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.     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.     

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

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.     

Effect of the electrochemical sodium gradient on alanine transport in 3T6 and CHO cells

The dependence of L-alanine uptake by 3T6 and CHO-K1 cells on Na+ electrochemical gradient has been studied. The Na+ chemical gradient was changed by a short-term (partial or complete) replacement of Na+ for choline. The membrane potential change was achieved by addition of potassium ionophore--valinomycin (10 microM) into the medium. It is determined that the value of Km for alanine uptake by 3T6 cells increases from 2 mM, with 140 mM Na+ in the medium, up to 30 mM, if the replacement of Na+ for choline is complete. Similar results are obtained for CHO cells. The membrane potential increase under the influence of valinomycin leads to the increase in the value of Vmax of the uptake. The data obtained are interpreted on the basis of the well known scheme of Na+ alanine complex transfer, where Na+ increases the affinity of the carrier to the amino acid, and the membrane potential increases the carrier mobility.     

Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.     

Mechanism of the stimulation of serine and alanine transport into isolated rat liver cells by bicarbonate ions.

1. Bicarbonate ions stimulate the transport of serine and alanine into isolated hepatocytes. 2. The effect of bicarbonate is to increase the Vmax. of the transport process without changing the apparent Km. 3. The intracellular pH was estimated from the distribution of the weak base methylamine and the weak acid 5,5'-dimethyloxazolidine-2,4-dione (DMO) across the plasma membrane. 4. The addition of bicarbonate to a cell suspension caused the internal pH to become more acid. 5. The initial rate of serine, alanine and glycine transport was a linear function of the initial difference in pH across the membrane. 6. It is concluded that bicarbonate activates the transport of these amino acids primarily by increasing the pH difference across the plasma membrane. 7. It is suggested that the uptake of serine together with Na+ ions occurs in exchange for H+ ions, which are translocated outwards on the same carrier system. Some preliminary evidence consistent with this model is presented.     

Gliotoxins disrupt alanine metabolism and glutathione production in C6 glioma cells: a 13C NMR spectroscopic study

Gliotoxins are a group of amino acids that are toxic to astrocytes, and are substrates of high-affinity sodium-dependent glutamate transporters. In the present study, C6 glioma cells were preincubated for 20 h in the presence of 400 μM L--aminoadipate, L-serine-O-sulphate, D-aspartate or L-cysteate, as well as in the presence of the poorly transported L-glutamate uptake inhibitor, L-anti-endo-methanopyrrolidine dicarboxylate. In experiments following [3-¹³C]alanine metabolism, all toxins caused a decreased incorporation of label into glutamate. Production of labelled lactate changed only when cells were incubated in the presence of L--aminoadipate or L-serine-O-sulphate. Incubation with L-anti-endo-methanopyrrolidine dicarboxylate caused no change in the amount of label incorporated into either glutamate or lactate. When glutathione production was followed using 1 mM [2-¹³C]glycine, differential effects of the gliotoxins were revealed. Most notably, both L-serine-O-sulphate and L--aminoadipate caused significant increases in labelling of glutathione. Once again, L-anti-endo-methanopyrrolidine dicarboxylate was without effect. Overall, we have shown that the gliotoxins cause disruption to alanine metabolism and glutathione production in C6 glioma cells, but that there are notable differences in their mechanisms of action. In the absence of any disruption to metabolism by L-anti-endo-methanopyrrolidine dicarboxylate, it is concluded that their mode of action involves more than inhibition of glutamate transport.     

Quantitative analysis of proton-linked transport systems. Glutamate transport in Staphylococcus aureus.

1. The magnitude of the protonmotive force in respiring Staphylococcus aureus was measured over the range of extracellular pH from 5.6 to 7.8. 2. The membrane potential remains constant at 150 mV, inside-negative, but the pH gradient decreases from 2.1 units, inside-alkaline, at pH 5.6 to zero at pH 7.5 and above. 3. The accumulation of glutamate in the soluble cell pool is pH-independent at a value equivalent to 100 mV. 4. The results of experiments studying co-transport of protons are consistent with a proton/glutamate stoichiometry of 2 and electrogenic transport across the pH range examined. 5. The amount of glutamate uptake is the result of a kinetic steady state between influx and efflux pathways. 6. Evidence is presented for the regulation of this kinetic steady state by the response of the initial rate of uptake to changes in the protonmotive force.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Control of alanine metabolism in rat liver by transport processes or cellular metabolism.

