Levels of dopamine and its metabolites in the extracellular medium of the striatum of newborn piglets during graded hypoxia.
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3,4-Dihydroxyphenylacetic Acid↗
Biomedical subjects
A Pastuszko
Publications and source records attributed to A Pastuszko.
Effect of neurocatin on the activity of monoamine oxidase B in rat brain synaptosomes.
Neurocatin, a small (about 2,000 Dalton) neuroregulator isolated from mammalian brain, is a powerful effector of monoamine oxidase B in rat brain synaptosomes. Incubation of intact synaptosomes with neurocatin caused an inhibition of the enzyme dependent on the concentration of neurocatin. This inhibition became statistically significant at a neurocatin concentration of 10 ng/200 microliters and was significant at all higher neurocatin concentrations. At 40 ng/200 microliters, neurocatin inhibited monoamine oxidase B activity by about 60%. This inhibitory effect was almost completely abolished by breaking the synaptosomal membrane by hypotonic buffer prior to incubation with neurocatin. In addition, incubation of the synaptosomes in calcium free medium almost completely abolished the inhibitory effect of neurocatin on monoamine oxidase B. The inhibition appeared to involve covalent modification of the enzyme mediated by a neurocatin receptor(s). Measurements of the kinetic parameters of the enzyme showed that 20 ng of neurocatin caused a statistically significant decrease in Vmax (by 20%) with no significant change in KM, compared to controls. Inhibition of monoamine oxidase by neurocatin is potentially of great clinical importance because this enzyme plays a major role in catabolism of the biogenic amines and alterations in its activity is believed to contribute to several neurological disorders.
Animals↗
Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure.
The present studies describes the relationship between extracellular dopamine in striatum of newborn piglets and cortical oxygen pressure. The extracellular level of dopamine was measured by in vivo microdialysis and the oxygen pressure in the cortex was measured by phosphorescence lifetime of oxygen probe in the blood. Controlled, graded levels of hypoxic insult to the brain of animals were generated by decreasing of the oxygen fraction in the inspired gas (FiO2) from 21% to 14%, 11%, and 9%. This resulted in decrease in the cortical oxygen pressure from 31-35 Torr to about 24 Torr, 15 Torr and 4 Torr, respectively. The changes in extracellular level of dopamine, DOPAC and HVA were dependent on changes in cortical oxygen pressure. Stepwise decrease in the cortical oxygen pressure (see above) caused increases in extracellular dopamine of about 80%, 200% and 550%, respectively. The levels of DOPAC and HVA progressively decreased and when cortical oxygen decreased to 4-6 Torr were about 50% and 70% of control, respectively. After return of FiO2 to control (21%), the cortical oxygen pressure rapidly increased to above normal, then returned to control values. The extracellular levels of dopamine, DOPAC, and HVA recovered more slowly, attaining control values in about 30 minutes. The data show that extracellular levels of dopamine increase with even very small decreases in oxygen pressure. Thus, there is no "oxygen reserve" which protects dopamine release and metabolism from decrease in oxygen pressure.
Analysis of Variance↗
3,4-dihydroxyphenylalanine (dopa) metabolism and retinoic acid induced differentiation in human neuroblastoma.
In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (dopa) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of dopa. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of dopa and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-69, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and enzyme expressions. The RA treatment modulated the activities of tyrosine hydroxylase and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.
Cell Differentiation↗
The effect of neurocatin on protein phosphorylation in striatal synaptosomes from rat brain.
Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of approximately 80 and approximately 60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to approximately 7.5 ng/100 microliters of suspension), incorporation of 32P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin > 7.5 ng/100 microliters caused progressive decrease in incorporation of 32P into many synaptosomal proteins; by a concentration of neurocatin of approximately 45 ng/100 microliters, the level of 32P incorporation into many proteins was < or = 70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of approximately 7.5 ng/100 microliters of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times > 2.0 min, showed progressive decrease in 32P incorporation. Removing extrasynaptosomal Ca2+ with EGTA attenuated the increased 32P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of 32P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.
Animals↗
Activity of tyrosine hydroxylase in the striatum of newborn piglets in response to hypocapnic hypoxia.
