bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Na+, K+‐ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum
Authors: Atterwill C. K. , Cunningham V. J. , Balázs R.
Published: October 2006

Abstract: The activities of certain properties of sodium, potassium‐activated adenosine triphosphatase (Na +, K+‐ ATPase; EC 3.6.1.3) were examined in cultures and peri‐ karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto‐ somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na +, K+‐ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15‐fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na +, K+‐ATPase (apparent Aj values of about 10–7M and 10–4‐10–5M, respectively), the high‐ affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na +,K + ‐ ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na+,K+‐ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na +, K+‐ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high‐ and the low‐affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na+, K+‐ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of 86Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K+ dependence of the Na+, K+‐ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as‐ trocyte preparations at 1 mM KCl, when only about one‐ third of the maximal activity was displayed by the cultured nerve cells.
Influence of Severe Hypoglycemia on Brain Extracellular Calcium and Potassium Activities, Energy, and Phospholipid Metabolism
Authors: Wieloch Tadeusz , Harris Robert J. , Symon Lindsay , Siesjö Bo K.
Published: October 2006

Abstract: In the cerebral cortices of rats, during insulininduced hypoglycemia, changes in the concentrations of labile phosphate compounds [ATP, ADP, AMP, and phosphocreatine (PCr)] and glycolytic metabolites (lactate, pyruvate, and glucose) as well as phospholipids and free fatty acids (FFAs) were studied in relation to extracellular potassium and calcium activities. Changes in extracellular calcium and potassium activities occurred at approximately the onset of isoelectricity. The extracellular calcium activity dropped from 1.17 ± 0.14 mM to 0.18 ± 0.28 mM and the potassium activity rose from 3.4 ± 0.94 mM to 48 ± 12 mM (means ± SD). Minutes prior to this ionic change the levels of ATP, PCr, and phospholipids were unchanged while the levels of FFAs remained unchanged or slightly elevated. Following the first ionic change the steady‐state levels of ATP decreased by 40%, from 2.42 to 1.56 μmol/g. PCr levels decreased by 75%, from 4.58 to 1.26 μmol/g. Simultaneously, the levels of FFAs increased from 338 to 642 nmol/g, arachidonic acid displaying the largest relative increase, 33 to 130 nmol/g. The first ionic change was followed by a short period of normalization of ionic concentrations followed by a sustained ionic change. This was accompanied by a small additional decrease in ATP (to 1.26 μmol/g). The FEA levels increased to 704 nmol/g. There was a highly sig nificant negative correlation between the levels of FFAs and the energy charge of the tissue. The formation of FFAs was accompanied by a decrease in the phospholipid pool. The largest relative decrease was observed in the inositol phosphoglycerides, followed by serine and ethanolamine phosphoglycerides. After 10 min of isoelectricity the levels of phospholipids had decreased by 5.12 μmol/g while the levels of FFAs had increased by 0.46 μmol/g, indicating oxidative metabolism or washout of the released FFAs. The attenuation of the rapid initial changes in the levels of the energy metabolites and FFAs as well as the correlation between the energy charge and the levels of FFAs suggests that a new steady state is established following the first ionic change. The importance of these reactions for the development of hypogiycemic neuronal damage is discussed.
A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia
Authors: Dóra Eörs
Published: October 2006

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose‐anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10‐3‐10‐1M) and cyanide (10‐5‐10‐2M) resulted in a concentration‐dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10‐2M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10‐1M induced approximately half of the NAD reduction evoked by 10‐2M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.
Alterations in the Retinal Dopaminergic Neuronal System in Rats with Streptozotocin‐Induced Diabetes
Authors: Nishimura Chihiro , Kuriyama Kinya
Published: October 2006

