Rat Cerebellar Synaptosomes Research Articles

Activation of α6 GABA A receptors on depolarized cerebellar parallel fibers elicits glutamate release through anion channels

Rat cerebellar synaptosomes labeled with [ 3 H] d-aspartate ( [ 3 H] d-ASP) were exposed in superfusion to muscimol. The GABA A receptor agonist did not affect [ 3 H] d-ASP basal release or the overflow provoked by 15 mM K +; muscimol potentiated the 35 mM K +-evoked overflow of [ 3 H] d-ASP or endogenous glutamate. Membrane potential measured by Rhodamine 6G fluorescence was −65 mV under resting conditions and −32 mV in the presence of 35 mM K +. The membrane potential was not significantly affected by muscimol. The muscimol effect on the K +(35 mM)-evoked [ 3 H] d-ASP overflow was not inhibited by omitting external Ca 2+ or by entrapping BAPTA to chelate cytosolic Ca 2+. Muscimol lost its ability to release glutamate following superfusion with d-aspartate to deplete cytosolic glutamate by heteroexchange suggesting that GABA A receptor activation elicits release of cytosolic glutamate. The non-transportable glutamate carrier blockers dihydrokainate or dl-TBOA did not reduce the muscimol potentiation. This was abolished by the anion channel blockers niflumic acid and NPPB. To conclude, when cerebellar parallel fiber terminals are sufficiently depolarized, activation of α6 GABA A receptors on these terminals mediates glutamate release in addition to that evoked by depolarization. This extra-release does not occur by exocytosis or transporter reversal but involves the opening of anion channels present on parallel fiber terminals.

Metabotropic Glutamate 1α and Adenosine A1 Receptors Assemble into Functionally Interacting Complexes

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.

L-[3H]nitroarginine and L-[3H]arginine uptake into rat cerebellar synaptosomes: kinetics and pharmacology.

Characteristics of the transport of the nitric oxide synthase substrate L-arginine and its inhibitor, NG-nitro-L-arginine (L-NOARG), into rat cerebellar synaptosomes were studied. Uptake of both L-arginine and L-NOARG was linear with increasing amount of protein (up to 40 micrograms) and time of incubation (up to 5 min) at 37 degrees C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of L-NOARG (650 pmol/mg of protein) was three to four times higher than that of L-arginine (170 pmol/mg of protein). L-NOARG uptake showed biphasic kinetics (Km1 = 0.72 mM, Vmax 1 = 0.98 nmol/min/mg of protein; Km2 = 2.57 mM, Vmax2 = 16.25 nmol/min/mg of protein). L-Arginine uptake was monophasic with a Km of 106 microM and a Vmax of 0.33 nmol/min/mg of protein. L-NOARG uptake was selectively inhibited by L-NOARG, NG-nitro-L-arginine methyl ester, and branched-chain and aromatic amino acids. L-Alanine and L-serine also inhibited L-NOARG uptake but with less potency. Uptake of L-arginine was selectively inhibited by NG-monomethyl-L-arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, L-NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas L-arginine is transported by the basic amino acid carrier system y+ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of L-NOARG and L-arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.

Interaction of SNARE Complexes with P/Q-type Calcium Channels in Rat Cerebellar Synaptosomes

P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.

Rapid and simple method for the determination of nitrite in synaptosomal superfusates by liquid chromatography with electrochemical detection

A rapid and simple technique using reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC—ED) was developed for the determination of nitrite. Standard solutions of sodium nitrite produced a linear current response over the concentration range 1.5–30 μ M. The chromatographic peaks of sodium nitrite were superimposable with peaks obtained after injection into the chromatograph of supernatants from rat cerebellar synaptosomes. The concentrations of nitrite in synaptosomes measured with HPLC—ED were similar to those found with a widely used colorimetric procedure. Therefore, this new method may represent a useful means of measuring nitrite in biological samples.