The effect of graded hypoxia induced by hyperventilation on the activity of tyrosine hydroxylase was measured in vivo by microdialysis. Microdialysis probes were inserted into the striatum of newborn piglets and perfused with medium containing 3-hydroxybenzylhydrazine, an inhibitor of L-aromatic amino acid decarboxylase. The level of 3,4-dihydroxyphenylalanine (DOPA) measured in the effluent dialysate was then an index of tyrosine hydroxylase activity. The oxygen pressure in the veins and capillaries of the cortex was measured, through a cranial window placed over the parietal cortex, by the phosphorescence lifetime of palladium-meso-tetra(4-carboxyphenyl)porphine added to the blood. After baseline measurements, PaCO2 was decreased from 38 torr (control value) to 19, 13, and 11 torr resulting in decreases in the cortical oxygen pressure from 40 +/- 6 torr to 26 +/- 3, 23 +/- 4, and 20 +/- 4 torr, respectively. Decrease in the oxygen pressure to 26 +/- 3 torr caused a statistically significant increase of 25-30% in the level of DOPA in the effluent perfusate. During the next step of increase in ventilator rate, when oxygen decreased only slightly, the level of DOPA remained at the higher level. Ventilation rates that lowered the oxygen pressure to below 20 torr, however, caused a progressive decrease in the level of DOPA. During recovery, the level of DOPA steadily increased, attaining 160% of control value after 1.5 h. When the oxygen pressure was decreased to 16 +/- 2 torr by a single increase in ventilator rate, the DOPA level decreased in the effluent to 15-20% below control.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of graded levels of tissue oxygen pressure on dopamine metabolism in the striatum of newborn piglets.
The effect of graded levels of tissue hypoxia on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid has been monitored in vivo by microdialysis. Reproducible levels of decreased oxygen in the brain were obtained by increasing the rate of ventilation from the control value of 25/min to as high as 95/min. With increasing ventilatory rate, the oxygen pressure in the cortex decreased from approximately 40 torr to 16 torr. As the oxygen pressure decreased stepwise from 40 to 27, 22, and 16 torr, the dopamine levels in the extracellular medium rose by 70, 90, and 150%, respectively, returning to baseline within a few minutes of return to control ventilation rates. Levels of the catabolic products 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid decreased with decreasing tissue oxygen. Unlike the dopamine levels, these catabolite levels continued to decrease through 30 min of recovery (to 50% of control), returning to baseline only after recovery periods of 1-2 h. These data suggest that hypoxia induces long-term alterations in the neurotransmitter turnover. The marked effects of mild tissue hypoxia (decrease of oxygen from 40 torr to 26 torr) on both the extracellular dopamine concentration and dopamine metabolism indicate that the metabolic consequences of decreased tissue oxygen pressure extend to higher values than generally appreciated.
3,4-Dihydroxyphenylacetic Acid↗
Microvascular damage in the cortex of cat brain from middle cerebral artery occlusion and reperfusion.
Oxygen-dependent quenching of phosphorescence provides an extraordinarily powerful method for examining the effects of ischemia/hypoxia on the cortex of the brain. Video camera technology has permitted imaging, through a window in the skull, of the phosphorescence of an oxygen probe, Pd meso-tetra-(4-carboxyphenyl)-porphine, bound to albumin in the blood of anesthetized animals. Images of the phosphorescence taken at different times after the flash of excitation light were used to generate high-resolution two-dimensional maps of the oxygen pressure. These maps show that cortical oxygenation is spatially heterogeneous and that there is dynamic time-dependent modulation of regional oxygen pressures. When the middle cerebral artery was occluded, the region for which it supplied blood became hypoxic, the severity of the hypoxia varying among animals. Release after 60 min of occlusion resulted in a rapid rise of the oxygen pressure to above-normal levels followed by onset of a delayed period of hypoxia. This period is characterized by generally low tissue oxygen pressures with local regions of more severe hypoxia. The delayed period of hypoxia appears to result from damage to the microvasculature, and this microvascular damage is proposed to be an important determinant of the extent of irreversible brain damage.
Animals↗
Effect of hyperventilation on oxygenation of the brain cortex of neonates.
