Regulation of Cl/HCO3 exchange in gastric parietal cells.

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.     

Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min- 1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.     

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.     

Furosemide-sensitive Na and K fluxes in human red cells. Net uphill Na extrusion and equilibrium properties

This paper reports experiments designed to find the concentrations of internal and external Na and K at which inward and outward furosemide-sensitive (FS) Na and K fluxes are equal, so that there is no net FS movement of Na and K. The red cell cation content was modified by using the ionophore nystatin, varying cell Na (Nai) from 0 to 34 mM (K substitution, high-K cells) and cell K (Ki) from 0 to 30 mM (Na substitution, high-Na cells). All incubation media contained NaCl (Nao = 130 or 120 nM), and KCl (Ko = 0-30 mM). In high-K cells, incubated in the absence of Ko, there was net extrusion of Na through the FS pathway. The net FS Na extrusion increased when Nai was increased. Low concentrations of Ko (0-6 mM) slightly stimulated, whereas higher concentrations of Ko inhibited, FS Na efflux. Increasing Ko stimulated the FS Na influx (K0.5 = 4 mM). Under conditions similar to those that occur in vivo (Nai = 10, Ki = 130, Nao = 130, Ko = 4 mM, Cli/Clo = 0.7), net extrusion of Na occurs through the FS pathway (180-250 mumol/liter cell X h). The concentration of Ko at which the FS Na influx and efflux and the FS K influx and efflux become equal increased when Nai increased in high-K cells and when Ki was increased in high-Na cells. The net FS Na and K fluxes both approached zero at similar internal and external Na and K concentrations. In high-K cells, under conditions when net Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional flux was found to be 2:3. In high-K cells, the empirical expression (Nai/Nao)2(Ki/Ko)3 remained at constant value (apparent equilibrium constant, Kappeq +/- SEM = 22 +/- 2) for each set of internal and external cation concentrations at which there was no net Na flux. These results indicate that in the physiological region of concentrations of internal and external Na, K, and Cl, the stoichiometry of the FS Na and K fluxes is 2 Na:3 K. In high-Na cells under conditions when net FS Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional fluxes was 3:2 (1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor

When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.     

HCO3-stimulated Cl efflux in the gulf toadfish acclimated to sea water.

Unidirectional efflux of Cl was examined in the Gulf toadfish, Opsanus beta, in artificial seawater solutions with modified concentrations of Cl and HCO3. Removal of Cl HCO3 reduced Cl efflux. Addition of HCO3 at typical seawater concentrations stimulated Cl efflux, independent of changes in the transepithelial potential. This active, HCO3-stimulated Cl efflux is saturable, with a Km of 2.4 mM, typical of the concentration of HCO3 found in sea water, and independent of external pH. Active extrusion of Cl offsets the net diffusional and oral gain of Cl faced by O. beta in sea water.     

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.     

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-

Mg(2+) efflux from rat erythrocytes was measured in NaCl, NaNO(3), NaSCN and Na gluconate medium. Substitution of extracellular and intracellular Cl(-) with the permeant anions NO(3)(-) and SCN(-) reduced Mg(2+) efflux via Na(+)/Mg(2+) antiport. After substitution of extracellular Cl(-) with the non-permeant anion gluconate, Mg(2+) efflux was not significantly reduced. In Na gluconate medium, an influence of the changed membrane potential and intracellular pH on Mg(2+) efflux could be excluded. The results indicate the existence of Cl(-)-independent Na(+)/Mg(2+) antiport and of Na(+)/Mg(2+) antiport stimulated by intracellular Cl(-). Intracellular Cl(-), as determined by means of (36)Cl(-), was found to stimulate Na(+)/Mg(2+) antiport through a cooperative effect according to a sigmoidal kinetics. The Hill coefficient for intracellular Cl(-) amounted to 1.4-1.8, indicating that two intracellular Cl(-) may be simultaneously active. With respect to specificity, Cl(-) was most effective, followed by Br(-), J(-), and F(-). Stimulation of Na(+)/Mg(2+) antiport by intracellular Cl(-) together with intracellular Mg(2+) may play a role during deoxygenation of erythrocytes and in essential hypertension.     

