Effects of glycine on dark-and ligh-induced pulvinar movements and modifications of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake

Glycine (1–50 mM) increases the rate of the dark-induced (scotonastic) movements and decreases the amplitude and the rate of the light-induced (photonastic) movements of the secondary pulvini of Mimosa pudica leaves. The uptake of glycine is accompanied by a long-lasting dose-dependent increase in the alkalinity of the bathing medium of the excised pulvini. The data are in agreement with a H⁺-glycine co-transport mechanism within the pulvinar cells. Fusicoccin (50 M), known to promote H⁺–K⁺ exchange, antagonizes the effects of glycine on the movements and the alkalization of the bathing medium of the excised pulvini. The present results argue for the hypothesis that proton fluxes mediate the scotonastic and photonastic pulvinar movements.Abbreviations Gly glycine - FC fusicoccin - P₁ primary pulvinus - P₂ secondary pulvinus     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

Glycine transport in rat brain and liver mitochondria

Transport of glycine by rat brain and liver mitochondria has been investigated by both [¹⁴C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoosmotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent K_m of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.     

pH-dependent glycine uptake in the presence and absence of sodium ions from rat small intestine

Intestinal uptake of glycine in rats was stimulated 15-20% in the presence of 120 mM Na at pH 6.0 and below but around neutral pH, the amino acid uptake was augmented to 60% compared to that in the Na-free medium. Glycine uptake was 30% more at pH 5.5 compared to that at pH 7.3 in the absence of Na. Kinetic analysis revealed a decrease in Kt for glycine uptake (9.62 mM) at pH 5.5 compared to that at pH 7.3 (Kt = 16.67 mM) with no change in maximal velocity (1.51 mumole/10 min/g tissue) in Na-free buffer. Addition of -SH group reacting reagents to the incubation medium produced 36-58% inhibition of glycine uptake in the presence of Na. However, in absence of Na, inhibition of the order of 21-35% and 8-23% was observed at pH 5.5 and 7.0, respectively. These findings suggest that glycine uptake in rat intestine is influenced by pH and -SH groups are implicated in the process(es).     

Glycine transport by hemolysed and restored pigeon red cells. Effects of a Donnan-induced electrical potential on entry and exit kinetics.

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl- with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined. In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the deprotonated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face. The V for glycine entry was determined for cells with their Cl- largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VC1, was 1.5-1.7. VT/VC1 rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl-, the analogous ratio (VGlu/VC1) was 1.7. The increase of VT/VC1 above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane. When cell Cl- was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl- was replaced by toluenedisulfonate therewas a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl- with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl-]/ medium [Cl-] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a "moving" charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl- medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+. The observed influences of the Donnan effect on V (glycine entry), on both components of Km (glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl-]/medium [Cl-] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it "across" the membrane and that no other steps involve charge movement. The properties of the system seem inconsistent with a translational ("ferry boat") mobile carrier.     

Hormonal and gravitropic specificity in the regulation of growth and cell wall synthesis in pulvini and internodes from shoots of Avena sativa L. (oat)

Segments can be cut from the peduncular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive leaf-sheath pulvinus and gibberellin-sensitive internodal tissue. Incorporation of [14C]glucose was used to monitor cell wall synthesis in these two tissues as affected by gravistimulus, indoleacetic acid (IAA), gibberellic acid (GA3), and fusicoccin (FC). Pulvinar cell wall synthesis was promoted by IAA and FC (both within about 1 h), as well as by gravistimulus (starting between 3 and 6 h), whereas GA3 had no effect on nongravistimulated pulvini. In contrast, GA3 and FC promoted internodal cell wall synthesis (initiated between 1 and 2 h), whereas IAA and gravistimulus caused a decrease in internodal uptake. FC preferentially promoted incorporation into the matrix component of the wall in both tissues. Gravistimulus failed to increase responsiveness of pulvinar tissue to IAA, whereas GA3 partially overcame gravistimulus-promoted incorporation into pulvinar cell wall, probably because of preferential movement of label into the rapidly elongating internode. The results demonstrate that these eight stimulus/tissue combinations can be examined easily in an isolated 10-mm stem segment, providing new opportunities for the comparative study of tissue- and stimulus-specific events in gene regulation and signal transduction in agronomically important cereals.     

Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus

This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high- and a low-affinity uptake mechanism. The high-affinity mechanism had a Km of 25.2-30.5 microM and a Vmax of 3.8-4.8 nmol/10 mg of cell water/5 min, whereas the low-affinity mechanism had a Km of 633-718 microM and a Vmax of 26.6-37.1 nmol/10 mg of cell water/5 min. At steady state, the high-affinity mechanism accumulated 10 microM [3H]glycine from the medium, achieving tissue concentrations that were 13-24 times that in the medium. The high-affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+-dependent release and the high-affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision.     