A new phosphorescence imaging method (Rumsey et al, Science (1988) 1649) has been used to continuously monitor the oxygen pressure in the blood of the cerebral cortex of newborn pigs. The animals' blood pressure was continuously measured and PaCO2, PaO2 and arterial blood pH were measured periodically. The oxygen pressure in the blood was quantitatively determined for regions of about 100 um square within the image (from a total field of about 3 mm diameter). It was observed that during hyperventilation, which lowered PaCO2 and increased pH of the blood, oxygen pressure decreased in proportion to the decrease in PaCO2. For example, hyperventilation which decreased PaCO2 from its normal value of 40 Torr to 10 Torr caused a rapid (within 5 minutes) decrease in oxygen pressure in the blood of capillaries and veins to approximately 1/4 of normal.
Animals↗
Oxygenation of the cortex of the brain of cats during occlusion of the middle cerebral artery and reperfusion.
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Animals↗
Activation of striatal tyrosine hydroxylase by neurocatin, a neuroregulator from mammalian brain.
Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of dopamine synthesis in striatal rat synaptosomes. Incubation of intact synaptosomes with neurocatin caused an increase in the rate of dopamine synthesis measured by accumulation of DOPA. The increase is rapid (within two minutes) and dependent on the concentration of added neurocatin. The stimulatory effect of neurocatin on dopamine synthesis occurred only in intact synaptosomes and was almost completely abolished by lysis of the synaptosomes with Triton X-100 or sonification prior to neurocatin addition. The kinetic parameters of tyrosine hydroxylase were measured in lysates prepared from synaptosomes preincubated with neurocatin. These showed that with increasing neurocatin concentration there was an increase in Vmax with no significant change in KM for the pteridine cofactor, compared to control. Activation of tyrosine hydroxylase by neurocatin is at least partially caused by a receptor mediated increase in phosphorylation of the enzyme. Protein kinase C and protein kinase II may be involved in this process.
Animals↗
Neurocatin-induced inhibition of monoamine oxidase A in rat brain synaptosomes.
Neurocatin is a small (about 2000 Da) neuroregulator isolated from mammalian brain. Earlier it was shown that addition of nanomolar concentrations of neurocatin to synaptosomes isolated from rat brain increased levels of serotonin and decreased catabolism of serotonin to 5-hydroxyindoleacetic acid (Fernandez-Novoa L and Pastuszko A. Neurosci Lett 122: 83-86, 1991). In the present study, we report that neurocatin addition resulted in a striking inhibition of monoamine oxidase A activity. This inhibition became statistically significant at a neurocatin concentration of approximately 5 nM and was significant at all higher neurocatin concentrations. Neurocatin at approximately 50 nM inhibited monoamine oxidase A activity by about 90%. The inhibitory effect of neurocatin on monoamine oxidase required its incubation with intact synaptosomes since addition after breaking the synaptosomes by hypotonic buffer or lysis by Triton X-100 almost completely blocked the inhibitory effect. Measurements of the kinetic parameters of the enzyme in lysates prepared from synaptosomes incubated with neurocatin showed a decrease in Vmax with no change in Km for the substrate (serotonin) compared to controls. Incubation of the synaptosomes with approximately 25 nM neurocatin resulted in an 80% decrease in the Vmax of monoamine oxidase A. Evidence that neurocatin is a powerful endogenous modulator of monoamine oxidase activity is particularly intriguing. This enzyme plays a major role in catabolism of the biogenic amines and is believed to contribute to several important neurological disorders.
Animals↗
Neurocatin induces changes in release and level of serotonin in synaptosomal fraction from rat brain.
A newly isolated factor from mammalian brain, neurocatin, is shown to increase both the level and release of serotonin in suspensions of synaptosomes isolated from rat brain. Incubation of synaptosomes for 10 min with approximately 20 nM neurocatin resulted in release of about 50% of the total pool of serotonin. Quantification of serotonin and its major metabolite, using an electrochemical detector, indicated that the presence of neurocatin also caused an increase in the absolute level of serotonin and decrease in its catabolism to 5-hydroxyindoleacetic acid (5-HIAA). The effect is dependent on the time of incubation and concentration of neurocatin. At a concentration of 20 nM, neurocatin increased about 60% the level of serotonin and decreased about 50% the level of 5-HIAA. Depolarization conditions--50 microM veratridine and 50 mM K+ medium--increased release of serotonin by 50% and 30% respectively without affecting the level of serotonin or its catabolism to 5-HIAA.
Animals↗
Effect of hyperventilation on oxygenation of the brain cortex of newborn piglets.
