Guanine nucleotides have a direct inhibitory effect on polyphosphoinositide turnover in rat cortical synaptosomes

1. The possible involvement of guanosine 5'-triphosphate (GTP)-binding proteins in the receptor mediated polyphosphoinositide (PPI) turnover event was investigated in rat cortical synaptosomes. 2. It was studied under the effects of guanine nucleotides on 32Pi incorporation into synaptosomal phospholipids in the absence or presence of carbachol. 3. The basal 32Pi incorporation into these phospholipids was altered by the presence of 1 mM carbachol: i.e. a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate and an increase in the incorporation of 32Pi into phosphatidylinositol and phosphatidic acid. 4. In the presence of guanine nucleotides: GTP, Gpp(NH)p and GDP at suitable concentrations, there was a general decreasing effect on 32Pi incorporation into all 4 phospholipids, which are all involved in PPI turnover cycle, either in the basal or carbachol-stimulated levels. 5. There was no selective effect among the guanine nucleotides studied on this PPI turnover event. It is, therefore, likely that these nucleotides have a direct inhibitory effect on PPI turnover, and this action may not act through a GTP-binding protein.     

Effects of anticonvulsant drugs on calcium transport and polyphosphoinositide metabolism in rat cortical synaptosomes

1. Anticonvulsants: phenytoin, phenobarbital, carbamazepine and valium at concentrations of 10-100 microM had a significantly inhibitory effect on both K+-stimulated Ca2+-uptake and 32Pi incorporation into phospholipids of rat cortical synaptosomes. 2. Other anticonvulsant, valproic acid, at concentration upto 100 microM had no effect on these two events. 3. Our results suggest that there is a link between Ca2+-influx and polyphosphoinositide turnover in synaptosomes, and this link may relate to the inhibitory effect of these drugs on neurotransmitter release mechanisms of this preparation.     

Inhibition of polyphosphoinositide turnover in rat cerebral cortex by clonidine

In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of alpha 1-adrenergic receptors, for example, increases polyphosphoinositide turnover, but stimulation of alpha 2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inositol and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an alpha 2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of alpha 2, 5HT2, H2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

Simple and complex synapses shown by freeze-etching of rat cortical synaptosomes

The shape of the synaptic sites (specialized contact areas) was examined on the synaptic membrane fracture faces of freeze-etched rat cortical synaptosomes. Simple and complex synapses were differentiated on the basis of the absence or presence of unspecialized areas in the synaptic sites. The particle-free nature of these areas is discussed.     

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.     

Presynaptic modulation by eicosanoids in cortical synaptosomes

In continuing experiments to determine the ionic basis of inhibitory presynaptic modulation, rat cortical synaptosomes were employed and receptor-activated K⁺ efflux was determined with a K⁺ sensitive electrode. When synaptosomes were sub-optimally depolarized by veratridine, the addition of agents that activated purinergic, ₂, muscarinic and opioid receptors all promoted K⁺ efflux. With 2-chloroadenosine as a model inhibitory presynaptic modulator, the increased K⁺ efflux evoked by this agent was blocked by the cyclooxygenase inhibitor indomethacin suggesting that arachidonic acid or its metabolites was an intermediary in opening the channel. When arachidonic acid and PGE₂ were tested, both promoted K⁺ efflux that was inhibited by dendrotoxin and mast cell degranulating peptide, two agents that are known to inhibit a delayed rectifier K⁺ current. Our results suggest that via eicosanoid second messengers, inhibitory presynaptic modulators open a sub-class of K channels that hyperpolarize nerve terminals, therefore less Ca²⁺ would enter per nerve impulse and thus the evoked release of neurotransmitters would be decreased.Abbreviations DTX dendrotoxin - MCDP mast cell degranulating peptide - NHGA norhydroguairetic acid - PGE₂ prostaglandin E₂     

Hypoxia affects the physiological behavior of rat cortical synaptosomes

Nerve cells, especially synaptosomes, are very susceptible to hypoxia and the subsequent oxidative stress. In this paper, we examined the effects of hypoxia (93% N(2):2% O(2):5% CO(2), v/v/v) on rat cortical synaptosomes by evaluating modifications of synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal mitochondrial Ca(2+) concentration ([Ca(2+)](i)), and desferoxamine-chelatable free iron and esterified F2-isoprostane levels after different periods of hypoxia and after 30 min of reoxygenation. Oxygen consumption decreased significantly during 120 min of hypoxia and was restored after reoxygenation. At the same time, ATP production decreased and remained significantly lower even after reoxygenation. This involved a depolarization of the synaptosomal mitochondrial membrane, although the [Ca(2+)](i) remained practically unchanged. Indeed, iron and F2-isoprostane levels, representing useful prediction markers for neurodevelopmental outcome, increased significantly after hypoxia, and there was a strong correlation between the two variables. On the whole our results indicate that synaptosomal mitochondria undergo mitoptosis after 2 h of hypoxia.     