1. Factors governing hepatic utilization of alanine were studied in vivo and in vitro in rats adapted to increasing dietary protein. 2. Hepatic alanine utilization was enhanced 5-fold with a 90%-casein diet, compared with a 13%-casein diet. The increased uptake resulted from enhanced fractional extraction in the presence of high concentrations of alanine in the portal vein. 3. The increase in alanine metabolism on high-protein diets was associated with an increase in alanine aminotransferase and in pyruvate utilization for gluconeogenesis. 4. The emergence of a high-affinity component appeared to be responsible for the enhanced transport of alanine with high-protein diets. 5. High extracellular concentrations after alanine loads resulted in a maximal rate of utilization and of accumulation of alanine by liver cells in vivo and in vitro. Alanine accumulation was particularly active with high-protein diets. 6. In starved rats, alanine transport was also increased, but low concentrations of alanine in afferent blood contributed to make transport limiting for alanine utilization. 7. In fed rats, the rates of transport and catabolism of alanine generally appear to undergo parallel changes; both processes thus play a fundamental role in the control of alanine utilization by the liver.     

Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles

The mechanisms of lactate and pyruvate transport across the plasma membrane of rat skeletal muscle under various pH and ionic conditions were studied in skeletal muscle sarcolemmal (SL) membrane vesicles purified from 22 female Sprague-Dawley rats. Transport by SL vesicles was measured as uptake of L(+)-[U-14C] lactate and [U-14C] pyruvate. Lactate (La-) transport is pH-sensitive; stimulations to fivefold overshoot above equilibrium values were observed both directly by a proton gradient directed inward, and indirectly by a monensin- or nigericin-stimulated exchange of Na+ or K+ for H+ across the SL. Isotopic pyruvate could utilize the transporter, and demonstrated pH gradient-stimulated overshoot and cis-inhibition characteristics similar to those of lactate. Overshoot kinetics were also demonstrated by pH gradient formed by manipulation of external media at pH 5.9, 6.6, and 7.4 and intravesicular media at 6.6, 7.4, and 8.0, respectively. Carbonyl cyanide m-chlorophenylhydrazone, an H+ ionophore, was used as a "pH clamp" to return all stimulated uptake courses back to equilibrium values. Lactate uptake was depressed when internal pH was lower than external pH. These data strongly suggest that La- and H+ are either cotransported by the carrier, or transported as the undissociated HLa, and can account for the majority of the lactate uptake at pH 7.4. The mechanism does not require cotransport of either K+ or Na+. However, an inwardly directed Na+ gradient without ionophore in the absence of a pH gradient doubled La- transport; treatment with amiloride, an inhibitor of the Na+/H+ exchanger, abolished this stimulation, suggesting that this transporter may be an important coregulator of intracellular pH, and could disrupt 1:1 H+ and La- efflux stoichiometry in vivo. We conclude that the majority of La- crosses the skeletal muscle SL by a specific carrier-mediated process that is saturable at high La- concentrations, but flux is passively augmented at low intracellular pH by undissociated lactic acid. In addition, a Na+/H+ exchange mechanism was confirmed in skeletal muscle SL, does affect both lactate and proton flux, and is potentially an important coregulator of intracellular pH and thus, cellular metabolism.     

