Ion Secretion and Isotonic Transport in Frog Skin Glands

The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of ²⁴Na⁺, ⁴²K⁺, and carrier-free ¹³⁴Cs⁺ in paired frog skins bathed on both sides with Ringer's solution, and with 10⁻⁵ m noradrenaline on the inside and 10⁻⁴ m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the ¹³⁴Cs⁺ flux ratio, J ^out _Cs/J ⁱⁿ _Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free ¹³⁴Cs⁺ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of ¹³⁴Cs⁺ indicates that a paracellular flow of water is dragging ¹³⁴Cs⁺ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs⁺ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na⁺ fluxes were measured as well, and it was found that also Na⁺ was subjected to secretion. The ratio of unidirectional Na⁺ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na⁺ and Cs⁺ do not take the same pathway through the glands. The flux ratio of unidirectional K⁺ fluxes indicated active secretion of K⁺. The time it takes for steady-state K⁺ fluxes to be established was significantly longer than that of the simultaneously measured Cs⁺ fluxes. These results allow the conclusion that — in addition to being transported between cells — K⁺ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na⁺, K⁺, Cl⁻ and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na⁺ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na⁺ entering the lateral space from returning to the serosal bath. Thus, Na⁺ is secreted into the outside bath. It has to be assumed then that the Na⁺ permeability of the basement membrane barrier (P ^BM _Na) is smaller than the Na⁺ permeability of the junctional membrane (P ^JM _Na), i.e., P ^JM _Na/P ^BM _Na > 1. The secretory paracellular flow of water further requires that the Na⁺ reflection coefficients (σ_Na) of the two barriers are governed by the conditions, σ^BM _Na > 0, and σ^BM _Na > σ^JM _Na. (iii) Na⁺ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na⁺ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na⁺ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996     

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley

This work investigated the importance of the ability of leaf mesophyll cells to control K⁺ flux across the plasma membrane as a trait conferring tissue tolerance mechanism in plants grown under saline conditions. Four wheat (Triticum aestivum and Triticum turgidum) and four barley (Hordeum vulgare) genotypes contrasting in their salinity tolerance were grown under glasshouse conditions. Seven to 10‐day‐old leaves were excised, and net K⁺ and H⁺ fluxes were measured from either epidermal or mesophyll cells upon acute 100 mM treatment (mimicking plant failure to restrict Na⁺ delivery to the shoot) using non‐invasive microelectrode ion flux estimation (the MIFE) system. To enable net ion flux measurements from leaf epidermal cells, removal of epicuticular waxes was trialed with organic solvents. A series of methodological experiments was conducted to test the efficiency of different methods of wax removal, and the impact of experimental procedures on cell viability, in order to optimize the method. A strong positive correlation was found between plants' ability to retain K⁺ in salt‐treated leaves and their salinity tolerance, in both wheat and especially barley. The observed effects were related to the ionic but not osmotic component of salt stress. Pharmacological experiments have suggested that voltage‐gated K⁺‐permeable channels mediate K⁺ retention in leaf mesophyll upon elevated NaCl levels in the apoplast. It is concluded that MIFE measurements of NaCl‐induced K⁺ fluxes from leaf mesophyll may be used as an efficient screening tool for breeding in cereals for salinity tissue tolerance.     

Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

The pH-dependent fluorescence quenching of acridine orange was used to study the Na⁺- and K⁺-dependent H⁺ fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na⁺-dependent H⁺ flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na⁺ concentration and was competitively inhibited by amiloride (K_i = 0.1 millimolar). The potential-dependent component of H⁺ flux showed an approximately linear dependence on Na⁺ concentration. In contrast, the K⁺-dependent H⁺ flux apparently consisted of a single component which showed an approximately linear dependence on K⁺ concentration, and was insensitive to amiloride. Based on the Na⁺- and K⁺-dependent H⁺ fluxes, the passive permeability of the vesicle preparation to Na⁺ was about half of that to K⁺.

The apparent K_m for Na⁺ of the electroneutral Na⁺/H⁺ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na⁺/H⁺ antiport which can account for the estimated in vivo accumulation ratio for Na⁺ into the vacuole.

