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.     

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108-116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na+/Cl-/glycine cotransport system is discussed.     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

beta-Amino acid transport across the renal brush-border membrane is coupled to both Na and Cl

Sodium-dependent beta-alanine uptake into dog renal brush-border membrane vesicles was studied. Kinetic analysis indicated a single transport system, highly specific for beta-amino acids, with Km = 35 microM at 100 mM NaCl. Sodium-dependent beta-alanine transport was markedly anion-dependent, being highest in the presence of chloride (Cl greater than Br greater than SCN greater than NO3 approximately I greater than F) and virtually nonexistent in the presence of gluconate and other nonphysiological chloride substitutes. In addition, it was observed that beta-alanine uptake could be driven against a concentration gradient by a chloride gradient. Similar results were found for sodium. Taken together, these observations provide strong evidence that beta-alanine transport across the renal brush-border membrane is coupled to both sodium and chloride. Studies of the dependence of beta-alanine flux on chloride and sodium concentrations indicated that one chloride ion and multiple sodium ions were involved in the beta-alanine transport event. beta-Alanine flux on chloride found to involve the net transfer of positive charge, consistent with these stoichiometric assignments. The hallucinogen harmaline inhibited beta-alanine uptake in a 1:1 fashion, presumably by acting at a single site on the transport molecule. The ability of harmaline to inhibit beta-alanine uptake was decreased when the chloride concentration was lowered but was unchanged when the sodium concentration was decreased. These results indicate that harmaline does not compete with sodium for a binding site on the carrier as has been suggested for other sodium-coupled transport systems, and that instead, chloride may be required for harmaline binding to the beta-alanine transporter.     

Ion exchange membrane bioreactor for selective removal of nitrate from drinking water: control of ion fluxes and process performance

An ion exchange membrane bioreactor (IEMB), consisting of a monoanion permselective membrane dialyzer coupled to a stirred anoxic vessel with an enriched mixed denitrifying culture, has been studied for nitrate removal from drinking water. The influence of nitrate and chloride concentrations on the selectivity of nitrate transport in the IEMB process was investigated. With appropriate dosing of chloride ions to the IEMB biocompartment, it was possible to regulate the net bicarbonate flux in the system, thus maintaining the bicarbonate concentration in the treated water at the desired level. The latter was not possible to achieve in Donnan dialysis, operated as a single process in which, besides the lower nitrate removal efficiency found, bicarbonate was co-extracted together with nitrate from the polluted water stream. Residual carbon source (ethanol) and nitrite were not detected in the treated water produced in the IEMB system. With a concentration of nitrate in the polluted water three times higher than the maximum contaminant level of 50 mg L(-1) allowed, the IEMB process was successfully operated for a period of 1 month before exceeding this limit.     

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

Structural and functional characterization of the human NBC3 sodium/bicarbonate co-transporter carboxyl-terminal cytoplasmic domain

The sodium bicarbonate co-transporter, NBC3, is expressed in a range of tissues including heart, skeletal muscle and kidney, where it modulates intracellular pH and bicarbonate levels. NBC3 has a three-domain structure: 67 kDa N-terminal cytoplasmic domain, 57 kDa membrane domain and an 11 kDa C-terminal cytoplasmic domain (NBC3Ct). The role of C-terminal domains as important regulatory regions is an emerging theme in bicarbonate transporter physiology. This study determined the functional role of human NBC3Ct and characterized its structure using biochemical techniques. The NBC3 C-terminal domain deletion mutant (NBC3DeltaCt) had only 12 +/- 5% of wild-type transport activity. This low activity is attributable to low steady-state levels of NBC3DeltaCt and almost complete retention inside the cell, as assessed by immunoblots and confocal microscopy, suggesting a role of NBC3Ct in cell surface processing. To characterize the structure of NBC3Ct, amino acids 1127-1214 of NBC3 were expressed as a GST fusion protein (GST.NBC3Ct). GST.NBC3Ct was cleaved with PreScission Protease and native NBC3Ct could be purified to 94% homogeneity. Gel permeation chromatography and sedimentation velocity ultracentrifugation of NBC3Ct indicated a Stokes radius of 26 and 30 angstroms, respectively. Shape modelling revealed NBC3Ct as a prolate shape with long and short axes of 19 and 2 nm, respectively. The circular dichroism spectra of NBC3Ct did not change over the pH 6.2-7.8 range, which rules out a large change of secondary structure as a component of pH sensor function. Proteolysis with trypsin and chymotrypsin identified two proteolytically sensitive regions, R1129 and K1183-K1186, which could form protein interaction sites.     

