Energy Coupling in H-Amino Acid Cotransport : ATP DEPENDENCE OF THE SPONTANEOUS ELECTRICAL REPOLARIZATION OF THE CELL MEMBRANES IN OAT COLEOPTILES

Experiments were undertaken in order to test the mechanism of energy coupling for amino acid uptake proposed in the cotransport hypothesis. According to the hypothesis an electrochemical potential difference in H⁺ is established by active H⁺ extrusion. That potential difference then drives the cotransport of H⁺ and amino acids into the cells. Application of amino acids to oat (Avena sativa var. Victory) coleoptiles induced transient depolarizations of the cell membrane electrical potentials considered to reflect the joint uptake of H⁺ and amino acids followed by an enhanced H⁺ extrusion. In the presence of KCN, cysteine induced strong depolarizations, but the rate of repolarization depended linearly upon the cyanide-adjusted ATP level of the tissue. At an ATP level 44% of normal, the membrane potential was 74% of normal, but the repolarization after cysteine-induced depolarization was practically nil. Sudden transitions from room temperature to temperatures below 15° C induced sharp depolarizations of the membrane which then repolarized within 3 min; the ATP content of the tissues was unaffected. Cysteine and alanine induced strong depolarizations at temperatures between 5 and 25°C, and the Q₁₀ for the rate of depolarization was 1.5 for cysteine and 1.6 for alanine. The Q₁₀ for the rate of repolarization was 3.0 for cysteine and 2.0 for alanine. These experiments support the prevailing view that the depolarizations are caused by the passive joint influx of H⁺ and amino acids and that the repolarizations depend upon the ATP-dependent extrusion of H⁺.     

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = −220 millivolts). In plants maintaining a low pd (−110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H⁺ cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H⁺ -extrusion pump.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na⁺/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na⁺ gradient. The uptake of l-carnitine was of high affinity (K_m=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na⁺/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H⁺ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [¹⁴C]TEA uptake, but unlabeled l-carnitine did not stimulate [¹⁴C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na⁺/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H⁺/organic cation antiporter.     

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

The cloned intestinal peptide transporter is capable of electrogenic H⁺-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K_0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K_0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H⁺/H₃O⁺ cotransport may vary depending on pH. Received: 19 August 1996/Revised: 10 October 1996     

Evidence supporting a model of voltage-dependent uptake of auxin into Cucurbita vesicles

The accumulation of [¹⁴C]indole-3-acetic acid (IAA), of [³H]tetra-phenyl phosphonium ion as a membrane potential probe, and of [¹⁴C]butyric acid as probe for pH gradients was studied with membrane vesicles from etiolated hypocotyls of Cucurbita pepo. Ion gradients (K⁺, H⁺) were applied in the presence and absence of specific ionophores e.g. valinomycin or carbonylcyanide m-chlorophenylhydrazone. In all cases tested, the accumulation of [¹⁴C]IAA equals neither potential probe nor pH-probe accumulation, but represents. an intermediate between the two. Auxin molecules seem to be taken up as positively charged ions and a pH gradient is required for accumulation. The uptake mechanism thus appears to be a specific, carrier-mediated cotransport of the anion of IAA and no less than two protons. The initial rates of auxin uptake by the saturable influx carrier, of permeation through the membrane, and of efflux by the phytotropin-affected efflux carrier were analysed.Abbreviations BA butyric acid - CCCP carbonylcyanid-3-chlorophenylhydrazone - CPD 2-carboxylphenyl-3-phenylpropan-1,3-dion - IAA indole-3-acetic acid - IAA anion of IAA - IAAH undissociated form of IAA - 2-NAA 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - TPP⁺ tetra-phenyl phosphonium ion     

Uptake of Acidic and Basic Sugar Derivatives in Lemna gibba G1

The uptake of acidic and basic sugar derivatives in Lemna gibba L. was studied. Uronic acids applied to the experimental solution (50 millimolar) induced a small decrease of the membrane potential (10 ± 1 millivolt galacturonic acid, and 20 ± 4 millivolt glucuronic acid). After incubation of the plants in a 0.1 millimolar solution of these substrates, no decrease in the concentration of reducing groups in the external solution was detected. Respiration increased by 31% with 50 millimolar galacturonic acid, whereas no effect was found with the same concentration of glucuronic acid. Glucosamine caused a considerable concentration-dependent membrane depolarization. (¹⁴C)glucosamine uptake followed Michaelis-Menten kinetics together with a linear component. Influx of this substrate was inhibited by glucose but the type of competition could not be clearly distinguished. Glucosamine, 50 millimolar, inhibited the respiration rate by 30%. The glucosamine uptake was pH-dependent, with maximum uptake at around pH 7. Lack of enhancement of uptake by low pH as well as the permanent membrane depolarization suggest a uniport mechanism for the charged species of the substrate and an electroneutral diffusion of the uncharged species.     