     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of sendai virus as fusogen

Bumetanide is a potent diuretic drug which has some structural features in common with furosemide. The steady-state exchange of K⁺ and Cl^? was investigated in Ehrlich ascites tumor cells treated with bumetanide. This agent did not alter the cellular content of K⁺ or Cl^? but the self-exchange of both ions was depressed. K⁺ self-exchange was inhibited by 55% at bumetanide concentrations as low as 10^?6 M. Cl^? self-exchange was less sensitive to this drug but at low concentrations (between 10^?6 and 10^?3 M) bumetanide was a more effective inhibitor of Cl^? transfer than furosemide. The steady-state K⁺ flux of cells equilibrated in NO₃^? media was compared with the K⁺ flux in cells treated with 10^?4 or 10^?3 M bumetanide; the Cl^? -sensitive K⁺ exchange was equivalent to the bumetanide-sensitive K⁺ exchange. Since the results suggested that a bumetanide-sensitive (Cl^?, K⁺) cotransport could be operative in steady-state cells, the stoichiometry of the bumetanide-sensitive fluxes was determined by measuring Cl^? and K⁺ fluxes simultaneously in the same cell suspension. At $\text{5 · 10}{}^{\mathrm{?4}}$ and 10^?3 M bumetanide concentrations, the ratio of these fluxes was $\text{0.98 ? 0.07 (}\text{S.E.}\text{)}\text{and}\text{1.04 ? 0.06}$, respectively, consistent with the postulated cotransport mechanism. At 10^?4 and 10^?5 M, however, the ratio of the bumetanide-sensitive Cl^?/K⁺ flux was significantly less than 1.0. Since the magnitude of the bumetanide-sensitive K⁺ flux at 10^?4 M was close to that of the Cl^?-sensitive flux, a ratio of less than 1.0 at this drug level indicates that Cl^? sensitivity and drug sensitivity may not reflect inhibition of the same process under all circumstances.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Sodium Recirculation and Isotonic Transport in Toad Small Intestine

Isolated small intestine of toad (Bufo bufo) was mounted on glass tubes for perfusion studies with oxygenated amphibian Ringer's solution containing glucose and acetate. Under open-circuit conditions (V _t =−3.9 ± 1.8 mV, N= 14) the preparation generated a net influx of ¹³⁴Cs⁺. The time course of unidirectional ¹³⁴Cs⁺-fluxes was mono-exponential with similar rate constants for influx and outflux when measured in the same preparation. The flux-ratio was time invariant from the beginning of appearance of the tracers to steady state was achieved. Thus, just a single pathway, the paracellular pathway, is available for transepithelial transport of Cs⁺. From the ratio of unidirectional Cs⁺-fluxes the paracellular force was calculated to be, 18.2 ± 1.5 mV (N= 6), which is directed against the small transepithelial potential difference. The paracellular netflux of cesium ions, therefore, is caused by solvent drag. The flux of ¹³⁴Cs⁺ entering and trapped by the cells was of a magnitude similar to that passing the paracellular route. Therefore, independent of the convective flux of ¹³⁴Cs⁺, every second ¹³⁴Cs⁺ ion flowing into the lateral space was pumped into the cells rather than proceeding, via the low resistance pathway, to the serosal bath. It is thus indicated that the paracellular convective flow of ¹³⁴Cs⁺ is driven by lateral Na⁺/K⁺-pumps. Transepithelial unidirectional ⁴²K⁺ fluxes did not reach steady state within an observation period of 70 min, indicating that components of the fluxes in both directions pass the large cellular pool of potassium ions. The ratio of unidirectional ²⁴Na⁺ fluxes was time-variant and declined from an initial value of 3.66 ± 0.34 to a significantly smaller steady-state value of 2.57 ± 0.26 (P < 0.001, N= 5 paired observations), indicating that sodium ions pass the epithelium both via the paracellular and the cellular pathway. Quantitatively, the larger ratio of paracellular Na⁺ fluxes, as compared to that of paracellular Cs⁺ fluxes, is compatible with convective flow of the two alkali metal ions through the same population of water-filled pores. With a new set of equations, the fraction of the sodium flux passing the basement membrane barrier of the lateral space that is recirculated through the cellular compartment is estimated. This fraction was, on average, 0.72 ± 0.03 (N= 5). It is concluded that isotonicity of the transportate can be maintained by producing a hypertonic fluid emerging from the lateral space combined with reuptake of salt via the cells. Received: 14 October 1998/Revised: 14 January 1999     

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.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Stromal pH and Photosynthesis Are Affected by Electroneutral K and H Exchange through Chloroplast Envelope Ion Channels

