Brain and Plasma Tetrahydroisoquinolines in Rats: Effects of Chronic Ethanol Intake and Diet

Brain concentrations of salsolinol (SAL), a simple tetrahydroisoquinoline (sTIQ) condensation product of dopamine (DA) and acetaldehyde, are reported to increase in chow-fed rats drinking ethanol/H2O ad libitum. However, our analyses showed that rat chow contains traces of SAL and, as previously reported, appreciable 3,4-dihydroxyphenylalanine (DOPA), a sTIQ precursor. To examine the effect of consumption of ethanol in a DOPA- and SAL-free diet on endogenous sTIQs, we analyzed two brain regions and blood plasma of rats undergoing prolonged intake (3 weeks and 23 weeks) of liquid diet containing 6.6% ethanol or isocaloric carbohydrate. SAL and three other DA-related sTIQs were quantitated using capillary gas chromatography/mass spectrometry in the selected ion mode with deuterated standards. In accord with studies on ethanol/chow-fed rats, sTIQ concentrations in hypothalamus were elevated after 3 weeks of ethanol, although after 23 weeks, hypothalamic sTIQs were either unchanged or reduced (O-methylated SAL). Furthermore, sTIQ concentrations in corpus striatum and, with one exception, plasma were not altered by ethanol ingestion for either duration. (However, 23 weeks of ethanol intake significantly reduced the striatal concentrations of DA and its acid metabolite, presumably reflecting neurotoxicity.) Reasoning that DOPA in diet might underlie the reported ethanol-dependent increases in striatal sTIQs, we found that L-DOPA supplementation (500 micrograms/rat/day) of EtOH/liquid diet-fed rats for 13 weeks tended to increase striatal SAL. Overall, the data indicate that elevations in endogenous sTIQ concentrations due to prolonged ethanol intake depend on the brain region, duration of intake, and even associated dietary constituents. In that regard, the higher striatal SAL concentrations in rats drinking ethanol ad libitum could have been facilitated by DOPA and perhaps SAL consumed in lab chow.     

Inhibition of Catechol-O-Methyltransferase by 6,7-Dihydroxy-3,4-Dihydroisoquinolines Related to Dopamine: Demonstration Using Liquid Chromatography and a Novel Substrate for O-Methylation

We report that 6,7-dihydroxy-3,4-dihydroisoquinolines related to dopamine are potent inhibitors of catechol-O-methyltransferase (COMT), but are not apparent substrates for the enzyme in vitro or in vivo. Three dihydroxy (catecholic) dihydroisoquinolines, including the 1-benzyl (DesDHP) and the 1-methyl (DSAL) analogs, were found to inhibit COMT activity in rat liver supernatant more effectively than the well-known inhibitor, tropolone. Inhibition of O-methylation was uncompetitive with substrate, and O-methylated products of the catecholic dihydroisoquinolines were undetectable. For these in vitro studies, a facile liquid chromatographic assay was developed utilizing as a site-specific substrate, 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline-1-carboxylate (salsolinol-1-carboxylate). This catechol produces only one phenolic product isomer when incubated with liver supernatant and S-adenosylmethionine. Following central injection of DSAL in rats, inhibition of brain COMT in vivo was indicated by the reduced brain levels of homovanillic acid, but not of 3,4-dihydroxyphenylacetic acid. Furthermore, O-methylated DSAL metabolites could not be detected in brain by liquid or gas chromatography. We suggest that 6,7-dihydroxy-dihydroisoquinolines are "nonmethylatable" COMT inhibitors because they exist as quinoidal tautomers resembling pyridones or tropolones rather than as catechols. Quinoid formation is supported by the fluorescence and ultraviolet spectra for DSAL and its O-methyl derivatives. The experiments reveal a new class of COMT inhibitors that may be of pharmacological and mechanistic value. Additionally, 3,4-dihydroisoquinolines could arise endogenously via oxidation of the 1,2,3,4-tetrahydroisoquinolines which are ingested or produced from cellular catecholamine condensations. However, it is unlikely that dihydroisoquinoline (e.g., DSAL) concentrations necessary to inhibit COMT significantly would be attained via endogenous pathways.     