Brain oxygen utilization is unchanged by hypoglycemia in normal humans: lactate, alanine, and leucine uptake are not sufficient to offset energy deficit

During hypoglycemia, substrates other than glucose have been suggested to serve as alternate neural fuels. We evaluated brain uptake of endogenously produced lactate, alanine, and leucine at euglycemia and during insulin-induced hypoglycemia in 17 normal subjects. Cross-brain arteriovenous differences for plasma glucose, lactate, alanine, leucine, and oxygen content were quantitated. Cerebral blood flow (CBF) was measured by Fick methodology using N(2)O as the dilution indicator gas. Substrate uptake was measured as the product of CBF and the arteriovenous concentration difference. As arterial glucose concentration fell, cerebral oxygen utilization and CBF remained unchanged. Brain glucose uptake (BGU) decreased from 36.3+/-2.6 to 26.6+/-2.1 micromol.100 g of brain(-1).min(-1) (P<0.001), equivalent to a drop in ATP of 291 micromol.100 g(-1).min(-1). Arterial lactate rose (P<0.001), whereas arterial alanine and leucine fell (P<0.009 and P<0.001, respectively). Brain lactate uptake (BLU) increased from a net release of -1.8+/- 0.6 to a net uptake of 2.5+/-1.2 micromol.100 g(-1).min(-1) (P<0.001), equivalent to an increase in ATP of 74 micromol.100 g(-1).min(-1). Brain leucine uptake decreased from 7.1+/-1.2 to 2.5 +/- 0.5 micromol.100 g(-1).min(-1) (P<0.001), and brain alanine uptake trended downward (P<0.08). We conclude that the ATP generated from the physiological increase in BLU during hypoglycemia accounts for no more than 25% of the brain glucose energy deficit.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Effect of hyper-osmotic stress on alanine content of Leishmania major promastigotes

Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. An increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions. Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. To estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-14C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of 14CO2 and 14C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Kinetic analysis of the protonation of a surface group of a macromolecule

High-field 31P-NMR studies of whole cells of Streptococcus faecalis have shown that delta pH can be formed by ATP hydrolysis and also by lactate transport. We have used 31P-NMR to measure the pH dependence of the variable stoichiometry of the proton/lactate carrier. At low external pH (pH approximately equal to 6.5) the influx stoichiometry was 1.1 H+/lactate, while at high pH (7.5) the ratio was almost 2; the apparent midpoint pH of this variable stoichiometry is 7. delta psi measurements support the electrogenic nature of lactate transport at high pH; the variable rate of membrane depolarization caused by lactate transport also had a midpoint near pH 7.0. The data is consistent with a symmetrical carrier operating with variable stoichiometry as proposed by Michels et al.     

Active transport of charged substrates by a proton/sugar co-transport system. Amino-sugar uptake in the yeast Rhodotorula gracilis.

1. In the yeast Rhodotorula gracilis several amino sugars were actively transported. Glucosamine, which is largely protonated at physiological pH (pK 7.75) was used as a model substrate. At pH 6.75 its half-saturation constant was 1 mM and the maximal velocity was 50 nmol/min per mg dry wt. 2. Amino sugars were taken up via the monosaccharide carrier. The transport of glucosamine was strongly restricted by monosaccharides. D-Xylose inhibited competitively the uptake of glucosamine. The inhibition constant was 1 mM. Cells preloaded with D-xylose showed exchange transport on subsequent addition of glucosamine. 3. Transport of glucosamine was energized by the membrane potential. Uncoupling agents such as carbonyl cyanide m-chlorophenyl-hydrazone and the lipophilic cation TPP+ (tetraphenylphosphonium ion) at concentrations that depolarized the membrane potential inhibited the uptake of glucosamine. Conversely the transport of glucosamine partly dissipated the membrane potential, which was monitored by radioactively labelled lipophilic cations. 4. The translocated charges were electrically compensated by the extrusion of protons and K+ (1 glucosamine molecule/0.85 H+ + 0.15 K+). 5. An increase of the pH in the range 4.75-8.75 lead to a decrease of the half-saturation constant from 5 mM to 1 mM and to an optimum of the maximal velocity at pH 6.75. We suggest that this fair constancy is due to the carrier not distinguishing between the protonated form of glucosamine (pH less than 7.75) and the deprotonated form (pH greater than 7.75). The increase of V(T) (maximal transport velocity) between pH 4.75 and 6.75 is due to the increase of the membrane potential: the decrease between pH 6.75 and 8.75 is due to the deprotonization of the carrier.     

Stimulation of alanine transport and metabolism by dibutyryl cyclic AMP in the hepatocytes from fed rats. Assessment of transport as a potential rate-limiting step for alanine metabolism

(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2-5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na+ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na+ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.