Abstract: Neurochemical alterations, which may be associated with the development of diabetic retinal dysfunction, were investigated using streptozotocin (STZ)‐induced hyperglycemia in rats. Young male Wistar rats, weighing 100–150 g, were made diabetic with daily intraperitoneal injections of STZ (30 mg/kg) for 5 days. This treatment caused a continuous hyperglycemia (400–600 mg/dl) and suppressed gain in body weight. Nine weeks after the STZ treatment, a significant increment in retinal valine and a decline in phenylalanine were noted, while the concentrations of other neuroactive amino acids, such as γ‐aminobutyric acid and aspartic acid, in the retina remained unchanged. On the other hand, the concentration of retinal dopamine (DA) was found to decrease significantly from the third week of hyperglycemia, when [3H]spiperone binding showed a tendency to increase in the retinal particulate fraction. However, the activities of tyrosine hydroxylase and aromatic l‐amino acid decarboxylase (AADC) and the uptake of [3H]tyrosine showed no alteration in the retina of diabetic rats. The accumulation rate of 3,4‐dihydroxyphenylalanine (DOPA) in vivo in the retina of diabetic rats, measured following the administration of the AADC inhibitor m‐hydroxybenzyl‐hydrazine (100 mg/kg i.p.), was also unchanged. Although [3H]DA uptake by retinal tissue was similar in control and diabetic animals, the spontaneous efflux of [3H]DA from the retina was found to be significantly accelerated in STZ‐treated animals. In addition, the release of preloaded [3H]DA, elicited by repeated photic stimulation, was significantly attenuated in retina from diabetic rats. These results suggest that an accelerated efflux of DA, possibly leading to the depletion of DA from the retinal DA system, may account for early retinal dysfunctions known to occur in diabetic subjects.
Changes of Ganglioside Pattern During Cerebellar Development of Normal and Staggerer Mice
Authors: Schaal Heiner , Wille Christiane , Wile Wolfgang
Published: October 2006

Abstract: The total amount of gangliosides per cerebellum of a wild‐type mouse increased 126‐fold during postnatal development. Although all major gangliosides were synthesized, the relative amount of individual ganglioside species changed during this period. In the developing wild‐type cerebellum a transient accumulation of GD3 occurred between birth and postnatal day 20 (P20) with the largest portion (23%) of the total ganglioside content at postnatal day 7 (P7). In the adult cerebellum GD3 was only a minor component (3.2%) of the ganglioside pattern. As demonstrated by immunofluorescence the accumulation of GD3 was predominantly associated with premigratory and early postmigratory granule cells. The ganglioside GD3 was found in two alkali‐stable forms in the young cerebellum, whereas the ganglioside species with the higher Rf value (migrating in the same position as the upper GD3 band) in the adult cerebellum was alkali labile. The cerebellum of the neurological mutant staggerer (sg/sg) was characterized by a low amount of GD1a in adult animals, due to the massive death of neurons in the postnatal cerebellar cortex. The neonatal loss of sialic acid residues from cerebellar cell surfaces in wild‐type mice and the maintenance of embryonic sialoglycoconjugates in the staggerer cerebellum cannot be explained by the alterations of ganglioside patterns observed during postnatal development.
Demonstration and Distribution of Kassinin‐Like Material (Substance K) in the Rat Central Nervous System
Authors: Shults C. W. , Yajima H. , Gullner H.‐G. , Chase T. N. , O'Donohue T. L.
Published: October 2006

Abstract: Antiserum was raised against kassinin in rabbits. Cross‐reactivity with other tachykinins was determined; these included substance K (100%) and substance P (0.1%). Peptides extracted from rat brain and synthetic tachykinins were chromatographed by reverse‐phase HPLC. The major peak of kassinin‐like material eluted at a time different from that of synthetic kassinin, eledoisin, physalaemin, neurokinin β, and substance P but coeluted with substance K. Measurement of kassinin‐like material in macrodissected and microdissected brain regions indicated that the distribution of kassinin‐like material was similar to that of substance P.
Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes
Authors: Bruce Gordon , Wainer Bruce H. , Hersh Louis B.
Published: October 2006

Abstract: A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50–60%). Purified ChAT was used to raise a monospecific anti‐human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.
Serotonin Agonists Reduce Dopamine Synthesis in the Striatum Only when the Impulse Flow of Nigro‐Striatal Neurons Is Intact
Authors: Spampinato U. , Esposito E. , Samanin R.
Published: October 2006