Effect of compounds structurally related to taurine and of taurine uptake inhibitors on the motility of hamster sperm in vitro

Several taurine-related compounds, taurine antagonists and taurine uptake inhibitors were tested for their effect on hamster sperm motility in vitro. Hypotaurine was approximately three times more effective than taurine. N-methyltaurine and taurocyamine were less effective. Inactive taurine-related compounds were not effective blockers of taurine's spermtimulating activity. However, 1-(4-nitrophenyl)-2-dimethylaminomethyl-l-propenone, a taurine uptake inhibitor, completely suppressed sperm motility at a molar concentration equal to, or less than, that of the taurine added to the incubation medium and also suppressed the motility-sustaining action of the cumulus oophorus.     

Oxygen uptake, acidification of medium and nitrate uptake induced by blue light in nitrate-starved Chlorella cells

Blue light-induced oxygen uptake of the colorless mutant of Chlorella kessleri (No. 9.80) was 30-40% higher in the presence of exogenous glycine than in its absence. None of the other amino acids tested had this effect. Moreover, mutant cells in which glutamine synthetase was inhibited by methionine sulphoximine, accumulated approximately 65% more ammonium ions under blue irradiation in the presence of exogenous glycine than in its absence. The protein kinase C inhibitors, staurosporine or K252a, reduced the enhancement of oxygen uptake by approximately 40%. The present results indicate that blue light-dependent deamination of endogenous glycine might be a prerequisite for enhanced oxygen uptake in Chlorella. This blue light-induced oxygen uptake was not influenced by the inhibitors of protein phosphatase, calyculin A or okadaic acid. On the contrary, calyculin A and okadaic acid had a marked effect on the acidification of the suspension medium and nitrate uptake induced by blue light in Chlorella cells. The different responses to the inhibitors of protein kinase and phosphatase suggest the presence of different pathways among the blue light signal transduction operating on oxygen uptake, acidification of the medium and nitrate uptake in Chlorella.     

Differences of affinity and energetics of the active transport systems for amino acids in Chang liver cells.

The plasma membrane of Chang liver cells was shown to have at least two distinct active transport systems, one with preferential affinity for glycine and one for leucine. The uptakes of glycine and leucine were specificially inhibited by Me-AIB and b-BCH, respectively. The uptake of glycine decreased remarkably within 10 min on incubation with DNP (2 mM), KCN (5 mM), and malonate (20 mM) under aerobic conditions, along with a decrease of cellular ATP concentration to as low as 1/4 of normal, while the uptake of leucine was not depressed under these conditions. Leucine uptake was, however, greatly reduced within 10 min on incubation with DNP plus ICH2CONH2 (5 mM), when the cellular ATP was estimated at about 0.066 mM. The active transport of leucine, but not that of glycine, was accompanied by further acidification of the intracellular fluid, which was lower in pH than the extracellular fluid by approximately 0.3 unit without addition of amino acid to the medium.     

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.     

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

Calmodulin Implication in the Glycine Uptake by Mimosa pudica Pulvinar Cells

Previous experiments have evidenced that calcium is functionallyimplicated in glycine uptake by pulvinar motor cells of Mimosapudica L. The present data show that compounds having anticalmodulinproperties, compound 48/80 and the sulfonamide W-7, inhibitedthe amino acid uptake suggesting that a step in this processmay be regulated by calmodulin. H⁺ excretion by the tissuesand transmembrane potential of the motor cells were not modifiedby these compounds, thus showing that the inhibition of aminoacid uptake was not an indirect consequence of a decrease inthe proton motive force energizing the glycine H⁺ cotransport.Therefore, the data argue for the implication of calmodulinin a specific Ca²⁺-regulated reaction. (Received March 2, 1994; Accepted May 6, 1994)     

Changes in ethanol, lactate and malate content in Acer pseudoplatanus cells: effects of fusicoccin and O2 availability

