Rat Brain Synaptic Terminals Research Articles

Localization of enolase in synaptic plasma membrane as an alphagamma heterodimer in rat brain.

Enolase, a glycolytic enzyme, is a multifunctional protein with location diversity. We revealed the intracellular distribution of enolase isozymes, such as alphaalpha-, alphagamma- and gammagamma-enolases, in rat brain synaptic terminals by biochemical and immunoelectron microscopic analyses. Specific activity of enolase of synaptic plasma membrane fraction (SPM2) obtained from synaptosomes was 23.2 +/- 4.4 x 10(-2) micromol/mg protein/min in the presence of 0.25% Triton X-100 and that of synaptosomal cytoplasm (LS) was 67.4 +/- 12.1 x 10(-2) micromol/mg protein/min. About half of enolase activity in synaptosomes was distributed to soluble fraction while the remaining stayed in particulate membrane fractions by ultracentrifugation. Immunoblot analysis of the fractions demonstrated both alpha and gamma subunits were distributed in SPM. In addition, immunoelectron microscopic analysis also revealed that both subunits were immunoreactive on the SPM. Using coimmunoprecipitation assay, we confirmed that the enolase was present not only as a homodimer form but also as an alphagamma hybrid form associated with membrane, where both subunits were coimmunoprecipitated from lysate of SPM2 in the presence of Mg(2+). These findings indicate that all forms (alphaalpha, alphagamma, and gammagamma) of enolase translocate to the plasma membrane and associate with some components in the SPM.

Adenosine 5′-tetraphosphate (Ap 4), a new agonist on rat midbrain synaptic terminal P2 receptors

The aim of this study was to see whether the compound adenosine 5′-tetraphosphate (Ap 4) is active in the central nervous system by examining its effect on isolated rat brain synaptic terminals. Ap 4 proved to be more resistant to ecto-enzymatic hydrolysis than adenosine triphosphate (ATP), showing only 2% hydrolysis after a 2-min incubation, compared to 75% for ATP. In addition, Ap 4 was able to produce concentration-dependent increases in intracellular Ca 2+ when applied extracellularly. This action was dependent upon the presence of extracellular calcium. Ap 4 acts through ionotropic ATP receptors (P2X receptors) and not through diadenosine polyphosphate receptors, since ATP abolished the response elicited by Ap 4 whereas Ap 5A did not. Ap 4, ATP and ATP- γ-S were of similar potency (EC 50≈20 μM) while 2MeSATP, α, β-meATP and ADP- β-S possessed slightly lower potency (EC 50≈50 μM). The P2-purinoceptor antagonists suramin and PPADS blocked the Ap 4 effect. The IC 50 values for these compounds were 35.5 and 7.8 μM respectively. Diinosine polyphosphates and inosine tetraphosphate inhibited the response elicited by Ap 4 with IC 50 values that varied between approximately 40 and 50 μM. These results show that Ap 4 is as good an agonist as ATP on synaptosomal P2X receptors, being more resistant to extracellular hydrolysis by ecto-nucleotidases.

Catabolism of Ap4A and Ap5A by rat brain synaptosomes.

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37 degrees C. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.

Dinucleotide receptor modulation by protein kinases (protein kinases A and C) and protein phosphatases in rat brain synaptic terminals.

The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.

A new family of dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5' adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a dinucleotide receptor has been described that is specific for adenine dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 +/- 0.10 microM, 8.33 +/- 0.22 microM, and 4.23 +/- 0.12 nM, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50 values of 100.08 +/- 5.72 microM for diinosine triphosphate, 29.51 +/- 1.40 microM for diinosine tetraphosphate and 27.75 +/- 1.65 microM for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.

Microwave Fixation and Localization of Calcium in Synaptic Terminals and Muscular Cells by Electron Probe X-Ray Microanalysis and Electron Energy-Loss Spectroscopy Imaging.

