Stoichiometry of sodium- and chloride-coupled glycine transport in synaptic plasma membrane vesicles derived from rat brain

The stoichiometric properties of the glycine transporter were studied in synaptic plasma membrane vesicles from rat brain. The present results, together with previous data from our laboratory, allow us to suggest a stoichiometry of 2 Na+ and 1 Cl- per glycine zwitterion for the translocation cycle catalyzed by the glycine carrier. We propose a kinetic model with an ordered mechanism for the binding/debinding of solutes.     

Ontogenetic studies on tryptophan transport into plasma membrane vesicles derived from rat brain synaptosomes: Effect of thyroid hormones

The uptake of tryptophan at various stages of development was examined in plasma membrane vesicles derived from rat brain. The total uptake has two components Na⁺-dependent and Na⁺-independent respectively. The Na⁺-dependent component of the transport system appears around the 5th postnatal day and increases with the age. TheK _m value of the system does not vary during development. The V_max increases five-fold between 14 and 35 day of postnatal life. Plasma membrane vesicles derived from T₃-treated rats are able to accumulate nearly three-fold more tryptophan than nontreated rats. The results support the idea that thyroid hormones at the earlier stages of life, promote the establishment of neurotransmission in the developing nervous system.     

Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes.

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.     

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl₂, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca²⁺]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K⁺]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

Interaction between phosphatidylserine vesicles and rat brain synaptosomes

Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated. These hybrid plasmids with the autonomous replication sequences from P. chrysosporium are maintained extra-chromosomally, are mitotically unstable and transform Ura3 deletion mutant of S. cerevisiae to Ura+ phenotype with high frequency. The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P. chrysosporium genomic DNA. Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning.     

Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain

Membrane vesicles from rat brain exhibit sodium-dependent uptake of L-[3H]glutamate in the absence of any transmembrane ion gradients. The substrate specificity of the process is identical with (Na+ + K+)-coupled L-glutamate accumulation. Although these vesicles are prepared after osmotic shock and are washed repeatedly, they contain about 1.5 nmol/mg of protein endogenous L-glutamate, apparently located inside the vesicles. The affinity of the process (Km approximately 1 microM) is similar to that of (Na+ + K+)-dependent accumulation by the L-glutamate transporter. Membrane vesicles have been disrupted by the detergent cholate, and the solubilized proteins have been subsequently reconstituted into liposomes. The reconstituted proteoliposomes also exhibit the above uptake--with the same characteristics--provided they contain entrapped cold L-glutamate. Counterflow is optimal when sodium is present on both sides of the membrane, but partial activity is still observed when sodium is present either on the inside or on the outside. Increasing the L-glutamate concentration above the Km results in counterflow completely independent of cis sodium. The initial rate of counterflow is 100-200-fold lower than that of net trans potassium dependent flux. The rate of net flux in the presence of trans sodium or lithium is about 10-fold lower than when choline or Tris are used instead. However, the rate of counterflow (no internal potassium present) was not stimulated by replacing internal sodium or lithium by internal choline. Therefore, optimal functioning of the transporter requires internal potassium while internal sodium and lithium are inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

Depolarization of plasma membrane of rat brain synaptosomes at extra- and intracellular acidification

To elucidate whether a decrease in pH_o and pH_i has the same effect on presynaptic plasma membrane depolarization, we compared the effects of 1 mM of amiloride which decreased pH_i down to 6.65 and incubation medium acidification down to pH^o 7.0 and 6.0 on DiSC3(5) dye fluorescence. Measurements were performed with and without the mitochondrial toxins: rotenone (10 μM) + oligomycin (5 μg/mL). In the presence of toxins the change in DiSC3(5) fluorescence was specific for plasma membrane potential. In the second case, DiSC3(5) signal represented the total potential of mitochondria and plasma membrane. It was shown that decreasing of pHo works both in the presence of mitochondrial toxins and also in their absence. Amiloride is able to increase DiSC3(5) fluorescence only in the presence of the mixture of rotenone + oligomycin. Therefore, unlike the mitochondrial potential, the plasma membrane potential is sensitive to a decrease in pH_i and pH_o.     

