Rat Cerebral Cortical Synaptosomes Research Articles

Plantainoside D Reduces Depolarization-Evoked Glutamate Release from Rat Cerebral Cortical Synaptosomes.

Inhibiting the excessive release of glutamate in the brain is emerging as a promising therapeutic option and is efficient for treating neurodegenerative disorders. The aim of this study is to investigate the effect and mechanism of plantainoside D (PD), a phenylenthanoid glycoside isolated from Plantago asiatica L., on glutamate release in rat cerebral cortical nerve terminals (synaptosomes). We observed that PD inhibited the potassium channel blocker 4-aminopyridine (4-AP)-evoked release of glutamate and elevated concentration of cytosolic Ca2+. Using bafilomycin A1 to block glutamate uptake into synaptic vesicles and EDTA to chelate extracellular Ca2+, the inhibitory effect of PD on 4-AP-evoked glutamate release was prevented. In contrast, the action of PD on the 4-AP-evoked release of glutamate in the presence of dl-TBOA, a potent nontransportable inhibitor of glutamate transporters, was unaffected. PD does not alter the 4-AP-mediated depolarization of the synaptosomal membrane potential, suggesting that the inhibitory effect of PD on glutamate release is associated with voltage-dependent Ca2+ channels (VDCCs) but not the modulation of plasma membrane potential. Pretreatment with the Ca2+ channel blocker (N-type) ω-conotoxin GVIA abolished the inhibitory effect of PD on the evoked glutamate release, as did pretreatment with the protein kinase C inhibitor GF109203x. However, the PD-mediated inhibition of glutamate release was eliminated by applying the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 or dantrolene, which inhibits Ca2+ release through ryanodine receptor channels. These data suggest that PD mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through N-type Ca2+ channels, subsequently reducing the protein kinase C cascade.

Inhibition of Na+, K+ -ATPase Activity by Phospholipase A2 and Several Lysophospholipids: Possible Role of Phospholipase A2 in Noradrenaline Release from Cerebral Cortical Synaptosomes

p-Bromophenacyl bromide (PBPB), quinacrine and indomethacin, which inhibit phospholipase A2 (PLA2; EC 3.1.1.4) activity in several tissues, caused a dose-dependent inhibition of prelabelled [3H]noradrenaline ([3H]NA) release evoked by high concentrations of K+ from rat cerebral cortical synaptosomes. Release of prelabelled [3H]NA was caused by natural lysophosphatidic acid (LPA; 10(-6)-10(-5) g mL-1) and lysophosphatidylcholine (LPC; 10(-6)-10(-5) g mL-1) and synthetic LPA (6 x 10(-6), 2 x 10(-5) M) and LPC (6 x 10(-6), 2 x 10(-5) M), but not by natural lysophosphatidylserine (LPS; 10(-5) g mL-1), lysophosphatidylethanolamine (LPE; 10(-5) g mL-1) and lysophosphatidylinositol (LPI; 10(-5) g mL-1). The release evoked by natural LPA and LPC could be inhibited only marginally by PBPB and quinacrine. Phosphatidic acid (PA)-specific and phosphatidylcholine (PC)-specific PLA2 activities from rat cerebral cortical synaptosomes were stimulated in incubation medium containing high concentrations of K+ or calcium ionophore A23187. Low concentrations of PLA2 (10(-6)-10(-8) g mL-1, from bee venom) inhibited the synaptic membrane Na+,K+-ATPase activity in incubation media with intracellular levels of free Ca2+. Several lysophospholipids (LPLs), metabolites of the PLA2 type, also inhibited the synaptic membrane Na+,K+-ATPase activity in a dose-dependent manner. The minimum effective concentrations of natural LPA, LPC, LPS, LPI and LPE were 10(-6), 4.7 x 10(-6), 10(-5), 4.7 x 10(-5) and 4.7 x 10(-5) g mL-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A proteomic survey of rat cerebral cortical synaptosomes

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

3H]adenosine transport in rat dorsal brain stem using a crude synaptosomal preparation