Electron microscopic demonstration of adenylate cyclase activity in rat cortical synaptosomes

Summary The electron cytochemical demonstration of adenylate cyclase activity was carried out in rat cortical synaptosomes. Reaction product was found in 60–70% of the synaptosomes in three predominant localizations: (i) on the postsynaptic density; (ii) on the outer aspect of the synaptosomal membrane; (iii) inside the synaptosome. Results suggest that in addition to postsynaptic localization adenylate cyclase activity is cytochemically demonstrable also at presynaptic sites.     

Proton permeability of the plasma membrane of rat cortical synaptosomes

1. Transmembrane pH gradients (acidic inside) and electrical gradients (negative inside) were estimated in cortical synaptosomes from the distribution of the weak base methylamine and the lipophilic cation tetraphenylphosphonium, respectively. 2. Acidic interior pH gradients were produced by outwardly directed K+ gradients in Na+-free media. External K+ accelerated the dissipation of preformed H+ gradients. The appearance of H+ in the medium was directly demonstrated by pH-stat titration of a weakly buffered medium. Amiloride failed to inhibit K+-induced H+ release. 3. Elevating K+ in the absence of Na+ did not affect the endogenous contents of noradrenaline, dopamine, and serotonin, as determined by high-performance liquid chromatography with electrochemical detection. 4. H+ diffusion potentials were generated when outwardly directed H+ gradients were imposed onto the plasma membrane indicating an electrogenic H+ efflux which is not coupled to other ions. 5. At low K+ in the Na+-free sucrose medium, the plasma membrane potential Em (derived from distribution of tetraphenylphosphorium cation) did not approach a value for EK, the K+ equilibrium potential (calculated from K+ gradients). The deviation of Em from EK could be quantitatively described by a modified constant-field equation, taking a relative H+/K+ permeability coefficient of 12,400 into consideration. 6. It is concluded that synaptosomes have a H+ conductance pathway in their plasma membrane in addition to the Na+/H+ antiporter. H+ influx is driven by and leads to a reduction of Em. K+/H+ exchange resulted from the electrical coupling of K+ and H+ fluxes via parallel K+ and H+ channels. Since the Na+/H+ antiporter counteracts passive equilibration of H+ under physiological conditions, a continuous cycling of H+ across the plasma membrane will take place. A possible physiological role of the H+ leak in pHi regulation is discussed.     

The effect of juglone on cholinergic mechanisms in rat brain synaptosomes

Juglone 5-hydroxy-1,4-napthoquinone exerts three activities on cortical synaptosomal preparations. It inhibits the release of acetylcholine and is an even more potent inhibitor of high-affinity choline transport into synaptosomes. In addition, as has already been shown by others using brain homogenates, juglone inhibits choline acetyltransferase.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17beta-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca2+-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10-11-10-6 M, showed an inverted U-shape potency in the regulation of Ca2+-ATPase activity. At physiologically relevant concentrations (10-8-10-9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10-11-10-6 M) and 17beta-estradiol (10-12-10-9 M) caused a dose-dependent increase in the hydrolytic ability of Ca2+-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Acetylation and phosphorylation of choline following high or low affinity uptake by rat cortical synaptosomes

Synaptosomal acetylcholine synthesis was found to be dependent on the presence of Na⁺-dependent HC-3 sensitive choline transport at low (5.5 mM) and high (35 mM) K⁺ concentrations. However, at 5, 20, and 100 M choline, choline phosphorylation was proportional to total choline uptake, in the presence or absence of high affinity transport. Only in the presence of eserine (50 M) did acetylcholine synthesis increase as the choline concentration was elevated from 20 M to 100 M, and this effect was observed at low and high K⁺ concentrations. Our results suggest that: 1) the synthesis of non-surplus synaptosomal ACh is dependent on high affinity choline transport; and 2) choline is equally likely to be phosphorylated after being taken up by low or high affinity transport.     