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H⁺ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H⁺/H₂PO₄⁻ stoichiometry of the cotransport is most likely to be 4.     

Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium.

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.     

Workshop 7: 2

Glutamine, the preferred precursor for neurotransmitter glutamate, is likely to be the principal substrate for the neuronal System A transporter SAT1 in vivo. By measuring currents associated with SAT1 expression in Xenopus oocytes, we found that SAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine, alanine and the System A‐specific analogue 2‐(methylamino) isobutyrate, each with K_0.5 of 0.3–0.5 mm . Amino acid transport is driven by the Na⁺ electrochemical gradient. Kinetic data indicates that Na⁺/cotransport comprises the ordered binding first of Na⁺ (a voltage‐dependent step), then alanine, then simultaneous translocation. Li⁺ (but not H⁺) can substitute for Na⁺ but results in reduced V_max. In the absence of amino acid, SAT1 mediates a cation leak with selectivity Na⁺, Li⁺, H⁺, K⁺. The temperature‐dependence of the leak current (E_a = 17 ± 3 kcal/mol) is consistent with carrier‐mediated Na⁺ uniport activity (cf 13 ± 2 kcal/mol for Na⁺/alanine cotransport) but the leak does not saturate at physiological [Na⁺], suggesting channel activity. Despite a Na⁺ Hill coefficient of 1, we obtained Na⁺/amino acid coupling coefficients greater than 1 from simultaneous measurement of charge and [³H]alanine or [³H]glutamine uptake. Interpretation of these data is model‐dependent and consistent with either (1) an all‐carrier model in which Na⁺/amino acid cotransport is thermodynamically coupled 2 : 1, cotransport is preferred over Na⁺ uniport, and in which there is little cooperativity between Na⁺ binding events, or (2) 1 : 1 coupling in parallel with an always‐on Na⁺ channel activity. In either scenario, the presence of SAT1 at the plasma membrane and resultant Na⁺ fluxes will place a significant energy burden on the cell.     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

The Hexose-Proton Cotransport System of Chlorella : pH-Dependent Change in Km Values and Translocation Constants of the Uptake System

The proton concentration in the medium affects the maximal velocity of sugar uptake with a K_m of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K_m of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V_max values at low substrate concentration versus proton concentration results in a K_m for protons of 0.14 µM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0–6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga.     

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

Effects of diclofop and diclofop-methyl on the membrane potentials of wheat and oat coleoptiles

Electrophysiological measurements were made on the mesophyll cells of wheat (Triticum aestivum L. cv Waldron) and oat (Avena sativa L. cv Garry) coleoptiles treated either with the herbicide diclofop-methyl (methyl 2-(4-(2′,4′-dichlorophenoxy)phenoxy)propanoate), or it's primary metabolite diclofop, (2-(4-(2′,4′-dichlorophenoxy)phenoxy)-propanoic acid). Application of a 100 micromolar solution of diclofop-methyl to wheat coleoptiles had little or no effect on the membrane potential (E_M), however in oat, E_M slowly depolarized to the diffusion potential (E_D). At pH 5.7, 100 micromolar diclofop rapidly abolished the electrogenic component of the membrane potential in both oat and wheat coleoptiles with half-times of 5 to 10 minutes and 15 to 20 minutes, respectively. The concentrations giving half-maximal depolarizations in wheat were 20 to 30 micromolar compared to 10 to 20 micromolar in oat. The depolarizing response was not due to a general increase in membrane permeability as judged from the E_M's response to changes in K⁺, Na⁺, Cl⁻, and SO₄²⁻, before and after treatment with diclofop and from its response to KCN treatment. In both plants, diclofop increased the membrane permeability to protons, making the E_M strongly dependent upon the external pH in the range of pH 5.5 to pH 8.5. The effects of diclofop can best be explained by its action as a specific proton ionophore that shuttles protons across the plasmalemma. The rapidity of the cell's response to both diclofop-methyl (15-20 minutes) and diclofop (2-5 minutes) makes the ionophoric activity a likely candidate for the earliest herbicidal event exhibited by these compounds.