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K- Cl cotransport. The anion exchange inhibitor 4,4''diisothiocyanato- 2,2''disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

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.     

Asymmetry in the mechanism for anion exchange in human red blood cell membranes. Evidence for reciprocating sites that react with one transported anion at a time

The kinetics of chloride and bromide transport were examined in intact human red blood cells and resealed ghosts. Because the influx and efflux of halide ions are almost equal (less than 0.01% difference), the stimulation of the exchange flux by external halides could be determined by measuring 36Cl or 82Br efflux. When the external halide concentration was increased by replacement of isoionic, isotonic solutions of sucrose and the nontransported anion citrate, the stimulation of the exchange flux was hyperbolic and was maximum at 20 mM halide externally. The K 1/2-out, the external concentration of chloride or bromide which stimulated the efflux to half of its maximum value, was 3 and 1 mM respectively, 15-fold smaller than K 1/2-in which we found to be about equal to the K 1/2 of halide self-exchange with nearly equal internal and external concentrations. Thus, the transport mechanism behaves asymmetrically with respect to these transported halides. Bromide flux was two-fold greater in bromide-chloride heteroexchange than in bromide-bromide self-exchange but it was still much smaller than the chloride self-exchange flux. The maximum influx and efflux of bromide in exchange for chloride were roughly eqal. Thus, since the maximum transport rates in the two directions are nearly equal, the kinetics of bromide equilibrium exchange with equal concentrations on the two sides are controlled on the inside where K 1/2 is greatest. The K 1/2-out Cl was a hyperbolic function of internal chloride concentration and was proportional to the maximum flux at each internal chloride concentration. These results are evaluated in terms of two broad categories of models. We conclude that, in contrast to other ion transport systems which have been shown to have kinetics of a sequential mechanism, anion exchange is compatible with a ping-pong mechanism in which a single site reciprocates between inside- and outside-facing orientations with asymmetric K 1/2 values.     

Fluid reabsorption by the ductuli efferentes testis of the rat is dependent on both sodium and chlorine

The role of Na(+) and Cl(-) in fluid reabsorption by the efferent ducts was examined by perfusing individual ducts in vivo with preparations of 160 mM NaCl in which the ions were replaced, together or individually, with organic solutes while maintaining the osmolality at 300 mmol/kg. Progressively replacing NaCl with mannitol reduced net reabsorption of water and the ions in a concentration-dependent manner, and caused net movement into the lumen at concentrations of NaCl less than 80 mM. The net rates of flux were lower for Na(+) than for Cl(-). In collectates, [Na(+)] was greater than [Cl(-)], indicating that Cl(-) transport is probably linked with another anion. Replacing either Na(+) or Cl(-) in perfusates (with choline and isethionate, respectively) while maintaining the other inorganic ion at 160 mM also reduced net rates of reabsorption in a concentration-dependent manner to zero when either ion was completely replaced. There were no significant differences in the osmolality of perfusate and collectate, and collectates contained a mean of 3.4 mM K(+), indicating a backflux of K(+) into the lumen. It is concluded that fluid reabsorption from the efferent ducts is dependent on the transport of both Na(+) and Cl(-) from the lumen (from a luminal concentration of at least 70-80 mM), and that Cl(-) transport is dependent on another anion. The epithelium is permeable to K(+) and has a higher permeability to a range of organic solutes (mannitol, choline, and isethionate) than epithelium in the proximal kidney tubules.     

Interactions between glutamine metabolism and cell-volume regulation in perfused rat liver

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism and cellular applications of a green fluorescent protein-based halide sensor

We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl(-), which increased to 7.86 at 150 mM Cl(-). At pH 7.5, YFP-H148Q fluorescence decreased maximally by approximately 2-fold with a K(D) of 100 mM Cl(-). YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and [Cl(-)]. Circular dichroism and absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating Cl(-)/YFP binding. Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or [Cl(-)], establishing a 1:1 YFP-H148Q/Cl(-) binding mechanism. Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP-H148Q quenching (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > Cl(-) > Br(-) > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions. The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore. YFP-H148Q transfected mammalian cells were brightly fluorescent with cytoplasmic/nuclear staining. Ionophore calibrations indicated similar YFP-H148Q pH and anion sensitivities in cells and aqueous solutions. Cyclic AMP-regulated Cl(-) transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl(-) channels was assayed with excellent sensitivity from the time course of YFP-H148Q fluorescence in response to extracellular Cl(-)/I(-) exchange. The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.     

