Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinson's disease with L-DOPA.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Effect of propofol on glutamate and gamma-aminobutyric acid release from rat hippocampal synaptosomes / 华中科技大学学报（医学）（英德文版）

To investigate the effect of propofol on the release of glutamate and gamma-aminobutyric acid (GABA) from rat hippocampal synatosomes, synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). With the experiment of Ca(2+)-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. For the observation of Ca(2+)-independent release of glutamate and GABA, no DHK, nipectic acid and Ca2+ were added from aCSF. The release of glutamate and GABA were evoked by 20 micromol/L veratridine or 30 mmol/L KCI. The concentration of glutamate and GABA in aCSF was measured by using high-performance liquid chromatography (HPLC). 30, 100 and 300 micromol/L propofol significantly inhibited veratridine-evoked Ca(2+)-dependent release of glutamate and GABA (P 0.05). Veratridine or elevated KCI evoked Ca(2+)-independent release of glutamate and GABA was not affected significantly by propofol (P > 0.05). Propofol could inhibit Ca(2+)-dependent release of glutamate and GABA. However, it has no effect on the Ca(2+)-independent release of glutamate and GABA.

Effect of thiopental sodium on the release of glutamate and gamma-aminobutyric acid from rats prefrontal cortical synaptosomes / 华中科技大学学报（医学）（英德文版）

To investigate the effect of thiopental sodium on the release of glutamate and gamma-aminobutyric acid (GABA) from synaptosomes in the prefrontal cortex, synaptosomes were made, the spontaneous release and the evoked release by 30 mmol/L KCl or 20 micromol/L veratridine of glutamate and GABA were performed under various concentrations of thiopental sodium (10-300 micromol/L), glutamate and GABA concentrations were determined by reversed-phase high-performance liquid chromatography. Our results showed that spontaneous release and evoked release of glutamate were significantly inhibited by 30 micromol/L, 100 micromol/L and 300 micromol/L thiopental sodium, IC50 of thiopental sodium was 25.8 +/- 2.3 micromol/L for the spontaneous release, 23.4 +/- 2.4 micromol/L for KCl-evoked release, and 24.3 +/- 1.8 micromol/L for veratridine-evoked release. But GABA spontaneous release and evoked release were unaffected. The study showed that thiopental sodium with clinically related concentrations could inhibit the release of glutamate, but had no effect on the release of GABA from rats prefrontal cortical synaptosomes.

Regulation of GTP-binding state in RalA through Ca2+ and calmodulin

RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

Effects of undernutrition during suckling on ATP and ADP hydrolysis by synaptosomes from the cerebral cortex of adult rats

Early undernutrition can cause permanent functional changes in the nervous system. Alterations in enzymes involved in neurotransmiter metabolism have been reported to result from early undernutrition. In a previous study, we demonstrated that undernutrition during suckling decreaseATP and ADP hydrolysis by synaptosomes from cerebral cortex by abouth 20% of the value found in 20-day-old well-nourished rats (j.B.T. Rocha, C.F. Melo, J.J.F.Sarkis and R.D. Dias, British Journal of Nutrition, 63:273-283, 1990). In the present study, we investigated whether this deficit persists in synaptosomes from cerebral cortex of nutritionally rehabilitated adult rats. rats were undernourished from birth to 25 days of life by feeding their dams a 7% casein (w/w) diet, while well-nourished offspring were fed by mothers maintained on a 28% casein diet. In contrast to the results previously obtained in young rats, the synaptosomes obtained from the cerebral cortex of early undernourished adult rats hydrolyzed ATP and ADP more efficiently than did those obtained from well-nourished rats. Specific activity (nmol min-1 mg protein-1, mean ñ SD) was 114.9ñ9.5 for undernourished rats (N=8) for ATP, and 50.4ñ6.1 (N=8) vs 38.8ñ4.5 (N=8) for ADP. These results suggest that the deficits found in young rats disappear in rehabilitation adult rats

Effect of tityustoxin and quabain on protein phosphorylation in a crude synaptosome fraction

1. The effect of titiyustoxin (TsTX) and ouabain on the incorporation of 32P into a protein of the same apparent molecular weight as synapsin I it described. 2. Tityustoxin-stimulated protein phosphorylation in a crude synaptosome fraction increased up to a concentration of 3.0 micron time of 15 s. 3. Trifluoperazizne (100 micronM) inhibited, while trifluoperazine sulphoxide (100 micronM) did not alter the effect on the protein phosphorylation induced by tityustoxin. 4. Unlike tityutixin, ouabain (100 micronM) hada no effect on protein phosphorylation even after incubation up to 20 min. 5. Ouabain at at 10 micronM, a concentration having no effect on rates of respiration and ATP hydrolysis in brain cortical slices, also had no effect on protein phosphorylation

Proline binding to mouse synaptosomes

La l-prolina ejerce unos efectos fisiológicos y conductúales los cuales sugieren su posible función como neuromodulador en el cerebro de los mamíferos. Se ha caracterizado parcialmente el enlazamiento de l-prolina a sinaptosomas de cerebro del ratón. Análisis preliminar de la cinética demustra la presencia de por lo menos dos sitios de enlazamiento en el rango submicromolar y de un sitio en el rango nanomolar. Aunque son necesarios estudios más detallados encaminados a esclarecer el significado biológico del enlazamiento de prolina a sinaptosomas de cerebro del ratón, estos resultados sirven de apoyo adicional a la posible función de prolina omo neuromodulador

Effect of Carbon Monoxide-Induced Hypoxia on Synaptosomal Uptake and Release of Dopamine in Rat Striatum

We studied the effect of carbon monoxide (CO)-induced hypoxia on synaptosomal uptake and release of dopamine (DA) in rat striatum. When the rats were intoxicated at a blood level of carboxyhemoglobin (HbCO), 60-70% for 3-4hrs, [3H] DA uptake was inhibited as much as 80% of control activity. This suppressed activity remained as long as 12 hrs after termination of the intoxication. After a week recovery period, the suppressed uptake activity was restored completely. When the rats were intoxicated maintaining a blood level of HbCO at 30-40% for 6-7hrs, the uptake was inhibited to 57% of the control actvity and this suppressed activity was restored within 12hrs. For the rats maintaining a blood level of HbCO at 15-25% for 6-7hrs, uptake inhibition was not shown. Acute CO intoxication(at 60-70% of HbCO for 3-4 hrs) caused an increase in K+-stimulated DA release to 147% of the control value. In conclusion, the diminished uptake and increased release of striatal DA in a CO intoxicated brain would cause an extraneuronal accumulation of DA with depletion of intraneuronal DA level, which may play a role in CO-induced hypoxic cell damage.