Potassium movement across the limiting membrane of the chloroplast inner envelope is known to be linked to counterex-change of protons. For this reason, K⁺ efflux is known to facilitate stromal acidification and the resultant photosynthetic inhibition. However, the specific nature of the chloroplast envelope proteins that facilitate K⁺ fluxes, and the biophysical mechanism which links these cation currents to H⁺ counterflux, is not characterized. It was the objective of this work to elucidate the nature of the system regulating K⁺ flux linked to H⁺ counterflux across the chloroplast envelope. In the absence of external K⁺, exposure of spinach (Spinacia oleracea) chloroplasts to the K⁺ ionophore valinomycin was found to increase the rate of K⁺ efflux and H⁺ influx. These data were interpreted as suggesting that H⁺ counterexchange must be indirectly linked to movement of K⁺ across the envelope. Studies using the K⁺ channel blocker tetraethylammonium indicated that K⁺ likely moves, in a uniport fashion, into or out of the stroma through a monovalent cation channel in the envelope. Blockage of K⁺ efflux from the stroma by exposure to tetraethylammonium was found to restrict H⁺ influx, further substantiating an indirect linkage of these cation currents. Further studies comparing the effect of exogenous H⁺ ionophores and K⁺/H⁺ exchangers suggested that K⁺ uniport through this ion channel likely is the main endogenous pathway for K⁺ currents across the envelope. These experiments were also consistent with the presence of a proton channel in the envelope. Movement of H⁺ through this channel was speculated to be regulated and rate limited by an electroneutral requirement for K⁺ countercurrents through the separate K⁺ uniport pathway. K⁺ and H⁺ fluxes across the chloroplast envelope were envisioned to be interrelated via this mechanism. The significant effect of cation currents across the envelope, as mediated by these channels, on photosynthetic capacity of the isolated chloroplast was also demonstrated.     

Effect of Insulin on Potassium Flux and Water and Electrolyte Content of Muscles from Normal and from Hypophysectomized Rats

It was reported previously that insulin hyperpolarized rat skeletal muscle and decreased K⁺ flux in both directions. The observations on K⁺ flux are now extended to take advantage of the greater sensitivity to insulin of hyperphysectomized rats. Insulin caused a shift of water from extracellular to intracellular space if glucose was present, but not in its absence. Insulin caused net gain of muscle fiber K⁺, though not necessarily an increase in K⁺ concentration in fiber water. It probably also decreased intrafiber Na⁺ and Cl^-. Insulin decreased K⁺ efflux. The effect was dose-dependent. Muscles from hypophysectomized rats were more sensitive to the action of insulin on K⁺ flux than were those from normal rats. The effect was demonstrable within the time resolution of the system, suggesting that insulin's action is on cell surfaces. K⁺ influx was also decreased by insulin. Bookkeeping suggests that some K⁺ influx be called active. Insulin seemed to decrease active K⁺ influx and passive K⁺ efflux. It is not resolved whether insulin has a true dual effect or whether it acts only on passive fluxes in both directions (the apparent action on active K⁺ influx being an artefact of incomplete definition of passive flux) or whether a single alteration in the membrane may affect both active and passive fluxes.     

Routes of epithelial water flow: aquaporins versus cotransporters

The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K⁺ and one Cl⁻, as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K⁺ ions in the presence of a transepithelial K⁺ gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P_f by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K⁺-H⁺ exchanger nigericin, indicating that KCC1 affects water transport solely by K⁺ extrusion. Intracellular K⁺ retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.     

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.     

The number of Na:K pump sites on red blood cells from HK and LK lambs

Lambs of known genotype with respect to the locus determining cation composition of red cells were obtained by selective matings. Numbers of K⁺ pump sites per cell were determined on HK and LK lambs 10–20 days postnatal by simultaneously determining [³H]ouabain binding and inhibition of active K⁺ transport. Red cells from HK lambs were indistinguishable from adult HK cells with regard to the K⁺ pump flux and number of pump sites. Cells from genetically LK lambs had pump fluxes and numbers of pump sites intermediate between those from adult HK and LK sheep. The results suggest that the change in cation composition and in the K⁺ pump during the first 60 days in genetically LK lambs can be correlated with a reduced number of K⁺ pump sites.     

Electrochemical gradients and k and cl fluxes in excised corn roots

The compartmental analysis method was used to estimate the K⁺ and Cl⁻ fluxes for cells of excised roots of Zea mays L. cv. Golden Bantam. When the measured fluxes are compared to those calculated with the Ussing-Teorell flux-ratio equation, an active inward transport of Cl⁻ across the plasmalemma is indicated; the plasmalemma K⁺ fluxes are not far different from those predicted for passive diffusion, although an active inward transport cannot be precluded. Whether fluxes across the tonoplast are active or passive depends upon the vacuolar potential which is unknown. Assuming no electropotential gradient, the tracer flux ratios are fairly close to those predicted for passive movement. However, if the vacuole is positive by about 10 millivolts relative to the cytoplasm, the data suggest active inward transport for K⁺ and outward transport for Cl⁻.     

Temperature dependence of active K+ transport in cation dimorphic sheep erythrocytes

Arrhenius diagrams of K⁺ pump fluxes measured between 15°C and 41°C were discontinuous in high K⁺ but not in low K⁺ sheep red cells. Exposure of low K⁺ cells to anti-L caused a bimodal temperature response of K⁺ pump flux with a transition temperature, $\text{T}\text{c}$, similar to that found in high K⁺ cells but with comparatively higher activation energies above $\text{T}\text{c}$.     

86Rb+ fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.