Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Studies on isolated subcellular components of cat pancreas

Summary Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na⁺K⁺)-ATPase, and 5-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membrane enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na⁺K⁺)-ATPase.     

Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [³H]GABA release induced by ouabain, veratridine and Na⁺ substitution by other monovalent cations (K⁺, Rb⁺, Li⁺, and choline). We observed that ouabain is unable to release [³H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [³H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na⁺ and it is inhibited by extravesicular Ca²⁺ (1 mM) under conditions which appear to permit its entry. However, veratridine-induced [³H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na⁺ by other monovalent cations promotes [³H]GABA release by altering the Na⁺ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li⁺, we observed that the fraction of [³H]GABA released relatively to the total amount of neurotransmitter released by K⁺ or Rb⁺ is about 28% and 68%, respectively. Since the replacement of Na⁺ by K⁺, Rb⁺, and Li⁺ causes different levels of membrane depolarization, and the replacement of Na⁺ by choline causes hyperpolarization of the vesicles, these results suggest that, in parallel to the [³H]GABA release, which is directly proportional to the level of membrane depolarization, this neurotransmitter can be released by decreasing the external Na⁺, which reflects an elevation of the Na⁺ concentration gradient (inout). Like veratridine-induced release, the depolarization-induced release of [³H]GABA by SPM vesicles is inhibited by Ca²⁺, which suggests that this divalent cation interfers with the cytoplasmic GABA release mechanism.Abbreviations used ATPase adenosine triphosphatase - GABA -aminobutyric acid - Mes 2 (N-morpholino)-ethanosulfonic acid - SPM synaptic plasma membranes - membrane potential     

Comparison of high-affinity binding of [3H]GABA to subcellular particles of rat brain and liver

The binding of [³H]GABA and retention of [¹⁴C]sucrose have been studied in freshly prepared synaptosomal-mitochondrial (P₂) fractions of rat cerebral cortex and liver using bicarbonate-buffered medium (containing 147 mEq/liter of N⁺), and in frozen/thawed crude membrane fractions of rat whole brain and liver using Na⁺-free Tris HCl medium. GABA-sensitive sites (GSS) and bicucul-line-methiodide-(BMI-) sensitive sites (BMI-SS) were defined as those amounts of [³H]GABA that were sensitive to the displacement by 10^–3 M unlabeled GABA or BMI. In the presence of added Na⁺, two high-affinity GABA-binding processes were detected in the P₂fraction of cerebral cortex. The lower-affinity process (likely related mainly to uptake sites) hadK _B10^–5 M,B _max for GSS3 nmol/mg protein, andB _max for BMI-SS0.5 nmol/mg protein, whereas the higher-affinity process (likely related to synaptic GABA receptors) hadK _B10^–7 M,B _max for GSS43 pmol/mg protein, andB _max for BMI-SS2 pmol/mg protein. Only the higher-affinity process was detected in the liver P₂ fraction and it hadK _B3.7×10^–8 M,B _max for GSS0.48 pmol/mg protein, andB _max for BMI-SS0.1 pmol/mg protein (i.e., about 1/100 and 1/20 the receptiveB _max values of cerebral cortex). This binding process of the liver P₂ fraction could represent sites involved in mitochondrial GABA transport. In Na⁺-free Tris HCl medium, high-affinity [³H]GABA binding appeared to exist in frozen/thawed membrane preparations of both brain and liver when data were expressed on a protein basis. However, this binding to liver membranes was not displaceable by 10^–3 M unlabeled GABA, and when these data were expressed on a weight basis and corrected for [³H]GABA present in trapped supernatant fluid of the pellets, no [³H]GABA binding was detected in the liver preparation.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.     

Lack of specificity in cation effects on solubilized benzodiazepine receptor

Receptors for benzodiazepines (BZ) and -carboline-carboxylic acid ethyl ester (-CCE) has been solubilized with decanoly-N-methylglucamide (DMG), a new kind of nonionic detergent. The apparent dissociation constants of diazepam and -CCE for solubilized receptor were similar to those for synaptic membranes. Sucrose density gradient centrifugation of the solubilized receptor protein revealed that the binding profile of [³H]-CCE essentially parallels that of [³H]diazepam and that both sedimentation coefficients were 10.5S. Co²⁺ and Ni²⁺, which increase [³H]diazepam binding and decrease [³H]-CCE binding to synaptic membranes, remarkably increased the binding of both to the solubilized receptor. Mg²⁺ and Ca²⁺, which had no effect on membrane receptor binding, also enhanced [³H]diazepam and [³H]-CCE binding to the solubilized receptor. The increase in binding in the presence of these divalent cations was due to a change in the apparent number of binding sites, with no change in binding affinities. The relative lack of specificity in divalent cation effects on solubilized BZ receptor may be caused by separation or destruction of the cation recognition site or channel of the BZ receptor complex by solubilization of the synaptic membrane with DMG.     