Uptake of adrenaline by rat brain synaptosomes. Effects of several psychotropic drugs]

Comparative study of the uptake of 3H-epinephrine (3H-EN) and 3H-norepinephrine (3H-NE) into rat brain crude synaptosomes and effect of psychotropic drugs of different classes on this process showed that isolated nerve terminals had their own transport system for EN. The crude synaptosomal fraction had two transport system's for EN; high-specific active uptake with high affinity (KM = 3.7 + 0.21 microM) and low-affinity uptake (KM2 = 98.0 + 47.5 microM). En accumulation was saturable, stereo-specific and inhibited by ouabain (3 X 10(-3) M), protoveratrine A and B (10(-4) M), NaN3 (2 X 10(-3) M), 2,4-dinitrophenol (2 X 10(-3) M), p-chloromercuribenzoate (10(-4) M). Actinomycin D had no effect on the uptake of 3H-EN. 3H-HE was accumulated by two uptake system: 1-high affinity uptake system with KM values of 0.49 + 0.13 microM, 2-low affinity uptake system with KM values of 21.1 + 7.71 microM. Amphetamine, mesocarb, chlorpromazine, fluphenazine and haloperidol were equally effective inhibitors of 3H-EN and 2H-HE uptake. Imipramine, phenazepam, diazepam and carbamazepine (5 X 10(-5) M) had no effect on the uptake of 3H-NE. Imipramine, zimelidine, norzimelidine and viloxazine (5 X 10(-5) M) were more potent inhibitors of the 3H-EN uptake than that of 3H-NE.     

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.     

Effect of hyperbaric oxygen on 2-deoxy-d-glucose andl-glutamate transport in rat cortical synaptosomes

Initial velocities of uptake ofl-glutamic acid and 2-deoxy-d-glucose (2-Dg) have been measured in cortical synaptosomes from rats which had been exposed to oxygen at high pressure (OHP) and compared to similar measurements in normobaric controls. Exposure to OHP had no significant effect on glutamate uptake at any combination of sodium and glutamate used. In contrast, OHP reduced 2-Dg uptake by an average of 17.5%. Although Kt was little affected, OHP exposure reduced apparent maximal transport capacity by 15%. Since hyperbaria with normal pO₂ had no significant effect on uptake, the effect of OHP is an oxygen effect, rather than a pressure effect. The effects of OHP on uptake do not parallel the effects of age; glutamate transport capacity was reduced in aged animals, while 2-Dg transport was unaffected.     

Synaptophysin is phosphorylated in rat cortical synaptosomes treated with botulinum toxin A.

Phosphorylation and dephosphorylation of neuronal proteins have been implicated in regulation of synaptic transmission. Studies were performed to determine if synaptophysin was phosphorylated or dephosphorylated during exposure of synaptosomes to botulinum toxin A (BoTX/A). Cholinergic-enriched synaptosomes were preincubated in the presence of 3H-choline to label newly synthesized acetylcholine (3H-ACh). This was followed by incubation with low or high potassium to stimulate release of newly synthesized 3H-ACh. BoTX/A inhibited total Ach release by 15-19% and inhibited release of newly synthesized 3H-ACh by 35%. A 165% increase in synaptophysin phosphorylation occurred in a dose-dependent manner over a range of doses (0.2 nM, 2 nM, 20 nM, 100 nM) of BoTX/A. When 4-Aminopyridine was added to synaptosomes that were BoTX/A treated, synaptophysin was dephosphorylated to control levels. Synaptosomes incubated with BoTX/A exhibited an inhibition of potassium stimulated ACh release and an increase in synaptophysin phosphorylation. Synaptophysin phosphorylation may be involved in inhibition of acetylcholine release.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17β-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca²⁺-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10^?11–10^?6 M, showed an inverted U-shape potency in the regulation of Ca²⁺-ATPase activity. At physiologically relevant concentrations (10^?8–10^?9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10^?11–10^?6 M) and 17β-estradiol (10^?12–10^?9 M) caused a dose-dependent increase in the hydrolytic ability of Ca²⁺-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Studies on the effects of acetylcholine and antiepileptic drugs on32Pi incorporation into phospholipids of rat brain synaptosomes

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated³²P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated³²P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the³²P labeling of phospholipids nor on the specific radioactivity of [³²P]ATP. Omission of Na⁺ drastically reduced both the³²P labeling of synaptosomal phospholipids and the specific radioactivity of [³²P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate Na⁺ and Ca²⁺ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca²⁺ and Na⁺ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.Abbreviations used ACh Acetylcholine - PA phosphatidic acid - PI phosphatidylinositol - poly PI polyphosphoinositides (diphosphoinositide and triphosphoinositide) - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - S.A. specific radioactivity     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.