presence of an inward Na⁺ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na⁺ gradient and in the presence of chloride gradient. l-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K⁺ or Cl^? did not show any translocation of net charge.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

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

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996