Abstract: The effects of 5‐methoxy‐N, N‐dimethyltryptamine (5‐MeO‐DMT) and m‐chlorophenylpiperazine (CPP), two 5‐hydroxytryptamine (5‐HT, serotonin) agonists, on the accumulation of 3,4‐dihydroxyphenylalanine (DOPA) were studied in the striatum of rats treated with γ‐butyrolactone (GBL). Unlike 2 mg/kg i.p. apomorphine, neither 5 mg/kg i.p. 5‐MeO‐DMT nor 2.5 mg/kg i.p. CPP significantly reduced the GBL‐induced increase in DOPA accumulation in the striatum. 5‐MeODMT and CPP significantly reduced DOPA accumulation in animals that had received the aromatic amino acid decarboxylase inhibitor Ro 4–4602 but not GBL. 5‐HT (10 μg in 0.5 μl) injected in the substantia nigra, pars compacta, like GBL, significantly increased Ro 4‐4602‐induced accumulation of DOPA in the striatum. The data indicate that 5‐HT agonists can reduce 3,4‐dihydroxyphenylethylamine (DA, dopamine) synthesis in the striatum of rats only when the impulse flow of DA neurons is intact. An indirect effect through mechanisms controlling DA synthesis in the striatum, for instance cholinergic and GABA‐ergic neurons, is suggested.
Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries
Authors: Betz A. Lorris
Published: October 2006

Abstract: Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7‐fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.
Atypical Distribution of Asymmetric Acetylcholinesterase in Mutant PC12 Pheochromocytoma Cells Lacking a Cell Surface Heparan Sulfate Proteoglycan
Authors: Inestrosa Nibaldo C. , Matthew William D. , Reiness C. Gary , Hall Zach W. , Reichardt Louis F.
Published: October 2006

Abstract: We studied the distribution of the molecular forms of acetylcholinesterase (AChE) in a stable variant (F3) of the rat pheochromocytoma cell line, PC12, that lacks a heparan sulfate proteoglycan on the cell surface. After treatment with nerve growth factor F3 cells synthesize less 4S enzyme, and more 10S and 16S enzyme than normal PC12 cells. This distribution is similar to that seen in normal cells after incubation with β‐d‐xylosides, molecules that interfere with proteoglycan assembly. Using collagenase treatment and membrane‐permeable and ‐impermeable inhibitors of AChE, we determined the cellular location of the AChE forms. Although in normal cells >90% of the 16S AChE is on the cell surface, approximately 60% is present in an internal pool in the variant. Following irreversible inhibition of all forms of AChE in the variant, the newly synthesized 16S AChE appears in the internal pool after a 1‐h lag, but is not detected on the cell surface until after 2.5 h. Our results thus show that 16S AChE is assembled internally within neuronal cells and that alterations in the synthesis and distribution of proteoglycans affect the total amount and cellular localization of the 16S AChE form.
Sidedness of Phosphatidylcholine‐Synthesizing Enzymes in Rat Brain Microsomal Vesicles
Authors: Arienti Giuseppe , Corazzi Lanfranco , Freysz Louis , Binaglia Luciano , Roberti Rita , Porcellati Giuseppe
Published: October 2006

The sidedness of CDP‐choline:1,2‐diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base‐exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury‐dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP‐choline:1,2‐diradylglycerol choline phosphotransferase and the choline base‐exchange activity are localized on the outer surface of rat brain microsomal vesicles.
Regional Acetylcholine Metabolism in Brain During Acute Hypoglycemia and Recovery
Authors: Ghajar J. B. G. , Gibson G. E. , Duffy T. E.
Published: October 2006