Alterations in Regional Brain Catecholamine Concentrations After Experimental Brain Injury in the Rat

Abstract: Although activation of brain catecholaminergic systems has been implicated in the cerebrovascular and metabolic changes during subarachnoid hemorrhage, cerebral ischemia, cortical ablation, and cortical freeze lesions, little is known of the response of regional brain catecholamine systems to traumatic brain injury. The present study was designed to characterize the temporal changes in concentrations of norepinephrine (NE), dopamine (DA), and epinephrine (E) in discrete brain regions following experimental fluid-percussion traumatic brain injury in rats. Anesthetized rats were subjected to fluid-percussion brain injury of moderate severity (2.2–2.3 atm) and killed at 1 h, 6 h, 24 h, 1 week, and 2 weeks postinjury (n = 6 per timepoint). Control animals (surgery and anesthesia without injury) were killed at identical timepoints (n = 6 per timepoint). Tissue concentrations of NE, DA, and E were evaluated using HPLC. Following brain injury, an acute decrease was observed in DA concentrations in the injured cortex ( p < 0.05) at 1 h postinjury, which persisted up to 2 weeks postinjury. Striatal concentrations of DA were significantly increased ( p < 0.05) only at 6 h postinjury. Hypothalamic concentrations of DA and NE increased significantly beginning at 1 h postinjury ( p < 0.05 and p < 0.05, respectively) and persisted up to 24 h for DA ( p < 0.05) and 1 week ( p < 0.05) for NE. These data suggest that acute alterations occur in regional concentrations of brain catecholamines following brain trauma, which may persist for prolonged periods postinjury.     

Enhanced Brain Cell Proliferation Following Early Adrenalectomy in Rats

We have previously demonstrated an increase in adult brain DNA content in rats adrenalectomized on postnatal day 11. The present studies examined cell proliferation in cerebral cortex, cerebellum, hippocampus, and midbrain-diencephalon following adrenalectomy at this age. Compared to sham-operated controls, adrenalectomized animals showed increased [3H]thymidine incorporation into DNA (measured at 1 h following a pulse injection) in all brain regions at 7 and 14 days postsurgery. In some areas, the effect was already present as early as 2 days following adrenalectomy. Chronic replacement with corticosterone prevented this increase in DNA labelling in a dose-dependent manner. When cell proliferation in the cerebral cortex and cerebellum was independently assessed by measuring changes in thymidine kinase activity, enzyme activity was significantly elevated in both areas at 7 and 14 days postsurgery. Finally, histological examination of the cerebellar cortex suggested a delayed disappearance of the external granular layer in several cerebellar lobules of adrenalectomized animals. Overall, these findings indicate that day-11 adrenalectomy leads to a prolonged stimulation of mitotic activity in areas where cell formation at this time is exclusively glial (i.e., cerebral cortex and mid-brain-diencephalon) as well as in areas where postnatal neurogenesis is also occurring (cerebellum and hippocampus). It is hypothesized that this stimulation results from the removal of a tonic inhibitory effect exerted by circulating glucocorticoids in the normal intact animal.     

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury ( p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site ( p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury ( p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury ( p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.     

A Regional Study of 4-Aminobutyrate Metabolism and Amino Acid Levels in Rat Brain Following Chronic Oral Administration of Ethanolamine O-Sulphate

Abstract: Ethanolamine O-sulphate (EOS) dissolved in the drinking water (5mg-ml⁻¹) was administered ad libitum to rats for 26 days. At the end of this period, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) activities, 4-aminobutyrate (GABA) concentration, and the levels of six other amino acids were measured in various brain regions. Significant inhibition of GABA-T accompanied by significant increases in GABA content were observed throughout the brain, although the magnitudes of these effects varied according to region. GAD activity was significantly reduced in most brain regions, although this effect was apparently not related to cofactor availability or the direct actions of EOS or increased GABA concentration. Glutamine levels were significantly reduced to approximately 72% of control values in all brain regions. Aspartate levels were significantly reduced to approximately 84% of control values in all regions except the striatum and cerebellum. Minor changes in other amino acid levels were also detected. These neurochemical changes which accompanied the primary effect of EOS on GABA-T are discussed in terms of indirect secondary metabolic changes rather than nonspecific enzyme inhibition by EOS.     