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC-fluorescence detection. Both basal and KCl-stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 +/- 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 +/- 83 pmol/mg protein/5 s. Ethanol caused a dose-dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol-stimulated release does not involve the dilazep-sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol-induced motor disturbances in the rat.

Effect of Acute Ethanol on Uptake of [3H]Adenosine by Rat Cerebellar Synaptosomes

Many classes of CNS-acting drugs have been suggested to act at least partially via inhibition of adenosine uptake. Synaptosomal uptake of [3H]adenosine and the effect of acute ethanol on it were studied in a rat brain area known to be involved in the coordination and modulation of normal motor activity, the cerebellum. Uptake of [3H]adenosine was found to be linear with time (about 40 sec) and increasing concentrations (up to 1.5 microM) of adenosine. The uptake of [3H]adenosine was inhibited by dilazep (IC50 = 2.5 x 10(-7) M) in a dose-dependent manner. Pharmacologically and/or toxicologically relevant concentrations of ethanol (2.5 to 100 mM) significantly inhibited the uptake of [3H]adenosine between 12 and 15%. Lineweaver-Burk plots indicated that both in vitro (25 mM) and in vivo (1.5 g/kg i.p.; 30 mM blood level) ethanol lowered Km as well as Vmax values for adenosine uptake to nearly the same extent. In the case of in vivo ethanol, no ethanol was present during the assay since synaptosome preparation would wash out residual ethanol. The results of the present study indicate possible membranal alterations by in vivo ethanol. It is concluded that the uptake of [3H]adenosine is inhibited by intoxicating concentrations of ethanol in vitro and by acute ethanol (1.5 g/kg) in vivo. This may partially explain the modulatory role of endogenous adenosine in ethanol-induced motor disturbances.

Release of endogenous glutamate from rat cerebellar synaptosomes: Interactions with adenosine and ethanol

The effects of ethanol and adenosine receptor agonist R-PIA and antagonist theophylline on release of endogenous glutamate were tested in rat cerebellar synaptosomal preparation. Release was carried out for 5 to 60 sec after which time the released glutamate was separated from the synaptosomal membranes by rapid filtration. The amount of released glutamate in the filtrate was measured by an enzyme-linked fluorometric assay. Basal endogenous glutamate release was estimated as 3.7 + 0.3 nmol/mg protein/5 sec and was stimulated by high K +. Glutamate release consisted of an initial rapid phase for the first 10 sec that was followed by a relatively slower phase. Both Ca 2+-dependent and Ca 2+-independent glutamate release were observed which suggested the involvement of both neuronal and glial constituents of the synaptosomal preparation, respectively. Pharmacologically relevant concentrations of ethanol (25–100 mM) caused a trend toward a dose-dependent inhibition of glutamate release. R-PIA and theophylline inhibited and stimulated, respectively, basal release of glutamate and R-PIA-inhibited release was blocked by theophylline. Ethanol (25 mM) blocked the stimulatory effect of theophylline and the results of experiments following the inclusion of adenosine deaminase suggested the involvement of adenosine in this effect of ethanol. The results support our previous findings that suggest an involvement of cerebellar adenosine in the motor disturbing effects of acute ethanol and extend those findings by indicating that ethanol inhibits glutamate release from granule cells of the cerebellar cortex through an adenosine-sensitive mechanism.

Serotonin inhibits the depolarization-evoked release of endogenous glutamate from rat cerebellar nerve endings

Glutamic acid (Glu) has been proposed as the neurotransmitter of cerebellar granule cells. 5-Hydroxytryptamine (5-HT) afferents project to the cerebellar cortex. The possible interaction between 5-HT and Glu was investigated by studying the effect of 5-HT on Glu release. The Ca 2+-dependent depolarization-evoked release of endogenous Glu from superfused rat cerebellar synaptosomes was potently inhibited by 5-HT. Methiothepin, but not ketanserin, cinanserin or methysergide, antagonized 5-HT. It is concluded that the release of Glu can be modulated by 5-HT through receptors sited on Glu terminals. These receptors belong to the 5-HT 1-type.