Amino acid specificity of the Na+/alanine cotransporter in pancreatic acinar cells

The method of tight-seal whole-cell recording was used to study the amino-acid specificity of the Na+/alanine cotransporter in pancreatic acinar cells. Single cells or small clusters of electrically coupled cells were obtained by enzymatic dissociation of mouse pancreas. Inward currents were measured under 'zero-trans' conditions, i.e., at finite concentrations of Na+ and amino acid at the extracellular side and vanishing concentrations at the cytoplasmic side. The cotransporter, which corresponds to 'system A', as previously defined in the literature, was found to exhibit a wide tolerance to neutral amino acids (L-cysteine, L-serine, L-alanine, glycine, L-phenylalanine). Competition experiments with 2-methylaminoisobutyric acid (MeAIB) indicate that for glycine a second electrogenic transport system exists in pancreatic acinar cells.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings

Summary Electrical currents associated with sodium-coupled alanine transport in mouse pancreatic acinar cells were studied using the method of whole-cell recording with patch pipettes. Single cells or small clusters of (electrically coupled) cells were isolated by collagenase treatment. The composition of the intracellular solution could be controlled by internal perfusion of the patch pipette. In this way both inward and outward currents could be measured under zero-trans conditions, i.e., with finite concentrations of sodium andl-alanine on one side and zero concentrations on the other. Inward andoutward currents for equal but opposite concentration gradients were found to be of similar magnitude, meaning that the cotransporter is functionally nearly symmetric. The dependence of current on the concentrations of sodium andl-alanine exhibited a Michaelis-Menten behavior. From the sodium-concentration dependence of current as well as from the reversal potential of the current in the presence of an alanine-concentration, gradient, a sodium/alanine stoichiometric ratio of 1:1 can be inferred. The finding that N-methylated amino acids may substitute, forl-alanine, as well as the observed pH dependence of currents indicate that the pancreatic alanine transport system is similar to (or identical with) the A-system which is widespread in animal cells. The transport system is tightly coupled with respect to Na⁺; alanine-coupled inward flow of Na⁺ is at least 30 times higher than uncoupled Na⁺ flow mediated by the cotransporter. The current-voltage characteristic of the cotransporter could be (approximately) determined from the difference of transmembrane current in the presence and in the absence ofl-alanine. The sodium-concentration dependence of the current-voltage characteristic indicates that a Na⁺ ion approaching the binding site from the extracellular medium has to cross part of the transmembrane electric field.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

The characterisation of two partially purified systems for Na+-dependent amino acid transport

Two systems mediating the transport of amino acids were studied in vesicles derived from protein-depleted membranes of pigeon erythrocytes. One system (ASC system) catalysed the Na⁺-dependent exchange of small neutral amino acids, such as alanine, serine and cysteine. The other system, also Na⁺-dependent, mediated the active transport of glycine. The ASC and glycine systems were distinguished by the sensitivity of the latter to the anion present, by the former's requirement for an exchangeable amino acid and by the inability of alanine to inhibit the transport of glycine. Preliminary results indicated that the influx of glycine was electrically silent. The only major integral protein retained in the vesicles was the band 3 protein, but that could not be unequivocally identified as the transporter.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Can elevated CO(2) improve salt tolerance in olive trees?

We compared growth, leaf gas exchange characteristics, water relations, chlorophyll fluorescence, and Na⁺ and Cl⁻ concentration of two cultivars (‘Koroneiki’ and ‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM) and elevated CO₂ (eCO₂) concentration (700 μL L⁻¹). The cultivar ‘Koroneiki’ is considered to be more salt sensitive than the relatively salt-tolerant ‘Picual’. After 3 months of treatment, the 9-month-old cuttings of ‘Koroneiki’ had significantly greater shoot growth, and net CO₂ assimilation (A_CO2) at eCO₂ than at ambient CO₂, but this difference disappeared under salt stress. Growth and A_CO2 of ‘Picual’ did not respond to eCO₂ regardless of salinity treatment. Stomatal conductance (g_s) and leaf transpiration were decreased at eCO₂ such that leaf water use efficiency (WUE) increased in both cultivars regardless of saline treatment. Salt stress increased leaf Na⁺ and Cl⁻ concentration, reduced growth and leaf osmotic potential, but increased leaf turgor compared with non-salinized control plants of both cultivars. Salinity decreased A_CO2, g_s, and WUE, but internal CO₂ concentrations in the mesophyll were not affected. eCO₂ increased the sensitivity of PSII and chlorophyll concentration to salinity. eCO₂ did not affect leaf or root Na⁺ or Cl⁻ concentrations in salt-tolerant ‘Picual’, but eCO₂ decreased leaf and root Na⁺ concentration and root Cl⁻ concentration in the more salt-sensitive ‘Koroneiki’. Na⁺ and Cl⁻ accumulation was associated with the lower water use in ‘Koroneiki’ but not in ‘Picual’. Although eCO₂ increased WUE in salinized leaves and decreased salt ion uptake in the relatively salt-tolerant ‘Koroneiki’, growth of these young olive trees was not affected by eCO₂.     

Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H⁺/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5′ and 3′ untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na⁺-independent uptake of proline, glycine, l-alanine and α-(methylamino)isobutyric acid. Proline uptake was maximal at pH?5.0 (K_m?2.2±0.7?mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na⁺ but in the presence of an inwardly directed H⁺ gradient. In the presence of Na⁺ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

The concentration of amino acids by yeast cells depleted of adenosine triphosphate

1. The ATP content of preparations of a strain of Saccharomyces carlsbergensis was lowered below 0.3nmol/mg of yeast by starving the yeast cells in the presence of both antimycin and 5mm-deoxyglucose. 2. When the depleted cells were put at pH4.5 with glycine up to about 20nmol of the amino acid/mg of yeast was absorbed without being chemically modified. The mechanism did not depend on an exchange with endogenous amino acids. 3. The concentration of the absorbed glycine could apparently reach 100–200 times that outside the cells. 4. Replacement of the cellular K⁺ by Na⁺ almost stopped amino acid absorption in the presence of antimycin and deoxyglucose, but not in their absence. 5. It is suggested that, when energy metabolism itself had stopped, a purely physical process, namely the movements of H⁺ and K⁺ into and out of the yeast respectively, served to concentrate the amino acids in the cells. Both ionic species appear to be co-substrates of the system transporting amino acids.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na⁺/HCO ₃ ^– cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na⁺ and HCO ₃ ^– , incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na⁺ and HCO ₃ ^– during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na⁺ and HCO ₃ ^– , PITC binds covalently to amino groups unprotected by substrates leaving the Na⁺/HCO ₃ ^– cotransporter available for specific labeling with FITC. Addition of NaHCO₃ to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4-diisothiocyanostilbene 2,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na⁺/HCO ₃ ^– cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

[Na] in the subsarcolemmal ‘fuzzy’ space and modulation of [Ca]i and contraction in cardiac myocytes

The strength of the heart beat depends on the amplitude and time course of the transient increase in [Ca²⁺] in the myocytes with each cycle. [Na⁺]_i modulates cardiac contraction through its effect on the Ca²⁺ flux through the Na/Ca exchanger. Cardiac excitation–contraction coupling has been postulated to occur in a microdomain or ‘fuzzy’ space at the junction of the T-tubules and the sarcoplasmic reticulum. This ‘fuzzy’ space is well described for the Ca²⁺ fluxes and the interaction between the L-type Ca²⁺ channel, the Ca²⁺ release channel of the sarcoplasmic reticulum and the Na/Ca exchanger. Co-localization of the Na⁺ transporters, in particular the Na/K pump and the Na⁺ channel, within this ‘fuzzy’ space is not as well established. The functional and morphological characteristics of the ‘fuzzy’ space for Na⁺ and its interaction with the Ca²⁺ handling suggest that this space is not strictly co-inciding with the Ca²⁺ microdomain. In this space [Na⁺] can be several-fold higher or lower than [Na⁺] in the bulk cytosol. This has implications for modulation of [Ca²⁺]_i during a single beat as well as during alterations in Na⁺ fluxes seen in pathological conditions.     

Involvement of Long-Distance Na<Superscript>+</Superscript> Transport in Maintaining Water Potential Gradient in the Medium-Root-Leaf System of a Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

The aim of this study was to determine the range of NaCl concentrations in the nutrient solution that allow Suaeda altissima (L.) Pall., a salt-accumulating halophyte, to maintain the upward gradient of water potential in the “medium-root-leaf” system. We evaluated the contribution of Na⁺ ions in the formation of water potential gradient and demonstrated that Na⁺ loading into the xylem is involved in this process. Plants were grown in water culture at NaCl concentrations ranging from zero to 1 M. The water potential of leaf and root cells was measured with the method of isopiestic thermocouple psychrometry. When NaCl concentration in the growth medium was raised in the range of 0–500 mM (the medium water potential was lowered accordingly), the root and leaf cells of S. altissima decreased their water potential, thus promoting the maintenance of the upward water potential gradient in the medium-root-leaf system. Growing S. altissima at NaCl concentrations f 750 mM and 1 M disordered water homeostasis and abolished the upward gradient of water potential between roots and leaves. At NaCl concentrations of 0–250 mM, the detached roots of S. altissima were capable of producing the xylem exudate. The concentration of Na⁺ in the exudate was 1.3 to 1.6 times higher than in the nutrient medium; the exudate pH was acidic and was lowered from 5.5 to 4.5 with the rise in the salt concentration. The results indicate that the long-distance Na⁺ transport and, especially, the mechanism of Na⁺ loading into the xylem play a substantial role in the formation of water potential gradient in S. altissima. The accumulation of Na⁺ in the xylem and acidic pH values of the xylem sap suggest that Na⁺ loading into the xylem is carried out by the Na⁺/H⁺ antiporter of the plasma membrane in parenchymal cells of the root stele.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 549–557.Original Russian Text Copyright © 2005 by Balnokin, Kotov, Myasoedov, Khailova, Kurkova, Lun’kov, Kotova.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.