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108-116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na+/Cl-/glycine cotransport system is discussed.     

Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

A platelet membrane preparation, enriched in plasma membrane markers, took up 45Ca2+ in exchange for intravesicular Na+ and released it after the addition of inositol 1,4,5-trisphosphate (IP3). The possibility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca2+ released by IP3 was eliminated by the addition of vanadate to inhibit Ca+-ATPase-mediated DTS Ca2+ sequestration and by the finding that only plasma membrane vesicles exhibit Na+-dependent Ca2+ uptake. Ca2+ released by IP3 was dependent on low extravesicular Ca2+ concentrations. IP3-induced Ca2+ release was additive to that released by Na+ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca2+ influx in addition to release from DTS membranes.     

Uptake and release of kyotorphin in rat brain synaptosomes

Uptake and release of kyotorphin (TyrArg) in rat brain synaptosomes were studied. Synthetic kyotorphin was taken up into crude synaptosomes (P₂), in a temperature-dependent manner. The Km and Vmax of the uptake were 1.31 ± 0.12 × 10⁻⁴M and 5.9 ± 0.5 pmol/mg protein/min, respectively. Metabolic inhibitors such as dinitrophenol and iodoacetamide and ouabain which is known as an inhibitor of Na⁺ dependent uptake mechanism significantly inhibited the uptake. When the synaptosomes previously preloaded with synthetic kyotorphin at 10⁻⁴M were exposed to high K⁺ medium, kyotorphin was released in a Ca²⁺-dependent manner. These findings support the view that kyotorphin plays a role as neurotransmitter/neuroregulator.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Depolarization-induced release of ATP from cholinergic synaptosomes is not blocked by botulinum toxin type A

We report here the effects of Botulinum Toxin type A on the release of ATP and Acetylcholine from Torpedo electric organ synaptosomes. Our results show that Botulinum Toxin type A inhibits specifically the K⁺-induced release of Acetylcholine from synaptosomes without affecting the release of ATP. Membrane potential and calcium uptake into cholinergic nerve terminals are not modified after Botulinum Toxin poisoning. It is suggested that either most of the ATP released during the depolarization of the cholinergic synaptosomes does not originate from cholinergic synaptic vesicles or that there are two populations of synaptic vesicles, Acetylcholine-enriched synaptic vesicles and ATP-enriched synaptic vesicles. However, the possibility that the ACh and ATP released could come from different intrasynaptosomal compartments cannot be excluded.     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

The structure of detergent-resistant membrane vesicles from rat brain cells

The size and the bilayer thickness of detergent-resistant membranes isolated from rat brain neuronal membranes using Triton X-100 or Brij 96 in buffers with or without the cations, K⁺/Mg²⁺ at a temperature of either 4 °C or 37 °C were determined by dynamic light scattering and small-angle neutron scattering. Regardless of the precise conditions used, isolated membrane preparations consisted of vesicles of ∼ 100 to 200 nm diameter as determined by dynamic light scattering methods, equating to an area of the lipid based membrane microdomain size of 200 to 400 nm diameter. By means of small angle neutron scattering it was established that the average thickness of the bilayers of the complete population of detergent-resistant membranes was similar to that of the parental membrane at between 4.6 and 5.0 nm. Detergent-resistant membranes prepared using buffers containing K⁺/Mg²⁺ uniquely formed unilamellar vesicles while membranes prepared in the absence of K⁺/Mg²⁺ formed a mixture of uni- and oligolamellar structures indicating that the arrangement of the membrane differs from that observed in the presence of cations. Furthermore, the detergent-resistant membranes prepared at 37 °C were slightly thicker than those prepared at 4 °C, consistent with the presence of a greater proportion of lipids with longer, more saturated fatty acid chains associated with the Lo (liquid-ordered) phase. It was concluded that the preparation of detergent-resistant membranes at 37 °C using buffer containing cations abundant in the cytoplasm might more accurately reflect the composition of lipid rafts present in the plasma membrane under physiological conditions.     

Lanthanides induce neurotransmitter release from the vesicular pool in rat brain synaptosomes

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.