Evidence for two conductance/exchange pathways for chloride in rat parotid secretory granules

Electron probe x-ray microanalysis was used to determine that bromide is localized to rat parotid secretory granules at early stages of an in situ Cl/Br washout experiment. Chloride efflux and bromide influx across the secretory granule membrane occurred with a time order of minutes. Since the Cl washout data indicated minimal Cl binding within the granule, and therefore minimal Br binding, the Br localization results suggest the presence of two or more anion conductance/exchange pathways in the granule membrane for the Cl (Br) ion.     

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.     

Cyclic variations in nitrogen uptake rate of soybean plants: effects of external nitrate concentration

Net uptake of NO3- by non-nodulated soybean plants [Glycine max (L.) Merr. cv. Ransom] growing in flowing hydroponic cultures containing 0.5, 1.0 and 10.0 mol m-3 NO3- was measured daily during a 24-d period of vegetative development to determine if amplitude of maximum and minimum rates of net NO3- uptake are responsive to external concentrations of NO3-. Removal of NO3- from the replenished solutions during each 24-h period was determined by ion chromatography. Neither dry matter accumulation nor the periodicity of oscillations in net uptake rate was altered by the external NO3- concentrations. The maxima of the oscillations in net uptake rate, however, increased nearly 3-fold in response to external NO3- concentrations. The maxima and minima, respectively, changed from 4.0 and 0.6 mmol NO3- per gram root dry weight per day at an external solution level of 0.5 mol m-3 NO3- to 15.2 and -2.7 mmol NO3- per gram root dry weight per day at an external solution level of 10.0 mol m-3 NO3-. The negative values for minimum net uptake rate from 10.0 mol m-3 NO3- solutions show that net efflux was occurring and indicate that the magnitude of the efflux component of net uptake was responsive to external concentration of NO3-.     

Endothelium modulates anion channel-dependent aortic contractions to iodide

Anion currents contribute to vascular smooth muscle (VSM) membrane potential. The substitution of extracellular chloride (Cl) with iodide (I) or bromide (Br) initially inhibited and then potentiated isometric contractile responses of rat aortic rings to norepinephrine. Anion substitution alone produced a small relaxation, which occurred despite a lack of active tone and minimal subsequent contraction of endothelium-intact rings (4.2 +/- 1.2% of the response to 90 mM KCl). Endothelium-denuded rings underwent a similar initial relaxation but then contracted vigorously (I > Br). Responses to 130 mM I (93.7 +/- 1.9% of 90 mM KCl) were inhibited by nifedipine (10(-6) M), niflumic acid (10(-5) M), tamoxifen (10(-5) M), DIDS (10(-4) M), and HCO(-)(3)-free buffer (HEPES 10 mM) but not by bumetanide (10(-5) M). Intact rings treated with N(omega)-nitro-L-arginine (10(-4) M) responded weakly to I (15.5 +/- 2.1% of 90 mM KCl), whereas hemoglobin (10(-5) M), indomethacin (10(-6) M), 17-octadecynoic acid (10(-5) M), and 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (10(-6) M) all failed to augment the response of intact rings to I. We hypothesize that VSM takes up I primarily via an anion exchanger. Subsequent I efflux through anion channels having a selectivity of I > Br > Cl produces depolarization. In endothelium-denuded or agonist-stimulated vessels, this current is sufficient to activate voltage-dependent calcium channels and cause contraction. Neither nitric oxide nor prostaglandins are the primary endothelial modulator of these anion channels. If they are regulated by an endothelium-dependent hyperpolarizing factor it is not a cytochrome P-450 metabolite.