Changes in the contents of ethanol, lactate and malate were determined at different activities of the plasma membrane H⁺ pump [in the presence and absence of fusicoccin (FC)] and at different O₂ availability in cultured cells of Acer pseudoplatanus L. FC induced acidification of the medium under all tested conditions of O₂ availability. At low O₂ concentrations both ethanolic and lactic fermentations occurred, and FC markedly stimulated lactate production but had no effect on ethanol production. There was also a small, stimulating effect of FC on malate production. At high O₂ concentrations no ethanol production was observed and lactate production was reduced. Under these conditions the stimulating effect of FC on lactate production decreased, while that on malate production increased. FC-induced synthesis of lactate and malate is interpreted as depending on the activation of lactate dehydrogenase (EC 1.1.1.27) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) (alkaline pH optima), respectively, due to the alkalinization of the cytoplasmic pH resulting from the stimulation of the H⁺ pump by FC. These results suggest that the balance between the two pH stat systems depends on the availability of O₂.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

Energetics of Amino Acid Uptake by Vicia faba Leaf Tissues

The uptake of [U-¹⁴C]threonine and of (α-¹⁴C]aminoisobutyrate (α-AIB) by Vicia faba leaf discs is strongly pH dependent (optimum: pH 4.0) and exhibits biphasic saturation kinetics. Kinetics of α-AIB uptake at different pH values indicate that acidic pH values decrease the K_m of the carriers while the maximal velocity remains nearly unaffected. Similar results were obtained for both system 1 (from 0.5 to 5 millimolar) and system 2 (from 20 to 100 millimolar).

After addition of amino acids to a medium containing leaf fragments, alkalinizations depending both on the amino acid added and on its concentration have been recorded.

The effects of compounds which increase (fusicoccin) or decrease (uncouplers, ATPase inhibitors, high KCl concentrations) the protonmotive force were studied both on the acidification of the medium and on amino acid uptake by the tissues. There is a close relationship between the time required for the effect of these compounds on the acidification and that needed for inhibition of uptake.

Studies with thiol inhibitors show that 0.1 millimolar N-ethylmaleimide preferentially inhibits uptake by the mesophyll whereas 0.1 millimolar parachloromercuribenzenesulfonate affects rather uptake by the veins.

New evidence was found which added to the electrophysiological data already supporting the occurrence of proton amino acid symport in leaf tissues, particularly in the veins.

     

Effect of High Physiological Temperatures on NAD+ Content of Green Leaf Mitochondria (Apparent Inhibition of Glycine Oxidation)

We observed a rapid decline in the rate of glycine oxidation by purified pea (Pisum sativum L.) leaf mitochondria preincubated at 40[deg]C for 2 min. In contrast, exogenous NADH and succinate oxidations were not affected by the heat treatment. We first demonstrated that the inhibition of glycine oxidation was not attributable to a direct effect of high temperatures on glycine decarboxylase/serine hydroxymethyltransferase. We observed that (a) addition of NAD+ to the incubation medium resulted in a resumption of glycine-dependent O2 uptake by intact mitochondria, (b) addition of NAD+ to the suspending medium prevented the decline in the rate of glycine-dependent O2 consumption by pea leaf mitochondria incubated at 40[deg]C, (c) NAD+ concentration in the matrix space collapses within only 5 min of warm temperature treatment, and (d) mitochondria treated with the NAD+ analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3[prime]-NAD+ retained high rates of glycine-dependent O2 uptake after preincubation at 40[deg]C. Therefore, we conclude that the massive and rapid efflux of NAD+, leading to the apparent inhibition of glycine oxidation, occurs through the specific NAD+ carrier present in the inner membrane of plant mitochondria. Finally, our data provide further evidence that NAD+ is not firmly bound to the inner membrane.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

Effect of butyric acid on the germination of the seeds of Phacelia tanacetifolia

The effect of a weak acid (butyric acid) on the germination of seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair) has been studied. Butyric acid inhibited the early phase of germination, and the inhibition was correlated to the amount of the per-meant undissociated form present in the incubation medium. The inhibition by butyric acid in the dark was also correlated to a decrease of dark fixation of CO₂ and to a more pronounced decrease in malic acid levels during the early phase of germination; suggesting that the uptake of the uncharged form of this weak acid was followed by a release of H+ into the cytoplasm, leading to a decrease in its pH. The inhibitory effect of butyric acid in the dark on many metabolic events (rise in respiratory activity, levels of reducing sugars, glucose-6–phosphate and malic acid, dark CO₂ fixation, transport activities and macromolecular synthesis) appeared similar to the one of light. Fusicoccin (FC), which directly stimulates the H⁺ pump at the plasmalemma level, ameliorated the effect of butyric acid, as well as that of light, on germination. The bulk of these data is interpreted as suggesting that the mechanism of light inhibition of germination of Phacelia tanacetifolia seeds might be the consequence of a general block of metabolic reactivation due to the presence of an unfavourable (acidic) cytoplasmic pH; which might be explained with the lack of the phytochrome-dependent activation of the H⁺ pump at the plasmalemma level.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.