Influence of Feeding Paradigm in Rats on Temporal Pattern of: II. Brain Serotoninergic and Catecholaminergic Systems

The relationship between the feeding paradigm (single diet versus food selection) and central idoleamines and catecholamines was studied. Temporal patterns of the brain parameters in response to presentation of a single diet of fixed composition (20% casein) or a choice between two isocaloric diets (0% and 60% casein) for 2 weeks under 8-h feeding cycles during the dark phase were measured in adult Sprague-Dawley rats. Groups of animals were then killed at the beginning and at 2-h intervals throughout the feeding period. The distribution and the temporal pattern of variation of the serotoninergic and the catecholaminergic parameters studied were significantly affected by the diet paradigms used. A different neurochemical equilibrium was observed before food intake and was characterized by a central serotoninergic predominance in subjects having a dietary selection experience but a central catecholaminergic predominance in animals adapted to a single diet. Hypothalamic and extrahypothalamic serotoninergic and catecholaminergic systems were found to intervene in an interdependent way, sometimes antagonistic according to the feeding paradigm and the related temporal changes in energy intake and macronutrient selection. These results suggest that central serotoninergic and catecholaminergic systems are influenced by the diet paradigm and display characteristic patterns of temporal variations during the feeding cycle. The feeding paradigm, per se, should then be considered as a potential synchronizer of central biological rhythms of monoamines, which in turn may affect food intake and appetite for macronutrients.     

Contribution of Sulfate Conjugation, Deamination, and O-Methylation to Metabolism of Dopamine and Norepinephrine in Human Brain

Abstract: The kinetic constants were determined for dopamine (DA) and norepinephrine (NE) metabolism by phenolsulfotransferase (PST), type A and B monoamine oxidase (MAO), and membrane-bound and soluble catechol- O - methyltransferase (COMT) in frontal lobe preparations of human brain. PST and membrane-bound COMT were found to have the lowest K _m, values for both catecholamines. By means of the appropriate rate equations and the calculated kinetic constants for each enzyme, the activity of each enzymatic pathway was determined at varying concentrations of DA and NE. Results indicate that deamination by MAO is the principal pathway for the enzymatic inactivation of DA whereas NE is largely metabolized by MAO type A and membrane-bound COMT under the in vitro assay conditions used. At concentrations less than 100 μ M , soluble COMT'contributes less than 5% to the total catabolism of either catecholamine. PST can contribute up to 15% of the total DA metabolism and 7% of NE metabolism.     

Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.     

Distribution of the Glucose Transporter in the Mammalian Brain

We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.     

Distribution of Brain-Derived Neurotrophic Factor in Rats and Its Changes with Development in the Brain

Abstract: A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.     

Delayed Neurologic Deterioration Following Anoxia: Brain Mitochondrial and Metabolic Correlates

Hyper- but not normoglycemic cats exposed to 8 min of anoxia show neurologic signs (fasciculations, myoclonic jerks, seizures) that develop after a symptom-free period. We examined brain mitochondrial function and metabolite concentrations at 0, 1, 3, and 5 h following exposure to anoxia, to correlate biochemical findings with the presence ("symptomatic") or absence ("presymptomatic") of neurologic signs. Brain mitochondria isolated postexposure only from symptomatic cats showed markedly decreased (-50%), state 3 (ADP-stimulated), and uncoupler-stimulated respiration rates with NAD- and FAD-linked substrates. Respiratory control and ADP/oxygen (ADP/O) ratios remained unchanged, indicating, respectively, that coupling and efficiency of ATP synthesis were preserved. Thus, inhibition of electron transport chain function, not phosphorylative activity, may be rate limiting for respiration. During anoxia, hyperglycemic cats showed higher brain lactate levels (26 versus 20 mumol/g), but similar ATP and phosphocreatine concentrations, compared with normoglycemic cats. After exposure, in all animals lactate and phosphocreatine were restored to control levels, whereas ATP remained at 85%. Cats that became symptomatic demonstrated four- to sixfold increases in lactate and 50% reductions in phosphocreatine. At 3 and 5 h postexposure, symptomatic animals showed significant reductions in ATP concentrations. We conclude that although initially asymptomatic, hyperglycemic cats exposed to anoxia undergo a neurologic deterioration over several hours following reoxygenation that is correlated with inhibition of mitochondrial respiration, increases in tissue lactate, and decreases in energy state.     