Action of External Divalent Ion Reduction on Sodium Movement in the Squid Giant Axon

Voltage clamp measurements of the sodium potential have been made on the resting squid giant axon to study the effect of variations in external divalent ion concentration upon net sodium flux. From these measurements the intracellular sodium concentration and the net sodium inflow were calculated using the Nernst relation and constant activity coefficients. While an axon bathed in artificial sea water shows a slow increase in internal sodium concentration, the rate of sodium accumulation is increased about two times by reducing external calcium and magnesium concentrations to 0.1 times their normal values. The mean inward net sodium flux increases from a mean control value of 97 pmole/cm² sec. to 186 pmole/cm² sec. in low divalent solution. Associated with these effects of external divalent ion reduction are a marked decrease in action potential amplitude, little or no change in resting potential, and a shift along the voltage axis of the curve relating peak sodium conductance to membrane potential similar to that obtained by Frankenhaeuser and Hodgkin (1957). These results implicate divalent ions in long term (minutes to hours) sodium permeability.     

Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles.

Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans-stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L-isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high-affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.     

Sodium-chloride transport in the medullary thick ascending limb of henle's loop: Evidence for a sodium-chloride cotransport system in plasma membrane vesicles

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell.     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.     

Driving forces for the uphill transport of amino acids into epidermal brush border membrane vesicles of the sea anemone,Anemonia sulcata (Cnidaria,anthozoa)

• 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
• 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na⁺-ions and Cl⁻-ions were required for the amino acid transport.
• 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
• 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
• 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.

     

Functional symmetry of the beta-galactoside carrier in Escherichia coli.

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K+/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the beta-galactoside transport protein is inherently symmetrical. One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K+/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.     

The Mechanism of Sodium and Chloride Uptake by the Gills of a Fresh-Water Fish, Carassius auratus : II. Evidence for NH4+ / NA+and HCO3- / Cl-exchanges

The addition of ammonium ions to the external medium results in an inhibition of the sodium influx and net uptake in Carassius auratus, while intraperitoneal injection of ammonium produces the opposite effect. The simultaneous chloride balance is not significantly affected by these treatments. The addition of bicarbonate ions to the external medium results in a reduction of the influx and net flux of chloride, while injection of bicarbonate produces the opposite effect. The simultaneous sodium balance is not significantly altered. The effects of the external additions are reversible after elimination of the excess ammonium or bicarbonate ions by rinsing. Inhibition of carbonic anhydrase in the gill by injection of acetazoleamide produces a simultaneous inhibition of both sodium and chloride exchanges. These results confirm the hypothesis of an exchange of sodium for ammonium, and of bicarbonate for chloride across the gill. A tentative schematic representation of the ionic absorption mechanisms in the branchial cell of the fresh-water teleosts is given. Similarities with other biological membranes and especially with the renal tubule are pointed out.     

Osmotic and ionic regulation during hypoxia in the medicinal leech, Hirudo medicinalis L.

The concentrations of inorganic and organic ions and osmolality in the blood of the medicinal leech, Hirudo medicinalis, were determined during normoxia and hypercapnic and hypocapnic hypoxia. In normoxic animals, the blood sodium concentration was 124.5 +/- 4.2 mmol/l and the total cation concentration was 132.2 +/- 4.3 mEq/l (mean +/- S.D.). Major anionic compounds were chloride (40.8 +/- 1.6 mmol/l), bicarbonate (8.4 +/- 1.3 mmol/l), and organic anions (42.5 +/- 2.3 mEq/l). Among the latter, malate accounts for 30.4 +/- 2.2 mEq/l. The nature of the remaining anion fraction, which balances cation and anion concentrations in leech blood, remains unknown. Within 96 h of hypercapnic hypoxia, the amount of organic osmolytes in leech tissue increased from the control level of 56.6 +/- 9.1 to 158.3 +/- 19.5 mumol/g dry weight. An even higher amount of organic acids was accumulated within 96 h of hypocapnic hypoxia (218.0 +/- 53.7 mumol/g dry weight). A possible reason for this is that lactate, which is a major end-product of hypocapnic hypoxia, cannot be excreted to the external medium as easily as propionate. The accumulation of blood organic acids generating osmotic stress in the animals was compensated by an equimolar decrease in sodium and chloride ion concentrations. In hypercapnic animals these changes resulted in a constant osmotic concentration of the blood (200 mosmol/kg H2O) during the experimental period. Between 24 and 96 h of hypocapnic hypoxia, however, the increase in the osmotic gradient between animal and medium was correlated with further net water uptake and the obvious deterioration of the volume- and ion-regulatory mechanisms in these animals.     

Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.