Insulin‐induced hypoglycemia in normothermic rats caused progressive neurological depression and differentially altered regional cerebral acetylcholine metabolism. Reductions of plasma glucose from 7.7 mM (control) to 2.5‐1.7 mM (moderate hypoglycemia associated with decreased motor activity) or 1.5 mM (severe hypoglycemia with lethargy progressing to stupor) decreased glucose concentrations in the cerebral cortex, striatum, and hippocampus to < 10% of control. Moderate hypoglycemia diminished acetylcholine concentrations in cortex and striatum (21% and 45%, respectively) and reduced [1‐2H2, 2‐2H2]choline incorporation into acetylcholine (62% and 41%, respectively). Severe hypoglycemia did not reduce the acetylcholine concentration or synthesis in cortex and striatum further. The concentrations of choline rose in the cortex (+ 53%) and striatum (+ 130%) of animals that became stuporous but a similar rise in [1‐2H2, 2‐2H2]choline left the specific activities of choline in these structures unchanged. Even severe hypoglycemia did not alter the hippocampal cholinergic system. In rats that developed hypoglycemic stupor and were then treated with glucose, the animals recovered apparently normal behavior, and the concentrations of acetylcholine and the incorporation of [1‐2H2, 2‐2H2]‐choline into acetylcholine returned to control values in the striatum but not in the cerebral cortex. Thus, impaired acetylcholine metabolism in selected regions of the brain may contribute to the early symptoms of neurological dysfunction in hypoglycemia.
Chromatin Proteins Share Antigenic Determinants with Neurofilaments
Authors: Wood John N. , Lathangue Nicholas B. , McLachlan Donald R. , Smith Bryan J. , Anderton Brian H. , Dowding Alan J.
Published: October 2006

Antigenic determinants common to distinct proteins may be unambiguously identified by the use of monoclonal antibodies. Some monoclonal antibodies to mammalian neurofilaments have recently been shown to cross‐react with the neurofibrillary tangles found at high density in the brains of senile dements with Alzheimers disease (SDAT). Here, we show that these antibodies also cross‐react with chromatin proteins, including the linker histones H1 and H10. Elevated levels of histone H10 have also been reported in SDAT brains.
Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex
Authors: Ravikumar B. V. , Sastry P. S.
Published: October 2006

Twenty‐two frontal cortices from normal human foetal brains of gestational ages ranging from 16 to 40 weeks and five postnatal brains ranging from 5 to 50 years were analysed for the ontogeny of muscarinic receptors using [3H]quinuclidinyl benzilate (QNB) as the ligand. QNB binding sites were shown to be stable up to 41/2 months of storage at – 70°C. QNB binding was characterized in frontal cortices of 28‐week‐old foetal brains as muscarinic receptors by the following criteria: (1) it was localised mainly in particulate fraction; (2) binding was saturable at a concentration of 1.5 nM; (3) the cholinergic antagonists atropine and scopolamine competed for the binding, with IC50 values of 1 and 0.8 nM, respectively. The agonists oxotremorine, carbachol, and pilocarpine gave IC50 values of 1, 15 and 18 μM, respectively. Nicotinic receptor ligands and noncholinergic drugs could not compete for the binding. Bimolecular association and dissociation rate constants for the reversible binding are 6.23 ± 108M−1± min−1 and 2.0 ± 10−2± min−1, respectively. The equilibrium dissociation constant is 33 pM. The KD obtained by saturation binding data is 103 pM. Ontogeny of muscarinic receptors showed three distinct phases: In phase I, they appear between 16 and 18 weeks [average concentration 109 fmol/mg protein of total particulate fraction (TPF)] and slowly increase up to 20 weeks (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 weeks at which time receptor concentration does not change perceptibly (average concentration 167 fmol/mg protein TPF). Phase III is a rapid increase of receptors occurring in the third trimester, reaching a maximum at birth (average concentration 450 fmol/mg TPF). After birth the receptor content declines and the values in adult frontal cortices are 50% of that at birth, indicating synapse elimination postnatally. Affinity of the receptor to QNB did not change during foetal development. Sodium chloride and GTP decrease the agonist affinity, as observed by carbachol competition of QNB binding. This effect was observed even at 16–18 weeks. The magnitude of sodium effect remained two‐ to threefold at all ages but the GTP effect increased from about 1.4‐fold at 16–18 weeks to two‐ to threefold at birth. These results suggest that the modulatory elements appear simultaneously with the receptors.
Human Blood—Brain Barrier Insulin Receptor
Authors: Pardridge William M. , Eisenberg Jody , Yang Jing
Published: October 2006

