bluuebunny/crossref_metadata_2025 huggingface dataset:
VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES
Authors: Spector Reynold
Published: October 2006

Abstract— The total concentrations of vitamin B6 (B6) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90μmol/l or kg respectively. The mechanisms by which B6 enters and leaves brain, choroid plexus and CSF were investigated by injecting [3H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [3H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [3H]B6 readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [3H]B6 into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [3H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [3H]PIN decreased the amount of [3H]B6 in brain and also decreased the percentage of [3H]B6 in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [3H]PIN (1 μm) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B6 from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.
ENDOGENOUS LEVELS OF NEUROTRANSMITTER CANDIDATES IN PHOTORECEPTOR CELLS OF THE TURTLE RETINA
Authors: Sarthy P. Vijay , Lam Dominic M. K.
Published: October 2006

Abstract—

The neurotransmitter used by vertebrate photoreceptors has not been identified, although aspartic acid, glutamic acid and acetylcholine have all been suggested as possible candidates. In the present study, we have measured the endogenous levels of these substances in isolated photoreceptors of the turtle retina. Fractions containing over 80% of identified photoreceptors were obtained by enzymatic dissociation of the retina followed by velocity sedimentation at unit gravity. The endogenous levels of free amino acids in these fractions were measured by amino acid analysis and the ACh content was measured by a radiochemical assay. In addition, identified photoreceptors were drawn into a micropipet individually under visual observation and their ACh content was determined. Our results show that while the concentrations of free aspartic acid and glutamic acid in isolated photoreceptors are similar to those found in the retina, the concentration of ACh in cone photoreceptors (0.1–0.2 mM) is 2‐ to 3‐fold higher than that in the turtle retina. This result combined with the findings of others suggests that turtle cone photoreceptors may be cholinergic
THE AFFINITY OF THE FUCOSE‐BINDING LECTIN FROM LOTUS TETRAGONOLOBUS FOR GLYCOPEPTIDES AND OLIGOSACCHARIDES ACCUMULATING IN FUCOSIDOSIS
Authors: Susz Jean Philippe , Dawson Glyn
Published: October 2006

Abstract— The affinity of the fucose‐binding lectin from Lotus tetragonolobus for fuco‐oligosaccharides accumulating in the brain and other tissues of a patient with fucosidosis was studied by two methods: by inhibition of the co‐precipitation of the lectin with porcine stomach mucin and by one‐step affinity chromatography on a column of the lectin bound to Sepharose‐4B. Both methods indicated that the lectin had greater affinity for the disaccharide Fuc(α, 1‐6)GlcNAc than for either the main fucosidosis storage material in brain, a fuco‐dekasaccharide, or the heterogeneous fuco‐glycopeptide fractions obtained from normal human and rat brain glycoproteins. Our results suggest that the fucose residue linked α(1‐6) to the N‐acetylglucosamine residue involved in the N‐glycosidic linkage to asparagine is not available to the lectin in the intact N‐glycosidic chains of normal brain glycopeptide fractions and that the lectin has poor affinity for the Fuc(α, 1‐3)GlcNAc linkage in rat brain glycoproteins.
METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS
Authors: Sun Grace Y. , Su Kwei L.
Published: October 2006

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1‐14C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half‐lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half‐life for turnover of synaptosomal lipids was 9 days.

The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl‐GPC and diacyl‐GPI, since radioactivity of other phosphoglycerides (diacyl‐GPS, diacyl‐GPE and alkenyl‐acyl‐GPE) continued to increase for 12‐24 h. In this fraction, half‐lives of 10‐14 h were obtained for the fast turnover pools of diacyl‐GPC and diacyl‐GPI, and slow turnover pools with half‐lives of 7 days for diacyl‐GPI and 10‐14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl‐GPI declined in a biphasic mode, thus exhibiting half‐lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl‐GPE and diacyl‐GPS in the synaptosomal fractions was observed for a period of 24 h. The half‐lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl‐GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.
TRANSPORT OF SUGARS INTO MICROVESSELS ISOLATED FROM RAT BRAIN: A MODEL FOR THE BLOOD‐BRAIN BARRIER1
Authors: Kolber Alan R. , Bagnell C. Robert , Krigman Martin R. , Hayward Jean , Morell Pierre
Published: October 2006

