The change from symmetry to asymmetry of a sodium transport system in red cell membranes

A study has been made with human red cells of sodium movements that are sensitive to the drug furosemide. The aim was to see if furosemide-sensitive movements that are symmetrical (exchange) became asymmetrical (net transport) on replacement of chloride with nitrate as the major external anion. Cells were incubated for 4 h at 37 degrees C with 140 mM sodium, and chloride or nitrate as the principal anion. Under a variety of conditions (presence and absence of ouabain or furosemide, or both) the cell sodium concentration was always higher when chloride was replaced with nitrate. The cells became leakier to sodium. Tracer studies indicated that, in contrast to the results in chloride medium, the decrease in sodium influx was greater than the fall in efflux when furosemide was added to cells in nitrate medium. The results confirm that the sensitivity of sodium efflux to furosemide depended on chloride. However, influx showed a different sensitivity in that furosemide still inhibited in cells incubated in nitrate medium. The stimulation of sodium influx with nitrate medium was independent of external potassium (10-50 mM) and the furosemide-sensitive influx was also constant. It is concluded that symmetrical transmembrane sodium movements with cells in chloride medium became downhill asymmetrical in nitrate medium, giving a net gain of cell sodium that was insensitive to ouabain and sensitive to furosemide. The drug thus partly retarded the gain of cell sodium that otherwise occurred in the somewhat leaky cells.     

Effect of reduction of culture medium sodium, using different sodium chloride substitutes, on the proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts

We have substituted choline chloride, tetramethylammonium chloride, sucrose, or glucose for culture medium sodium chloride. When culture medium sodium is reduced below physiological levels (143 mM) by replacement of graded concentrations of sodium chloride with equivalent concentrations of choline chloride, normal fibroblasts approach proliferative inactivity in the presence of 90 mM Na, while their Rous sarcoma virus (RSV)-infected counterparts proliferate actively; both normal and neoplastic cells die with further sodium reduction. When culture medium NaC; is replaced with tetramethylammonium chloride, however, both normal and RSV-infected fibroblasts alike approach proliferative inactivity in the presence of 110 mM Na and both die off in the presence of 90 mM Na. When culture medium NaCl is replaced with sucrose or glucose yet another set of results is obtained: both normal and RSV-infected fibroblasts proliferate at reduced, although significant, rates in the presence of 42 mM Na. It is clear from our experimental results that the effects of reduction of culture medium sodium on cell proliferation differ markedly with the use of different sodium chloride substitutes. Caution must be exercised, therefore, in drawing inferences concerning the role of sodium in mitogenesis from experimental studies based on the tactic of reduction of external sodium.     

Further Studies of Sodium Transport in Feline Red Cells

The transport of radioactive sodium in high sodium cat red blood cells has been studied under various experimental conditions. It was found that iodoacetate (IAA) and iodoacetamide (IAM) inhibit Na influx by 50% whereas NaF has no effect. Reversible dyes, such as methylene blue (Mb), also inhibit this influx by 60%. Both IAA and Mb effects show a lag period of about 40 min. Cell starvation abolishes the volume-dependent Na influx which is generally observed in these cells. IAA reduces significantly the volume-dependent Na influx but does not inhibit it completely. 5 mM magnesium chloride produces a twofold increase in Na influx. On the other hand, MgCl₂ has no effect on Na transport in human red cells or on potassium or sulfate transport in cat red cells. The effect of MgCl₂ is quite rapid and does not interfere with the volume-dependent Na influx. This effect is abolished in starved cells. Reincubation of previously stored cells in buffered solutions containing glucose and MgCl₂ causes more than one order of magnitude increase in Na influx. These several observations are discussed in terms of the possibility of a link between Na transport and Na-Mg-activated ATPase.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Sodium fluxes in sweet pepper exposed to varying sodium concentrations

The sodium transport and distribution of sweet pepper (Capsicum annuum L.) under saline conditions were studied after transferring the plants to a sodium-free nutrient solution. Sodium stress up to 60 mM did not affect the growth of sweet pepper, as it appears able to counteract the unfavourable physiological effects of sodium efficiently. Sodium was particularly accumulated in the basal pith cells of the stem and in the root cells, while almost no sodium was directed to the leaves or the fruits. The sodium concentration in the pith cells and xylem sap gradually decreased towards the shoot tip. Removal of sodium from the medium resulted in a 50% release of sodium from the plant after 1 week without affecting the gradient in the pith cells. In contrast, the concentration profile in the xylem sap was completely changed: the sodium concentration in the xylem sap at the stem base was similar to that at the top.Phloem transport was studied in a split root experiment, in which both portions of the roots were exposed to 15 mM NaCl and one part was fed with additional ²²NaCl. During continuous exposure to 15 mM NaCl no label was detected in unlabelled root parts. However, after transferring the plants to a sodium-free solution, ²²Na was rapidly released from the unlabelled roots, indicating a downward phloem transport.It was concluded that pith cells, the intermediates between the xylem and phloem, play a decisive role in the recirculation of sodium throughout the plant. Release of sodium from the plants following transfer to a sodium-free solution may be explained by changes in the diffusion resistance for passive sodium efflux from the cells.Key words: Xylem, phloem, sodium, fluxes, sweet pepper      