A new phosphorescence imaging method (Rumsey et al. Science Wash. DC 241: 1649-1651, 1988) has been used to continuously monitor the PO2 in the blood of the cerebral cortex of newborn pigs. A window was prepared in the skull and the brain superfused with artificial cerebrospinal fluid. The phosphorescent probe for PO2, Pd-meso-tetra(4-carboxyphenyl)porphine, was injected directly into the systemic blood. The phosphorescence of the probe was imaged, and the lifetimes were measured using flash illumination and a gated video camera. The PO2 in the blood of the veins and capillary beds of the cortex was calculated from the lifetimes. Systemic blood pressure was continuously monitored while the systemic arterial PCO2, PO2, and blood pH were measured periodically. The PO2 in the blood was quantitated for 60- to 200 microns2 regions within the image (from a total field of approximately 3 mm diam). The PO2 in the microvasculature was not uniform across the viewing field but increased or decreased in each region independently of the other regions. Thus at any point in time the PO2 in a region could be substantially above or below the average value. During hyperventilation, which lowered arterial PCO2 and increased pH of the blood, the average PO2 decreased in proportion to the decrease in arterial PCO2. For example, hyperventilation, which decreased arterial PCO2 from its normal value of 40 Torr to 10 Torr, caused a rapid (within 5 min) decrease in PO2 in the blood of capillaries and veins to approximately one-third of normal.
Animals↗
Effect of neurocatin, a new neuroregulatory factor from brain, on the metabolism of catecholamines in CHP-134 human neuroblastoma cells.
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Brain↗
Purification of neurocatin, a neuroregulatory factor from brain.
A neuroregulatory factor (neurocatin) has been isolated from bovine brain. Neurocatin is a powerful affector of catecholamine metabolism in synaptosomes isolated from rat brain, causing increased formation of norepinephrine (NE) and decreased formation of 3,4-dihydroxyphenylacetic acid (DOPAC). The ratio NE/DOPAC in synaptosomes has been used to measure the neurocatin content of samples during isolation. Neurocatin has now been purified to a single chromatographic peak by high-resolution HPLC and a preliminary amino acid content measured. It appears to be a small peptide with a molecular weight in the range of 2,000-2,500 Da.
3,4-Dihydroxyphenylacetic Acid↗
Modulation of catecholamine metabolism in synaptosomes by a neuroregulatory factor from mammalian brain.
Incubation of synaptosomes from rat brain with bovine brain extract caused inhibition of oxidative deamination of dopamine, decreased formation of 3,4-dihydroxyphenylacetic acid, increased formation of norepinephrine and its N-methyl derivatives and increased release of catecholamines. Omission of Ca2+ from the extrasynaptosomal medium completely blocked the brain extract induced release of [3H]catecholamines and decreased, by about 80%, the effect on changes in catecholamine metabolism. Chromatography of the brain extract on Sephadex-G25 columns resulted in an active compound eluting at the position expected for compounds with molecular weights in the region of 1500 to 2500 Da.
3,4-Dihydroxyphenylacetic Acid↗
Effects of DL-2-amino-5-phosphonovalerate on metabolism of catecholamines in synaptosomes from rat brain.
Incubation of synaptosomes from rat brain with DL-2-amino-5-phosphonovalerate (APV) stimulated an increased release of dopamine, and this effect was strictly dependent on the extrasynaptosomal calcium level. APV increased biosynthesis of dopamine from tyrosine by 30%, whereas monoamine oxidase activity was inhibited by 30%. When synaptosomes were incubated with radioactive dopamine, APV caused a large decrease in incorporation of label into 3,4-dihydroxyphenylacetic acid but greatly increased incorporation into norepinephrine and its N-methyl derivatives. Quantification of dopamine and its metabolites in synaptosomes, using electrochemical detection, indicated that the presence of APV resulted in changes in the absolute levels of the aforementioned dopamine metabolites similar to the changes in radiolabel incorporation. Omission of Ca2+ from the extrasynaptosomal medium greatly diminished the APV-induced changes in catecholamine metabolism. The metabolic changes appear to largely result from an increased intrasynaptosomal Ca2+ level due to the APV-induced increase in calcium permeability of the plasma membrane.
2-Amino-5-phosphonovalerate↗