A new model system for characterizing the human brain capillary, which makes up the blood–brain barrier (BBB) in vivo, is described in these studies and is applied initially to the investigation of the human BBB insulin receptor. Autopsy brains were obtained from the pathologist between 22–36 h postmortem and were used to isolate human brain microvessels which appeared intact on both light and phase microscopy. The microvessels were positive for human factor 8 and for a BBB‐specific enzyme marker, γ‐glutamyl transpeptidase. The microvessels avidly bound insulin with a high‐affinity dissociation constant, KD= 1.2 > 0.5 nM. The human brain microvessels internalized insulin based on acid‐wash assay, and 75% of insulin was internalized at 37°C. The microvessels transported insulin to the medium at 37°C with a t1/2=$70 min. Little of the 125I‐insulin was metabolized by the microvessels under these conditions based on the elution profile of the medium extract over a Sephadex G‐50 column. Plasma membranes were obtained from the human brain microvessels and these membranes were enriched in membrane markers such as γ‐glutamyl transpeptidase or alkaline phosphatase. The plasma membranes bound 125I‐insulin with an ED50= 10 ng/ml, which was identical to the 50% binding point in intact microvessels. The human BBB plasma membranes were solubilized in Triton X‐100 and were adsorbed to a wheat germ agglutinin Sepharose affinity column, indicating the BBB insulin receptor is a glycoprotein. Affinity cross‐linking of insulin to the plasma membranes revealed a 127K protein that specifically binds insulin. These studies demonstrate (a) the biochemistry of the human BBB may be investigated using the human brain microvessel model system, and (b) the human BBB insulin receptor has structural characteristics typical of the insulin receptor in peripheral tissues and may be part of a combined endocytosis‐exocytosis system for the transport of the peptide through the BBB in man.
Uridine Transport and Metabolism in the Central Nervous System
Authors: Spector Reynold
Published: October 2006

Abstract: The mechanisms by which uridine enters and leaves brain, choroid plexus, and cerebrospinal fluid (CSF) were investigated in the isolated choroid plexus in vitro and by injection [3H]uridine intravenously and intraventricularly. Consistent with its postulated role in transporting uridine from blood into CSF, the isolated rabbit choroid plexus concentrated uridine with 10 μM uridine in the medium. [3H]Uridine, with and without unlabeled uridine, was infused at a constant rate into conscious adult rabbits. At 180 min, [3H]uridine entered CSF, choroid plexus, and brain more rapidly than mannitol. In brain, approximately 60–80% of the nonvolatile radioactivity was [3H]uridine phosphates. The addition of 2.1 mmol/kg of unlabeled uridine to the infusion syringe decreased the relative entry of [3H]uridine into brain by approximately 75% due mainly to the decreased formation of [3H]uridine phosphates and increased formation of [3H]uracil. Two hours after the intraventricular injection of [3H]uridine. [3H]uridine was cleared from CSF more rapidly than mannitol, in part to brain, where approximately 75% of the [3H]uridine was converted to [3H]uridine phosphates. The intraventricular injection of 42μmol unlabeled uridine with the [3H]uridine decreased the phosphorylation of [3H]uridine in brain significantly and also decreased the clearance of [3H]uridine from the CSF. From blood, uridine enters CSF and the extracellular space of brain. Uridine then can enter brain cells, be phosphorylated to uridine phosphates, and subsequently incorporated into RNA or be catabolized to uracil.
N‐Acetylation of L‐Aspartate in the Nervous System: Differential Distribution of a Specific Enzyme
Authors: Truckenmiller M. E. , Namboodiri M. A. A. , Brownstein M. J. , Neale J. H.
Published: October 2006

Abstract: L‐Aspartate N‐acetyltransferase, a nervous system enzyme that mediates the synthesis of N‐acetyl‐L‐aspartic acid, has been characterized. In the presence of acetyl‐CoA, L‐aspartate was acetylated 10‐fold more efficiently than L‐glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10‐fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10–15 pmol/min/mg tissue) and retina the lowest detectable activity (1–1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane‐associated and was solubilized by treatment with Triton X‐100.
Effect of Passive Avoidance Training on In Vitro Protein Synthesis in Forebrain Slices of Day‐Old Chicks
Authors: Schliebs Reinhard , Rose Steven P. R. , Stewart Michael G.
Published: October 2006