The neuromodulator adenosine, has been shown to have the highest density of central uptake sites in the nucleus tractus solitarius in the dorsal brain stem. The nucleus tractus solitarius is involved in the central regulation of reflex cardiovascular activity suggesting that adenosine may also be involved in central cardiovascular control. Thus, the present study has characterized the transport of [ 3H]adenosine into rat dorsal brain stem synaptosomes. The process was found to be saturable and highly dependent on temperature. Furthermore, [ 3H]adenosine transport in rat dorsal brain stem synaptosomes was far more sensitive to the removal of sodium ions than has been previously reported for rat cortical synaptosomes. In addition transport was rapid, being linear for at least 30 s at 37°C, reaching equilibrium within 1 min and had an apparent K m value of 2.7 ± 0.2 μM ( n = 4) and a V max value of 135.5 ± 17.8 pmol/mg protein/min ( n = 4). These kinetic parameters are within an order of magnitude of adenosine uptake processes found in rat cerebral cortical synaptosomes. Transport of [ 3H]adenosine was significantly inhibited by an excess of unlabelled adenosine (1 mM) and the adenosine uptake inhibitor S(4-nitrobenzyl)-6-thioinosine (100 μM) while morphine (150 μM) and flurazepam (150 μM) were largely ineffective as inhibitors of the process, in contrast with previous findings in rat cortical synaptosomes. The present findings demonstrate the presence of a high affinity transport system for adenosine in the rat dorsal brain stem which appears to differ in some properties to uptake processes found in rat cortex.

Ca(2+)-dependent inactivation of P-type calcium channels in nerve terminals.

Rapid Ca2+ signals evoked by K+ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca2+ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca2+ concentrations revealed that the responsible voltage-dependent Ca2+ channels, previously identified as P-type Ca2+ channels, inactivate owing to the rise in intracellular Ca2+ levels. At millimolar extracellular Ca2+ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca2+, thus limiting the Ca2+ signal amplitudes to 500-600 nM. A slower, probably voltage-dependent regulation appears to be effective at lower Ca2+ influx rates, leading to submaximal Ca2+ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca2+ levels appears to be responsible for the different inhibition characteristics of Cd2+ versus omega-agatoxin IVa.

Effects of membrane peroxidation on [3H]acetylcholine release in rat cerebral cortical synaptosomes.

[3H]Acetylcholine (ACh) release, malonaldehyde formation and 45calcium-uptake were measured in rat cerebral cortical nerve terminal that were exposed to various concentrations of ferrous and ascorbate ions. At a constant molar ratio of 25:1, ferrous:ascorbate, these ions increased malonaldehyde (MA) synthesis in a concentration-dependent manner. Treatment with these ions in the same ratio also induced a dose-related inhibition of the K(+)-depolarization-induced release of newly synthesized [3H]ACh. Combined exposure to Fe2+/ascorbate also reduced calcium ionophore A23187-induced [3H]ACh release. Neither ferrous nor ascorbate ions alone altered depolarization- or ionophore-induced [3H]ACh release over this concentration range. Depolarization- and A23187-induced 45calcium uptake were not affected by peroxidation, suggesting that membrane peroxidation influenced some process in the release-process subsequent to calcium influx in a manner similar to what is observed during aging.

Analysis of rapid calcium signals in synaptosomes

A combination of the stopped-flow technology with dual channel spectrofluorometry of Ca(2+)-indicators was utilized for the measurement of rapid Ca(2+)-signals in rat cerebral cortical synaptosomes evoked by K(+)-depolarization. There was no observable contribution of Ca(2+)-ions from intracellular stores to the rise in [Ca2+]i. The kinetics of the fast increase in intracellular Ca2+ concentration was analysed in relation to the depolarization strength. The maximal increase in [Ca2+]i and the time course of Ca(2+)-channel inactivation were determined for depolarizations obtained by different extracellular K(+)-concentrations ([K+]o). An apparent threshold was observed at about 18 mM [K+]o; a maximal Ca(2+)-signal amplitude was estimated at about 40 mM [K+]o. Pharmacological properties of the involved Ca(2+)-channels were determined using selective Ca(2+)-channel blockers (Dihydropyridines, omega-Conotoxin, omega-Agatoxins); the results suggest that a P-type voltage-dependent Ca(2+)-channel is the relevant channel type, generating the evoked Ca(2+)-signals in rat cerebral cortical synaptosomes.