Abstract— Microvessels (primarily capillaries) were isolated from the brains of rats 25‐35 days of age. This preparation was characterized by light, transmission, and scanning electron microscopy. Transmission electron microscopy revealed that the endothelial cell membranes were intact and were impermeable to horseradish peroxidase. However, scanning electron microscopy revealed that damage to the membrane occurred during isolation. The isolated microvessel preparations were metabolically competent as demonstrated by their ability to metabolize [14C]glucose.

Aliquots of microvessel preparation were incubated with radioactive non‐metabolizable analogs of D‐glucose at various concentrations. The kinetics of accumulation of radioactivity in the capillaries were analyzed according to a model for carrier‐mediated diffusion and affinity constants for 3‐O‐methyl‐ D‐glucose and 2‐deoxyglucose were calculated (about 18 mM at 20°C in each case). These affinity constants are somewhat greater than that expected from whole animal experiments reported by other laboratories. This discrepancy is probably accounted for by the presence of a passive diffusion component. However, despite this complication, the primary mechanism for entry of D‐glucose analogues at physiological concentrations is compatible with carrier‐mediated transport since: the uptake of sugar analogs was shown to be saturable, to exhibit competition for uptake between structurally similar molecules, and to be non‐concentrative. In contrast, the uptake of glycerol, mannitol, and L‐glucose by isolated microvessels obeyed the kinetics of simple passive diffusion and was not saturable.

Our results are compatible with the concept that the capillary is the anatomic locus of the blood‐brain barrier and that this structure contains the carrier‐mediated transport system for monosaccharide penetration into brain.
KINETICS OF BLOOD‐BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS
Authors: Cremer Jill E. , Cunningham Vincent J. , Pardridge William M. , Braun Leon D. , Oldendorf William H.
Published: October 2006

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age‐groups. Data were analysed in terms of the Michaelis‐Menten equation with a component to allow for non‐saturable diffusion. Values are given for Km, Vmax and Kd. In suckling rats (15‐21 days) the Vmax values for both pyruvate and lactate were 2.0 μmol g−1 min−1. In 28‐day‐old rats the Vmax values had fallen to one‐half and in adults they were less than one‐tenth. Km, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28‐day‐old and adults although there was no difference with age in the Km values.

The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post‐natal development.
α‐MELANOCYTE‐STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN
Authors: Barnea Ayalla , Cho Gloria , Porter John C.
Published: October 2006

Abstract— The amount of α‐melanocyte‐stimulating hormone (α‐MSH) in the entire hypothalamus as well as the amount of α‐MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1‐24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso‐osmotic sucrose solution, and free granules and synaptosomes containing α‐MSH were fractionated by means of continuous sucrose density gradient centrifugation. α‐MSH was quantified by radioimmunoassay. The total amount of α‐MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α‐MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5‐24‐month‐old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α‐MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.
EFFECTS OF GABA ANALOGUES OF RESTRICTED CONFORMATION ON GABA TRANSPORT IN ASTROCYTES AND BRAIN CORTEX SLICES AND ON GABA RECEPTOR BINDING
Authors: Schousboe A. , Thorbek P. , Hertz L. , Krogsgaard‐Larsen P.
Published: October 2006