Abstract: Slices from the forebrains of day‐old chicks represent a highly active in vitro protein‐synthesising system. The in vitro incorporation of l[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell‐soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (>40 μm from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04–1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20–50% inhibition of incorporation of radioactive l‐leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one‐trial passive avoidance paradigm, l‐[14C]leucine incorporation was 23% higher (p < 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.
Restricted Transport of Vitamin D and A Derivatives Through the Rat Blood‐Brain Barrier
Authors: Pardridge William M. , Sakiyama Roland , Coty William A.
Published: October 2006

Abstract: The present studies measure the transport of retinol, retinoic acid, 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3], and 25‐hydroxyvitamin D3 [25‐(OH)D3] through the rat brain capillary endothelial wall, i.e., the blood‐brain barrier (BBB). The vitamin A and D derivatives bind both to albumin and to specific high‐affinity binding proteins in plasma. In the presence of physiologic concentrations of plasma proteins, the extraction by brain of all four compounds was 5% or less.
Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase in C6 Glioma Cells
Authors: McMorris F. Arthur , Smith Thomas M. , Sprinkle Terry Joe , Auszmann Jeanine M.
Published: October 2006

Abstract: In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine‐labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half‐life of the enzyme (∼33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or α‐amanitin. Thus, cyclic AMP induces the synthesis of CNP.
Monitoring the Effect of a Tryptophan Load on Brain Indole Metabolism in Freely Moving Rats by Simultaneous Cerebrospinal Fluid Sampling and Brain Dialysis
Authors: Hutson P. H. , Sarna G. S. , Kantamaneni B. D. , Curzon G.
Published: October 2006

Abstract: Rats were given L‐tryptophan, 50 mg/kg i.p., and its concentration in the CNS was monitored in individual freely moving animals using repeated sampling of cisternal CSF and concurrent striatal dialysis. The 5‐hydroxytryptamine metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) was also measured. Results were compared with changes of central tryptophan and 5‐HIAA concentrations in brains of rats killed at various times after administration of L‐tryptophan, 50 mg/kg i.p. Tryptophan changes in CSF were proportionate to those in whole brain and followed essentially identical time courses. Results for the striatal dialysate and whole striatum also paralleled each other. Similarly, results for 5‐HIAA showed proportionality between CSF and brain and between dialysate and striatum. The data obtained were used to determine pharmacokinetic data for individual rats, i.e., areas under curves for both tryptophan and 5‐HIAA and half‐lives for the decline of tryptophan. Kinetic parameters varied considerably from rat to rat. However, mean half‐lives for tryptophan in CSF, brain, dialysate, and striatum were all comparable. Results in general show the value of repeated CSF sampling and intracerebral dialysis for concurrent monitoring of changes of indole metabolism in the whole brain and a specific brain region, respectively. The methods should be suitable for the continuous monitoring of changes of central transmitter metabolism in parallel with observation of behavior following environmental or dietary changes or drug administration. They also should be of use in the investigation of drug kinetics in the CNS.
Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition
Authors: Inouye Hideyo , Ganser Allen L. , Kirschner Daniel A.
Published: October 2006

Abstract: We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x‐ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4–9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02–0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20–30% higher than normal; non‐hydroxycerebroside and sphingomyelin are 15–20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.
Acetylcholine Transport and Drug Inhibition Kinetics in Torpedo Synaptic Vesicles
Authors: Bahr Ben A. , Parsons Stanley M.
Published: October 2006

Abstract: Steady‐state initial velocity uptake of [3H]acetylcholine ([3H]ACh) by purified Torpedo electric organ synaptic vesicles was studied. Transport specific activity decreased at higher vesicle concentration. Michaelis‐Menten type kinetics describe [3H]ACh active transport at constant vesicle concentration with no evidence of cooperativity or transporter heterogeneity. The ACh dissociation constant is about 0.3 mM, transport has a maximal velocity of about 1.6 nmol/min/mg protein, and both are dependent on the vesicle preparation. Nonradioactive ACh was a competitive inhibitor with respect to [3H]ACh. The potent transport inhibitor dl‐trans‐2‐(4‐phenylpiperidino)cyclohexanol (AH5183) is a non‐competitive inhibitor with respect to [3H]ACh, with an inhibition constant of 41 ± 7 nM. Inhibition by AH5183 is reversible. The results suggest that AH5183 does not bind to the ACh transporter recognition site on the outside of the vesicle membrane, and thus it might inhibit allosterically.
Activation of β‐Adrenergic Receptors Stimulates Release of an Inhibitory Transmitter from Astrocytes
Authors: Shain William , Madelian Vergine , Martin David L. , Kimelberg Harold K. , Perrone Mark , Lepore Rhonda
Published: October 2006