Protein kinase C and the regulation of glutamate exocytosis from cerebrocortical synaptosomes.

The role of protein kinase C (PKC) in the regulation of transmitter glutamate release from rat cerebral cortical synaptosomes is investigated. Two depolarization protocols are used: first, elevated KCl, which produces a clamped depolarization, and second, 4-aminopyridine, which evokes spontaneous "action potentials" allowing any potential modulation of Na+ or K+ channels to influence release. Although the PKC inhibitor Ro 31-8220 prevents both the depolarization-evoked and phorbol dibutyrate (PDBu)-evoked phosphorylation of the major presynaptic PKC substrate, myristoylated alanine-rich C kinase substrate, it is without effect on KCl-evoked Ca(2+)-dependent glutamate release. Ro 31-8220 totally inhibits the Ca(2+)-dependent 4-aminopyridine-evoked release of glutamate in the presence and absence of PDBu and again decreases the phosphorylation of myristoylated alanine-rich C kinase substrate. Ro 31-8220 strongly inhibits the 4-aminopyridine-evoked increase in [Ca2+] both in the presence and absence of PDBu and antagonizes the PDBu enhancement of depolarization. This indicates that PKC isoforms activatable by PDBu and sensitive to Ro 31-8220 play no discernable role in Ca(2+)-secretion coupling per se in cerebral cortical glutamatergic nerve terminals, but that the kinase plays a major role in regulating the depolarization of the terminal.

High d-glucose concentrations increase GABA release but inhibit release of norepinephrine and 5-hydroxytryptamine in rat cerebral cortex

The effect of high d-glucose concentrations on the K + (15–25 mM)-evoked release of [ 3H]GABA, [ 3H]norepinephrine ([ 3H]NE), [ 3H]5-hydroxytryptamine ([ 3H]5-HT) and [ 3H]acetylcholine ([ 3H]ACh) was investigated in rat cerebral cortical slices and synaptosomes superfused with Krebs' solution which normally contains 11.1 mM glucose. In slices, the release of [ 3H]GABA was enhanced by increasing d-glucose by 32–320 mM, the release of [ 3H]NE and [ 3H]5-HT was inhibited when d-glucose was increased by 60–320 mM but the release of [ 3H]ACh was not affected. The increase of [ 3H]GABA release and the inhibition of [ 3H]NE release were mimicked by d-fructose and NaCl at similar osmolarities, whereas dimethylsulfoxide (DMSO; a freely membrane-permeable drug) at equimolar concentrations failed to affect the release of [ 3H]GABA and [ 3H]NE. The GABA B receptor antagonist p-(3-aminopropyl)-p-diethoxymethyl-phosphinic acid (CGP 35348) abolished the inhibitory effect of an increase in d-glucose by 60 and 100 mM on [ 3H]NE release but only tended to diminish the inhibition caused by an increase by 320 mM. In synaptosomes, the K +-evoked release of 3H-GABA was enhanced by increasing d-glucose by 60–320 mM. The K +-evoked release of [ 3H]NE was only inhibited when d-glucose was increased by 320 mM, whereas it was not affected by an increase by 100 mM. In conclusion, high d-glucose differentially influences neurotransmitter release in the brain cortex. Hyperosmolarity in the extracellular fluid together with an osmotic gradient extra- versus intracellular probably plays a role in the mechanism(s) underlying the changes observed. The inhibition of [ 3H]NE release induced by an increase in d-glucose by up to 100 mM is probably due to the enhancement of [ 3H]GABA release from GABAergic interneurons, thus leading to an increased activation of inhibitory GABA B receptors either on the noradrenergic axon terminals themselves or on an unknown excitatory neuron which innervates the noradrenergic nerve terminals. The effects of d-glucose on neurotransmitter release may contribute to the neurologic disorders during diabetic coma.