Tissue Distribution and Immunocytochemical Localization of Neurotrophin-3 in the Brain and Peripheral Tissues of Rats

Abstract: The tissue distribution of neurotrophin-3 (NT-3) was investigated in rats at 1 month of age using a newly established, sensitive two-site enzyme immunoassay system for NT-3, as well as the immunocytochemical localization of this protein. The immunoassay for NT-3 enabled us to quantify NT-3 at levels > 3 pg per assay. In the rat brain, NT-3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT-3 was widely distributed in peripheral tissues. Appreciable levels of NT-3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT-3 bound to pyramidal cells in the CA2-CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT-3-immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT-3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT-3 not only functions as a classical target-derived neurotrophic factor but also can play other roles.     

Effect of Pargyline on Brain N-tele-Methylhistamine in Portocaval-Shunted Rats: Relation to Amine Neurotransmitters

Abstract: HPLC determination of histamine, serotonin, dopamine, and noradrenaline in the brain tissue of rats with portocaval anastomoses (PCA) has revealed a selective increase in histamine concentration. In the posterior hypothalamus, the steady-state level of the amine metabolites showed an inverse pattern; N-tele -methylhistamine(t-MeHA), as estimated by gas chromatography-mass spectrometry, was not changed significantly by portocaval shunting, whereas 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid were more than doubled. Interestingly, the net increase in t-MeHA concentration in response to pargyline (80 mg/kg i.p.) was almost the same for PCA and sham-operated rats. This implies that the great enhancement of the histamine level in this area might be a consequence of the persistent stimulation of its synthesis and the unchanged activity of histaminergic neurons. In the rest of the brain, on the other hand, the steady-state level of t-MeHA was higher after PCA (3.8-fold), as were the levels of 5-HIAA and homovanillic acid. Surprisingly, t-MeHA remained unchanged after monoamine oxidase blockade. Of the pargyline-induced alterations in the concentrations of indoles and catechols, the most pronounced were those in the serotonin level; serotonin was elevated more than twofold in hypothalamus and more than 12-fold in the rest of the brain, with a concomitant 80% decrease in 5-HIAA. The dopamine and, to a much smaller extent, noradrenaline levels were also increased, and the levels of homovanillic acid and 3,4-dihydroxyphenylacetic acid fell below the detection limit. The study suggests that at least two different mechanisms operate in the brains of PCA rats to counteract the excessive synthesis of neuromediators, e.g., increased deposition and increased metabolism.     

A Bioluminescence Method for the Demonstration of Regional Glucose Distribution in Brain Slices

Abstract: Regional glucose distribution in brain slices was assessed by a bioluminescence technique. The reaction is based on light emission of luminiferous marine bacteria, Vibrio fischeri , induced by NADPH. Freeze-dried brain slices were covered by a solution which contained: (a) enzymes and substrates for glucose oxidation and NADPH formation and (b) an extract of Vibrio fischeri for the bioluminescence reaction. Glucose-induced bioluminescence was recorded on photographic film. Patterns of regional decrease in glucose concentration were demonstrated in cat brains after occlusion of the left middle cerebral artery. This decrease correlated well with a concomitant depletion of ATP and an increase in NADH-fluorescence.     

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.     

Brain α-Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.     

Regional Distribution and Production Rate of 3-Methoxy-4-Hydroxyphenylethyleneglycol Sulphate (MHPG-SO4) in Rat Brain

The levels of noradrenaline (NA) and 3-methoxy-4-hydroxyphen-ylethyleneglycol sulphate (MHPG-SO₄) in 15 brain regions showed a parallel distribution in male Wistar rats. The differences in regional distribution of MHPG-SO₄ were similar to those in the rate of NA turnover reported by other investigators. The accumulation rates of MHPG-SO₄ during 45 and 90 min after probenecid injection significantly correlated to the steady state levels of MHPG-SO₄ in nine regions studied. With the results, the regional levels of MHPG-SO, either in untreated or in probenecid-treated rats, are considered to be a useful index of NA turnover.     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.