—The effects of a variety of acyclic or heterocyclic GABA analogues on GABA receptor binding and on high affinity transport of GABA in cultured astrocytes and mini‐slices of brain cortex were studied. The receptor and transport sites were found to be stereospecific and they exhibited opposite stereoselectivity for (R)‐ and (S)‐trans‐4‐amino‐4‐methylcrotonic acid and (R)‐ and (S)‐β‐proline. The most potent inhibitors of GABA binding were (RS)‐4, 5‐dihydromuscimol, muscimol, GABA, isoguvacine and isonipecotic acid with IC50values of, respectively, 0.009, 0.006, 0.033, 0.037 and 0.33 μM. Under the present experimental conditions the following compounds inhibited preferentially the glial transport system: (3RS, 4SR)‐4‐hydroxynipecotic acid, guvacine, (RS)‐N‐methylnipecotic acid, (RS)‐β‐proline and β‐alanine (IC50 values 10, 25, 70, 320 and 1000 μM, respectively vs. 200, 100, 300, 1200 and >5000 for neuronal transport). On the other hand, (R)‐trans‐4‐amino‐4‐methylcrotonic acid, (3RS, 4SR, 5SR)‐4‐hydroxy‐5‐methymipecotic acid and (RS)‐3‐hydroxy‐5‐aminovaleric acid preferentially inhibited neuronal transport as studied in mini‐slices of brain cortex (IC50 values 160, 300 and 430 μM, respectively vs. 500, > 5000 and 1400 μM for glial transport).
The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes
Authors: Bender A. S. , Wu P. H. , Phillis J. W.
Published: October 2006

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [3H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m, was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent Km's of 1 μam (“high‐affinity A”) and 5 μm (“high‐affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4‐dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high‐affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the Km values and kinetic properties of papaverine action in the synaptosomal high‐affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.
Actions of Tremorgenic Fungal Toxins on Neurotransmitter Release
Authors: Norris P. J. , Smith C. C. T. , De Belleroche J. , Bradford H. F. , Mantle P. G. , Thomas A. J. , Penny R. H. C.
Published: October 2006

Abstract: The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterised by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or intraperitoneal injection 45 min prior to killing (rat) and synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA (γ‐aminobutyric acid) and aspartate by 213%, 455% and 277%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1300% and 1200%, respectively, but not that of GABA from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the veratrine (75 μm) stimulated release of glutamate, aspartate and GABA from cerebrocortical synaptosomes by 33%, 46% and 11%, respectively and the stimulated release of glycine and GABA from spinal cord/ medulla synaptosomes by 67% and 32% respectively. Verruculogen pretreatment did not alter the veratrine‐induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate and GABA by 68%, 62% and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300–400%) in the miniature‐end‐plate potential (m.e.p. p.) frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5–7 min and declined to the control rate over a similar period. No change in the amplitude of the m.e.p. p. ‘s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.
Cholinergic Neuronotrophic Factors: Fractionation Properties of an Extract from Selected Chick Embryonic Eye Tissues
Authors: Manthorpe Marston , Skaper Stephen , Adler Ruben , Landa Keith , Varon Silvio
Published: October 2006

Abstract: An aqueous extract derived from selected intraocular tissues of 15‐day chick embryos contains a soluble macromolecular agent which is capable of ensuring the survival of 8‐day chick embryonic ciliary ganglionic neurons in monolayer culture. When this ciliary neuronotrophic factor (CNTF) was concentrated using ultrafiltration and subjected to Sephadex G100 and G200 chromatography, activity was detected in most of the eluted fractions. A peak of the most active fractions was eluted in a region corresponding to a molecular weight of 35–40 ± 103 and contained about 20‐30% of the applied protein. CNTF activity bound readily to DE‐52 cellulose resin at neutral pH and was eluted with NaCl in a narrow region containing about 20‐40% of the applied protein. Gel electrophoretic staining profiles of the active DE52 fraction indicated considerable (but still only partial) simplification in protein composition. While significant CNTF activity losses were incurred in response to each of the above treatments, an active material could be conveniently generated in one working day in milligram amounts having a specific activity of 60,000 trophic units/mg protein. This trophic activity is in the same range as that of the only other known neuronotrophic factor, Nerve Growth Factor.
Multiple α2‐Noradrenergic Receptor Sites in Rat Brain: Selective Regulation of High‐Affinity [3H] Clonidine Binding by Guanine Nucleotides and Divalent Cations
Authors: Rouot Bruno M. , U'Prichard David C. , Snyder Solomon H.
Published: October 2006