Abstract: Activation of β‐adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP‐mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2′‐dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the β‐adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS—receptor‐mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.
Effects of Phenylalanine on the Release of Endogenous Dopamine from Rat Striatal Slices
Authors: Milner J. D. , Irie K. , Wurtman R. J.
Published: October 2006

Abstract We examined the effect of phenylalanine (50–400 μM) on the electrically stimulated release of endogenous 3,4‐dihydroxyphenylethylamine (dopamine or DA) from superfused rat striatal slices. In the absence of tyrosine, phenylalanine (25 μM) partially sustained DA release, but less well than an equimolar concentration of tyrosine. In the presence of tyrosine (50 μM), phenylalanine (in concentrations of ≥200 μM) inhibited DA release into the superfusate. This inhibition was not associated with changes in tissue levels of tyrosine or DA, nor was it mimicked by addition of high concentrations of tyrosine or leucine to the medium. We conclude that phenylalanine is a less effective precursor of DA in rat striatum than tyrosine and that it can also act to inhibit DA synthesis, depending on its concentration.
Regional Differences in 5‐Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures
Authors: Kimelberg H. K. , Katz D. M.
Published: October 2006

Abstract The uptake of 3H‐labelled 5‐hydroxytryptamine (5‐HT, serotonin) norepinephrine ([3H]NE), and 3,4‐dihy‐droxyphenylethylamine ([3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+‐dependent uptake showed marked regional differences. For [3H]5‐HT the magnitude of uptake was corpus striatum ≥ cerebral cortex ≥ hippocampus, whereas for [3H]NE the order was hippocampus ≥ corpus striatum ≥ cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+‐dependent uptake. [3H]5‐HT uptake was specifically inhibited by 10‐7M flu‐oxetine whereas [3H]NE uptake was preferentially inhibited by 10‐7M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.
Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats
Authors: Fuglsang Annette , Lomholt Margrethe , Gjedde Albert
Published: October 2006

Abstract: Little is known of the selectivity of the blood–brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D‐glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood–brain permeabilities of the hexoses were 25–50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10‐fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady‐state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half‐saturation concentration of glucose transport across the blood–brain barrier was considerably higher than in adult rats, about threefold, suggesting that net blood–brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.
Characterization of a Translational Inhibitor Isolated from Rabbit Brain Following Intravenous Administration of d‐Lysergic Acid Diethylamide
Authors: Fleming Scott W. , Brown Ian R.
Published: October 2006

Abstract: Intravenous administration of d‐lysergic acid diethylamide (LSD) to rabbits results in a transient inhibition of brain protein synthesis in vivo and in vitro. A translational inhibitor that appears in the postribosomal supernatant fraction of cerebral hemispheres following LSD administration was partially purified by gel filtration on Sephadex G‐150 and precipitation with 60% ammonium sulfate. This inhibitor, which was proteinaceous, reduced the translational capacity of an initiating cell‐free protein synthesis system derived from brain. It also inhibited a messenger RNA‐dependent reticulocyte lysate programmed with brain polysomes and a globin‐synthesizing reticulocyte lysate system. Addition of the partially purified inhibitor to a brain cell‐free protein synthesis system resulted in the decreased formation of ternary complexes as well as 40 and 80S initiation complexes, suggesting that the inhibitor affects an early step in the initiation of protein synthesis in brain.
Activation of Complement by Myelin: Identification of C1‐Binding Proteins of Human Myelin from Central Nervous Tissue
Authors: Vanguri Padmavathy , Shin Moon L.
Published: October 2006