The tyramine binding site in the central nervous system: An overview

The [³H]Tyramine (TY) binding site is proposed as a high affinity marker of the membrane carrier for dopamine (DA) in synaptic vesicles from DA-rich brain regions. Under precise assay conditions, there is neither a consistent association of TY with the neuronal, cocaine-sensitive DA transporter, nor with mitochondrial or microsomal targets. TY-labeled sites have a high affinity for selected toxins such as the Parkinsonian agent MPP⁺ (1-methyl-4-phenylpyridinium ion), or drugs such as diphenylalkylamine Ca²⁺-channel antagonists. The MPP⁺/TY site interaction, which in the striatum leads to depletion of vesicular DA, occurs in dopaminergic as well as in noradrenergic regions, though with different kinetic profiles. TY-labeled carriers for DA and noradrenaline (NA) in respective vesicles seem to be different entities, which might result in a region-specific rate of toxin sequestration and/or release from heterogeneous vesicles. Whereas MPP⁺ is a potent competitive-type inhibitor of [³H]TY binding, prenylamine-like Ca²⁺-channel antagonists can compete with TY for the vesicle site, in a tetrabenazine- or reserpine-like manner, and also inhibit TY binding thanks to the extra-channel directed impairment of membrane bioenergetics they are proposed to provoke. This follows from the generally-accepted assumption that similar mechanisms are operational for secretory organelles in adrenals and CNS, and from the marked sensitivity of TY binding to miscellaneous energy-disrupting agents. A model is therefore proposed, depicting the TY-, DA- or MPP⁺-labeled, vesicle carrier, as a dimeric protein which may switch from the cytoplasm-oriented, recognition state, to the vesicle-oriented, transport state, thanks to the establishment of an H⁺-ATPase-supported, membrane protein electrochemical gradient.     

Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Potent,extra-channel influence of several calcium-channel modulators on striatal binding of [3H]tyramine

A number of Ca²⁺-, K⁺-, and Na⁺-channel modulators has been tested with respect to their effects on [³H]tyramine (TY) binding, as a putative marker for the vesicular dopamine (DA) transporter in striatal membrane preparations containing vesicle ghosts. Among organic Ca²⁺-channel modulators, the diphenylalkylamines tested consistently inhibited TY binding: the order of potency was prenylamine>lidoflazine>flunarizine>cinnarizine, with K_i values of 0.1, 0.2, 0.5 and 1.2 M, respectively. Low (up to 100 nM) concentrations of prenylamine did competitively inhibit TY binding, and higher concentrations provoked a mixed-type inhibition. Furthermore, LIGAND-analysis of competition curves revealed a high- and a low-affinity binding site for prenylamine and flunarizine. The TY binding process was also sensitive to selected K⁺- and Na⁺-channel modulators. Since several Ca²⁺-antagonists are known to affect H⁺-ATPase and the bioenergetics of catecholamine storage vesicles in chromaffin granules, thus affecting monoamine storage, the energy requirements for the formation of the TY/carrier complex were here assessed, assuming similarity between chromaffin granules and synaptic vesicles. TY binding, though not reflecting endovesicle-sequestered TY, was indeed strongly sensitive (with K_i coefficients in the fM or low nM range) to the dissipation of the vesicular transmembrane proton concentration ( pH), electrical ( ), and proton electrochemical ( H⁺) gradients, provoked by a number of specifically targeted agents. It is concluded that Ca²⁺-channel agents of the diphenylalkylamine group may directly affect striatal TY binding due to an extrachannel-regulated competition with TY for the vesicular carrier of DA, as well indirectly, by disruption of the transmembrane energization of the reserpine-sensitive carrier.     

Purification and characterization of plasma membrane fractions from cultured pituitary cells

Highly purified plasma membrane fractions have been prepared from GH₃ pituitary cells grown in suspension cultures. These membrane fractions have been obtained by differential and sucrose gradient centrifugation and were characterized in terms of their lipid content, marker enzyme analysis and the binding of ³H-labelled thyrotropin-releasing hormone (TRH) to its receptor. Alkaline phosphatase and 5′-nucleotidase activities were enriched 12- to 15-fold in the plasma membrane fraction with somewhat greater enrichment (28-fold) of the specific binding component for [³H]TRH, with a specific activity of 2286 fmol [³H]TRH bound per mg protein. A single class of binding sites for TRH was observed with an apparent dissociation constant of 18 nM, a value similar to that observed for intact cells. No detectable TRH binding to the nuclear fraction was observed that could not be ascribed to residual plasma membrane contamination. By electron microscopy, these fragments appeared to be sealed vesicles with an average diameter of approximately 1800 Å. The binding of ¹²⁵I-labelled wheat germ agglutinin was used as a marker for plasma membrane purification. In addition to specific binding to this membrane fraction, specific binding was also observed in the nuclear fraction. Studies with fluorescein-labelled wheat germ agglutinin revealed that, in fixed cells, fluorescence was restricted to the plasma membrane. However, if the cells were treated with Triton before labelling, most of the fluorescence was then associated with the cell nucleus. Hence, the use of wheat germ agglutinin binding as a specific plasma membrane marker must be reevaluated.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