Glutamate exocytosis evoked by 4-aminopyridine is inhibited by free fatty acids released from rat cerebrocortical synaptosomes

The Ca 2+-dependent release of glutamate induced by 4-aminopyridine (4-AP) in rat cerebral cortical synaptosomes was reduced by removal of bovine serum albumin (BSA) from the incubation medium. The decrease in the glutamate release in the absence of BSA was consistent with a reduction in the rise in cytosolic free [Ca 2+] after depolarization with 4-AP. Contrarily, neither the glutamate release nor the elevation in cytosolic free [Ca 2+] after depolarization with 30 mM KCl was altered by the removal of BSA. The inhibitory action of the free fatty acids released during the incubation of synaptosomes was also observed when exogenous free fatty acids were added to the medium in the presence of BSA. The highest inhibition of 4-AP-induced release of glutamate was observed in the presence of arachidonic acid. The results strongly suggest an inhibitory action of free fatty acids by decreasing Ca 2+ entry and glutamate release in rat cerebrocortical synaptosomes.

Comparison of GABA uptake by brain and kidney preparations

The uptake ofγ-aminobutyric acid (GABA) was measured in rat cerebral cortical synaptosomes and rat kidney brush-border membrane vesicles (BBMV). Three GABA uptake systems (Km = 1.3, 50 and 3246µM respectively) were present in synaptosomes, but only two uptake systems (Km = 11 and 1203µM respectively) were detectable in BBMV. The uptake systems in the two types of tissue preparations were similar in that every system was inhibited byp-hydroxymercuribenzoate and by the action of neuraminidase, thereby indicating that, irrespective of the tissue, both sulfhydryl and sialyl groups were necessary components of all the GABA uptake systems. In contrast, differences were observed between synaptosomal and BBMV uptake systems with respect to their sensitivity to inhibition by GABA structural analogs. While the synaptosomal GABA uptake systems were strongly inhibited by nipecotic acid, diaminobutyric acid,β-alanine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), none of these compounds significantly inhibited the BBMV uptake systems. It is therefore concluded that the GABA uptake systems in brain and kidney tissues are quite different entities. While the role of GABA transport in the inactivation of the neurotransmitter function of GABA is well documented, the role of the kidney GABA uptake system is unclear, and the possibility exists that the latter systems are present in kidney tissues primarily to transport compounds other than GABA. The present study does however highlight a difference in drug susceptibility of the brain and kidney uptake systems, a phenomenon which may have some therapeutic relevance with respect to the use of GABA analogs as anticonvulsant agents.

Histidine release from brain slices and synaptosomes.

The release of radioactive histidine was studied in a superfusion system by using preloaded rat cerebral cortical slices and guinea pig synaptosomes. ATP/ADP ratios in both preparations were used to assess the energy levels and thus the oxygenation of brain slices and synaptosomes. ATP/ADP ratios similar to the intact brain and nerve terminals were found in slices and synaptosomes even after hours of superfusion with oxygenated buffer. Depolarization by 30 or 50 mM K+ induced release of histidine from slices and synaptosomes. The results confirm that under physiological energetic conditions, histidine is released both from slices and synaptosomes after depolarization with potassium.

Dipalmitoylphosphatidylcholine liposomes reduce [3H]acetylcholine levels in an eserine-sensitive manner in rat cerebral cortical synaptosomes.

We investigated the effects of various phospholipids on the presynaptic levels of newly synthesized [3H]acetylcholine (ACh) in rat cerebral cortical synaptosomes. When administered as small unilamellar vesicles (200-500 A diameters) dipalmitoylphosphatidylcholine (DPPC) reduced [3H]ACh levels in concentration and time-related manners, while increasing the efflux of labelled choline to a similar extent. The reductions in synaptosomal [3H]-ACh levels induced by DPPC (3 mg/ml) were found in the cytosolic S3 but not microsomal P3 fraction, arguing for a cytoplasmic, non-vesicular site of action. DPPC-induced reductions in [3H]ACh levels were blocked by 100 microM eserine, a tertiary amine cholinesterase inhibitor, but not with 100 microM neostigmine, a quaternary ammonium inhibitor. Large unilamellar vesicles (2000-5000 A diameters) consisting of soybean-phosphatidylcholine reduced [3H]ACh levels to the same extent that small vesicles did at the same concentration (3 mg/ml). Taken together, these results suggest that DPPC can fuse with membranes to increase the hydrolysis of cytoplasmic ACh via a small intra-terminal subpopulation of cholinesterases.