The distribution of calcium ions in the rat brain synaptic terminals, in their synaptic vesicles, membranes and mitochondria; and in the sarcoplasmic reticula, mitochondria and their endosacs of skeletal or cardiac muscle cells, and in the caveolae of endothelial cells of blood capillaries was investigated with a two-step inorganic chemical precipitation method using potassium oxalate followed by potassium antimonate.During the process, calcium ions were precipitated with potassium oxalate containing aldehyde fixative as an insoluble precipitate of calcium oxalate under simultaneous computerized microwave irradiation fixation. Then the precipitates were chemically changed to calcium antimonate during the postfixation procedure with an osmium tetroxide fixative containing potassium antimonate. The chemical natures and elemental binding ratios of this precipitate of calcium and antimony in the tube test and in sections were investigated by computerized electron probe X-ray microanalysis using an energy dispersive type X-ray detector (EDX) and electron energy-loss spectroscopy (EELS) imaging. The EDX analyzed chemical nature and calculated values of precipitates, in both atomic binding ratios (AT, %) and molecular weight ratios (WT, %), were exactly the same as those of the theoretical calculated values based upon the chemical formula of calcium antimonate. The data indicated that calcium ions exsisted in the site of precipitate.Electron energy-loss spectroscopy imaging showed the distribution of digital net calcium images at electron microscopic resolution. Calcium exsisted in the nerve terminals, synaptic vesicles, mitochondria and synaptic membranes; in the skeletal and cardiac muscular endosacs of sarcoplasmic reticula, mitochondria, and in the endocytotic vesicles of cardiac sarcoplasmic membranes, or in the caveolae of blood vessel capillary endothelial cells.High magnification O-loss images and EELS spectra confirmed differences in the fine precipitates in a section, as most of the precipitates were Ca-antimonate showing a clot of fine needle-like crystals, but in some cases we found antimonic acid showing an electron opaque foamy spot, or just contamination with an irregularly shaped homogeneous opacity. They were found mixed together, and it was not easy to differentiate them under EDX analysis.Summarized data were reported in the 10th International Congress on Histochemistry and Cytochemistry, Kyoto, Japan, 1996 [24] and XXlth International Congress & International Academy of Pathology, and 12th World Congress on Academy of Enviromental Pathology, Budapest, Hungary, 1996 [26].

Possible functional role of diadenosine polyphosphates: Negative feedback for excitation in hippocampus

Diadenosine polyphosphates (Ap 4A and Ap 5A) are present in secretory granules of chromaffin cells 8 as well as in the rat brain synaptic terminals. 6 Their contribution to the exocytosis of the total synaptosomal content is considerable, ranging from 7% to 12%. Ap 4A and Ap 5A are released from synaptosomes in a Ca 2+-dependent manner. 6 There are indications on the high affinity of diadenosine polyphosphates to P 2 receptors. 5,7 but their action on P 1 receptors remains unclear. Here we report that both substances induce a blocking action on excitatory synaptic transmission in the rat hippocampus. This action is elicited via the A 1 (subclass of P 1) receptors and differs in some respects from the action of adenosine.

Ap4A and ADP-beta-S binding to P2 purinoceptors present on rat brain synaptic terminals.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]-Ap4A was used for binding studies a Kd value of 0.10 +/- 0.014 nM and a Bmax value of 16.6 +/- 1.2 fmol mg-1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 +/- 0.09 microM. 2. Displacement of [3H]-Ap4A by non-labelled Ap4A and P2-purinoceptor ligands showed a displacement order of Ap4A > adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki values of 0.017 nM, 0.030 nM, 0.058 nM and 0.147 nM respectively for the high affinity binding site and values of 0.57 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 +/- 0.022 nM and 3.9 +/- 2.1 fmol mg-1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively. The second binding site potency order was Ap5A> Ap4A > Ap6A,with Ki values of 0.28 microM, 0.53 microM and 5.32 microM respectively.5. Displacement studies of [35S]-ADP-beta-S with P2-purinoceptor agonists showed the following potency pattern: ADP-beta-S > AMP-PNP >alpha,beta-MeATP with Ki values of 0.021 nM, 0.029 nM 0.215 nM respectively in the high affinity binding site. 2-Methylthio-adenosine 5'-triphosphate (2MeSATP) was unable to displace [35S]-ADP-beta-S in this binding site. The second binding site showed a profile of ADP-beta-S> a,beta-MeATP> AMP-PNP > 2MeSATP and Ki values of 0.0 18 microM, 0.212 microM, 0.481 microM and 18.04 microM respectively.6. These studies suggest the presence of a new P2-purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P2d.

Presence of diadenosine polyphosphates— Ap 4A and Ap 5A— In rat brain synaptic terminals. Ca 2+ dependent release evoked by 4-aminopyridine and veratridine

The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap 4A and Ap 5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap 4A content per mg of protein was 169 ± 25 pmol and 159 ± 22 pmol for Ap 5A. The study of the exocytotic release of these compounds was carried out with 100 μM 4-aminopyridine or 10 μM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 ± 3.0 pmol/mg protein of Ap 4A and 11.6 ± 2.4 pmol/mg protein of Ap 5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 ± 3.0 and 16.6 ± 2.8 pmol/mg of protein of Ap 4A and Ap 5A respectively. The ratios of exocytosis were close to 7–9% and 10–12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.