Effects of nitrite exposure on blood respiratory properties,acid-base and electrolyte regulation in the carp (Cyprinus carpio)

Summary Adult carp were subjected to 1 mM environmental nitrite for 48 h and nitrite uptake and changes in blood respiratory properties, extracellular electrolyte composition and acid-base status were examined.A constant influx of nitrite caused an accumulation of NO ₂ ^– in plasma to 5.4 mM in 48 h. The fraction of methaemoglobin rose with plasma [NO ₂ ^– ] to 83%, and the arterial oxygen content decreased to extremely low values. Arterial increased as a compensation to this O₂-shortage, whereas the O₂ saturation of the functional (unoxidized) haemoglobin decreased, revealing a reduction in its O₂ affinity.Blood haematocrit decreased as a result of red cell shrinkage, which caused very high red cell haemoglobin (Hb) concentrations. The erythrocytic nucleoside triphosphate (NTP) concentration showed a parallel increase whereby NTP/Hb, as well as the relative contributions of ATP and GTP to NTP, remained unchanged.Plasma [Cl^–] declined by 15 mM in 48 h, off-setting the plasma [NO ₂ ^– ] increase, minor changes in plasma [HCO ₃ ^– ] and a considerable increase in plasma [lactate]. Arterial pH and [HCO ₃ ^– ] rose slightly during the first 24 h of nitrite exposure, but returned to control values at 48 h. The rise in plasma [lactate] was not reflected in an extracellular metabolic acidosis. Plasma [K⁺] increased by 94% in 48 h, revealing an uncompensated extracellular hyperkalemia, whereas plasma [Na⁺] decreased, and plasma [Ca⁺⁺] was unchanged. Plasma osmolality remained essentially constant.The NO ₂ ^– accumulation could be reversed by transfer of the fish to NO ₂ ^– -free water, but nitrite off-loading was slower than the preceding NO ₂ ^– loading.Abbreviations Hb hemoglobin - NTP nucleoside triphosphate - Hct hematocrit - fractional saturation of Hb with oxygen     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Synaptosomal non-mitochondrial ATPase activities and drug treatment

Energy-using non-mitochondrial ATPases were assayed in rat cerebral cortex synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The following enzyme activities were evaluated: Na⁺, K⁺-ATPase; high- and low-affinity Ca²⁺-ATPase; basal Mg²⁺-ATPase; Ca²⁺, Mg²⁺-ATPase. The evaluations were performed after four week-treatment with saline [controls] or -adrenergic agents (-yohimbine, clonidine), energymetabolism interfering compound (theniloxazine), and oxygen-partial pressure increasing agent (almitrine), in order to define the plasticity and the selective changes in individual ATPases. In rat cerebral cortex, the enzyme adaptation to four-week-treatment with -yohimbine or clonidine was characterized by increase in both high- and low-affinity Ca²⁺-ATPase activities. The action involves the enzyme form located in the synaptic plasma membranes. The enzyme adaptation to the subchronic treatments with theniloxazine or almitrine was characterized by increase in Na⁺, K⁺-ATPase or Mg²⁺-ATPase activities, respectively. The action involves the enzymatic forms located in the synaptic plasma membranes. Thus, the pharmacodynamic effects of the agents tested should also be related to the changes induced in the activity of some specific synaptosomal nonmitochondrial ATPases.     

Vesicular and carrier-mediatied depolarization-induced release of [3H]GABA: Inhibition by amiloride and verapamil

The Ca²⁺-dependent, presumably exocytotic fraction of the [³H]GABA released by depolarization is dissected from the depolarization-induced Na⁺-dependent, carrier-mediated fraction of [³H]GABA release in mouse brain synaptosomes. GABA homoexchange is prevented by the [³H]GABA carrier blocker, DABA. The absence of external Na⁺ completely abolishes the release of the carrier-mediated, presumably cytoplasmic release of [³H]GABA induced by homoexchange and heteroexchange with GABA and DABA, respectively. The carrier-mediated, Na⁺-dependent fraction of the depolarization-induced release of [³H]GABA is resistant to tetrodotoxin (TTX) but is sensitive to amiloride and verapamil. The Ca²⁺-dependent fraction of the [³H]GABA released by high K⁺ depolarization is also completely abolished by amiloride (from 300 M) and sensitive to verapamil (30 M), but in contrast is insensitive to the absence of external Na⁺ and to DABA. On the basis of these results we conclude that amiloride and verapamil inhibit high K⁺-induced release of [³H]GABA by antagonizing the entrance of Ca²⁺ (and possibly Na⁺ when external Ca²⁺ is absent) through a population of voltage sensitive presynaptic Ca²⁺ channels activated by depolarization.Depto. de Biología Molecular Instituto de Investigaciones Biomédicas UNAM.