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization, veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [3H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [3H]acetylcholine release in a concentration-dependent manner (5-10 mM); a high concentration (10 mM) also elevated [3H]choline efflux. However, the 35 mM K+ induced release of [3H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K+ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 microM). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K+ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.

Occurrence of a large Ca2+-independent release of glutamate during anoxia in isolated nerve terminals (synaptosomes).

Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+-dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+-independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+-independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.

Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.

Protein phosphorylation in nerve terminals: comparison of calcium/calmodulin-dependent and calcium/diacylglycerol-dependent systems

Rat cerebral cortical synaptosomes that had been prelabeled with 32P-orthophosphate were exposed to either (1) K depolarization which causes Ca2+ influx and hence would be expected to activate Ca2+-dependent enzymes, including Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases (Ca/CaM kinases and protein kinase C, respectively); or (2) phorbol esters or 1-oleoyl-2-acetyl-glycerol (OAG), which selectively activate protein kinase C. Proteins whose state of phosphorylation was affected by these treatments could be divided into 3 classes. Class A includes 5 phosphoproteins that showed rapidly increased phosphorylation by synaptosomal depolarization but not by OAG or phorbol ester. Four of these proteins, synapsins Ia and Ib and proteins IIIa and IIIb, are neuron-specific, synaptic vesicle-associated proteins known to be substrates for Ca/CaM kinases I and II. These phosphoproteins were rapidly dephosphorylated upon synaptosomal repolarization. Class B is composed of 2 phosphoproteins that showed rapidly increased phosphorylation by either synaptosomal depolarization or treatment with phorbol ester or OAG. These 2 acidic proteins of Mr87 and 49 kDa are known from in vitro studies to be specific substrates for protein kinase C. Thermolytic peptide mapping indicated that the 87 kDa protein in synaptosomes was phosphorylated by protein kinase C in situ. These 2 phosphoproteins were slowly dephosphorylated upon synaptosomal repolarization. Class C comprises 4 phosphoproteins that were rapidly dephosphorylated upon synaptosomal depolarization and may be substrates for Ca2+-activated protein phosphatase(s). These data suggest that Ca2+ influx into nerve terminals activates Ca/CaM kinases I and II, protein kinase C, and unidentified protein phosphatase(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Norepinephrine Uptake by Adenine Nucleotides and Divalent Cations: Role for Extracellular Protein Phosphorylation

This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high-affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5'-adenylylimidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+-stimulated uptake 1 was blocked by App(NH)p; Mg2+-stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca-omission, and inhibition of the phosphorylation of one specific, 39,000-dalton, Ca2+-dependent, protein component in synaptosomes. This 39K protein was distinct from the alpha subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.

Ubiquitin‐Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.

Effects of ovariectomy and estradiol benzoate on high affinity choline uptake, ACh synthesis, and release from rat cerebral cortical synaptosomes

Several presynaptic processes were studied in cerebral cortical synaptosomes prepared from intact adult female rats or from ovariectomized animals that received 3 subcutaneous injections of either estradiol benzoate (10 μg/kg) or vehicle. Injections were given 1/day, every other day, and animals were sacrificed 1 h after the last injection. High affinity choline uptake and coupled acetylcholine (ACh) synthesis were reduced by ovariectomy, and restored to control levels by the estradiol benzoate injections. In contrast, low affinity choline uptake and depolarization-induced [ 3H]ACh release were unaffected by either ovariectomy or estradiol benzoate injections. These results suggest that changes in estradiol levels may alter the high affinity transport process regulating ACh synthesis in this tissue.