Chloride-dependence of amino acid transport in rabbit ileum

Chloride-dependence of influx across the brush-border membrane of distal rabbit ileum was examined for beta-alanine, 2-methylaminoisobutyric acid (MeAIB), leucine, lysine, proline and D-glucose. Influx of leucine at 2 mM and of D-glucose at 0.5 mM was chloride-independent indicating that substitution of isethionate for chloride has no unspecific effect on sodium gradient driven transport processes. In contrast influx of beta-alanine and MeAIB was totally dependent on the presence of chloride ions. In the absence of chloride, proline transport was reduced to 20% of its control level. This remaining transport can be accounted for by the function of the carrier of alpha-amino-monocarboxylic acids. Transport of leucine at 0.1 mM was reduced by absence of chloride. This is in accordance with the observation of leucine transport by the beta-alanine carrier. The kinetics of chloride and sodium activation of transport of MeAIB were examined at 1 mM MeAIB. Chloride activation was characterized by a Hill coefficient of 1 and a K1/2 of 23.5 mM, and sodium activation by a Hill coefficient of 2 and a K1/2 of 51 mM. Thus cotransport of chloride with an imino acid would be compatible with the known rheogenic nature of this transport. This study adds the imino acid carrier and the beta-alanine carrier to the group of chloride-dependent, epithelial amino acid transport systems.     

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.     

Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes.

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

Sodium fluxes in roots of Eleocharis uniglumis, a brackish water species

Abstract Fluxes of sodium across the plasmalemma and tonoplast of the roots of Eleocharis uniglumis have been measured using ²²Na. E. uniglumis (one glumed spike rush) was collected from an estuarine habitat where it was growing in a wide range of salinities (1 mM-50 mM Na). Compartmental analysis was used to determine sodium concentrations in the cytoplasm and the vacuole. Application of the Ussing-Teorell equation revealed the presence of sodium pumps in the plasmalemma and the tonoplast. Active sodium transport into the cytoplasm from the bathing medium was found to occur in most of the external sodium concentrations investigated. There also appeared to be active transport of sodium into the cytoplasm from the vacuole. In contrast to halophytes, high levels of sodium appeared to be accumulated in the cytoplasm of E. uniglumis roots.     

In Vivo Substitution of Choline for Sodium Evokes a Selective Osmoinsensitive Increase of Extracellular Taurine in the Rat Hippocampus

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.     

Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.     

The Effect of Sodium Chloride on Heat Resistance and Recovery of Heated Spores of Bacillus stearothermophilus

Increasing concentrations (2, 4 and 8% w/v) of sodium chloride in the heating medium progressively reduced the heat resistance of spores of Bacillus stearothermophilus. Storage at 4° in water or in sodium chloride solutions had little effect on viable counts of unheated spores, but with the increase in sodium chloride concentration there was a reduction in the heat activation effect and a small decrease in heat resistance of the spores. Increasing the severity of heat treatment rendered spores increasingly sensitive to sodium chloride in the plating medium.     

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.     

Lysis of Escherichia coli mutants by lactose.

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.     

The Role of Potassium in Active Transport of Sodium by the Toad Bladder

Studies were carried out on the isolated urinary bladder of the toad, Bufo marinus, in order to explain the dependence of active sodium transport on the presence of potassium, in the serosal medium. Attempts to obtain evidence for coupled sodium-potassium transport by the serosal pump were unsuccessful; no relation between sodium transport and uptake of K⁴² from the serosal medium was demonstrable. Rather, the predominant effect of serosal potassium appeared to be operative at the mucosal permeability barrier, influencing the permeability of this surface to sodium. The mucosal effects of serosal potassium were correlated with effects on cellular cation content. When sodium Ringer's solution was used as serosal medium, removal of potassium resulted in significant decrease in tissue potassium content, commensurate increase in tissue sodium content, and marked depression of mucosal permeability and sodium transport. When choline replaced sodium in the serosal medium, removal of potassium resulted in only slight alterations of tissue electrolyte content, and effects on mucosal permeability and sodium transport were minimal.