Abstract: Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b‐9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1‐activating protein, myelin was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti‐myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56–58‐kilodalton band comigrated with the W2 protein and an approximately 45–47‐kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.
Autoradiographic Evidence of [3H]SCH 23390 Binding Site; in Human Prefrontal Cortex (Brodmann's Area 9)
Authors: Dawson Ted M. , McCabe R. Tyler , Stensaas Suzanne S. , Wamsley James K.
Published: October 2006

Abstract: The distribution of dopamine D‐1 receptors has been determined in human prefrontal cortex (Brodmann's area 9) by an in vitro light microscopic autoradiographic method. Dopamine D‐l receptors were localized by using [3H]SCH 23390 as a ligand. Our results indicated that [3H]‐SCH 23390 binding to slide‐mounted tissue sections of human brain is specific, saturable, and of high affinity. Lamina Va contained the highest density of D‐l receptors, with a Bmax value of 11.2 × 1.3 fmol/mg tissue. The KD values for [3H]SCH 23390 in all laminae ranged from 2.6 to 3.2 nM. Competition studies performed with [3H]SCH 23390 indicated a pharmacologic profile consistent with labeling of the D‐l receptor.
Effect of Lactacidosis on Pyridine Nucleotide Stability During Ischemia in Mouse Brain
Authors: Welsh Frank A. , Sakamoto Tetsuya , McKee Ann E. , Sims Renée E.
Published: October 2006

Abstract: Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/ kg. In a third model (unilateral hypoxia‐ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.
Photoaffinity Labeling of Adenylate Cyclase‐Linked Serotonin Receptors in Aplysia Neurons
Authors: Saitoh T. , Shih J. C.
Published: October 2006

Abstract: Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 μM. Under the same conditions, 1 ‐[2‐(4‐aminophenyl)ethyl]4‐(3‐trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kactof 20 μM. The azido derivative of this compound, 1‐[2‐(4‐azidophenyl)ethyl]4‐(3‐trifluoromethylphenyl)piperazine, or of serotonin, (4‐amino, 3‐nitrophenylazido‐serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact < 10−4M). Irradiation of the membranes in the presence of 100 μM 1‐[2‐(4‐azidophenyl)ethyl]4‐(3‐trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 μM serotonin. Under the same conditions, 100 μM 4‐amino, 3‐nitrophenylazido‐serotonin did not inhibit serotonin‐stimulated adenylate cyclase activity. When [3H] 1 ‐[2‐(4‐azidophenyl)ethyl]4‐(3‐trifluoromethylphenyl)piperazine (20 μM) was irradiated with membranes for 5 min at 4°C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate‐polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mMserotonin during photolysis. These peptides may be related to serotonin receptors.
Cerebral Glucose Utilization: Comparison of [14C]Deoxyglucose and [6‐14C]Glucose Quantitative Autoradiography
Authors: Collins Robert C. , McCandless David W. , Wagman Iris L.
Published: October 2006

Abstract: The [14C]deoxyglucose [Sokoloff et al., J. Neurochem.28, 897–916 (1977)] and [6‐14C]glucose [Hawkins et al., Am. J. Physiol.248, C170‐C176 (1985)] quantitative autoradiographic methods were used to measure regional brain glucose utilization in awake rats. The spatial resolution and qualitative appearance of the autoradiograms were similar. In resting animals, there was no significant difference between the two methods among 18 gray and three white matter structures over a fourfold range in glucose utilization rates (coefficient of correlation = 0.97). In rats given increasing frequencies of photoflash visual stimulation, the two methods gave different results for glucose utilization within visual pathways. The linearity of the metabolic response was studied in the superior colliculus using an on‐off checkerboard stimulus between 0 and 33 Hz. The greatest increment in activity occurred between 0 and 4 Hz stimulation with both methods, probably representing recruitment of neuronal elements into activity. Above 4 Hz, there was a progressive increase in labeling with [14C]de‐oxyglucose up to 1.7 times control at 33 Hz. With [6‐14C]‐glucose, there was no further increment in change above a 30% increase seen at 4 Hz. Measurement of tissue glucose revealed no drop in the visually stimulated structures compared to control. We interpret these results to indicate that, with increasing rates of physiological activity, the products of deoxyglucose metabolism accumulate progressively, but the products of glucose metabolism are removed from brain in 10 min.