Both kinetic and equilibrium experiments indicate the presence of distinct high‐ and low‐affinity [3H]clonidine binding associated with α2‐ noradrenergic receptors in rat cerebral cortex membranes. Guanine nucleotides selectively decrease the numbers of high‐affinity [3H]clonidine binding sites, with no influence on the low‐affinity sites. The influence of nucleotides is exerted by GTP, its nonmetabolized analogue, guanyl‐5′‐yl imidodiphosphate, and GDP–but not by GMP, ADP, ATP, and AMP. In contrast, divalent cations increase high‐affinity [3H]clonidine binding‐an effect not evident at low‐affinity sites. Manganese is the most potent of the divalent cations, while magnesium and calcium are less active. Divalent cations and guanine nucleotides appear to elicit interactive rather than simply additive influences upon α2‐receptors. The inhibitory influence of sodium upon α2‐receptor binding of [3H]clonidine is exerted to the same extent upon both high‐ and low‐affinity [3H]clonidine binding.
Interactions of Divalent Cations and Guanine Nucleotides at α2‐Noradrenergic Receptor Binding Sites in Bovine Brain Mechanisms
Authors: U'Prichard David C. , Snyder Solomon H.
Published: October 2006

Guanine nucleotides selectively decrease binding of the agonist ligands [3H]clonidine and [3H]epinephrine to α2‐noradrenergic receptors in calf cortex membranes. In the presence of 1.0 mM‐calcium,‐magnesium, ormanganese, inhibition by GTP of [3H]clonidine binding is reversed, so that low concentrations of GTP increase [3H]clonidine binding, whereas high GTP concentrations cause a secondary decrease. In the presence of divalent cations, low concentrations of guanyl‐5′‐yl imidodiphosphate [Gpp(NH)p], unlike GTP, do not increase binding. Differences between effects of GTP and Gpp(NH)p in the presence of divalent cations are also observed with [3H]epinephrine binding to rat and calf cortex α2‐receptors. In reversing the inhibition of α2‐agonist binding by GTP, manganese, with an ED50 of 21 μM, is much more potent and effective than magnesium or calcium. Manganese by itself increases [3H]clonidine binding by 20% an effect which is irreversible, while the interactive effects of manganese and GTP are reversible. Divalent cations also antagonize the sodium‐induced inhibition of α2‐agonist binding in calf cortex membranes, and manganese has similar ED50 values in antagonizing both sodium and GTP.
The Lipid Composition of Rat CNS Axolemma‐enriched Fractions
Authors: DeVries George H. , Zmachinski Catherine J.
Published: October 2006

The lipid composition is reported for rat CNS axolemma‐enriched membrane fractions isolated from rat CNS white matter via a purified preparation of myelinated axons. The more dense axolemma‐enriched fraction is 51.5% lipid, comprised of 21.3% cholesterol, 11.3% galactolipid (cerebrosides and sulfatides in a molar ratio of 1.3:1.0), 24.2% ethanolamine glycerophospholipid (41.0% in the plasmalogen form), 27.8% choline glycerophospholipid (14.7% in the plasmalogen form), 5.3% serine glycerophospholipid, 2.8% inositol glycerophospholipid, and 2.0% sphingomyelin. The less dense axolemma‐enriched fraction has a lipid composition that is intermediate between that of the more dense axolemma‐enriched fraction and the concomitantly isolated myelin; this myelin has a lipid composition consistent with that previously reported for myelin isolated by an alternative procedure. A concomitantly isolated and operationally defined myelin‐free axon fraction has a lipid composition distinct from that which we have previously reported for a rat CNS myelin‐free axon preparation. The more dense axolemma‐enriched fraction contains 11.94 μg ganglioside NeuNAc per mg dry weight in contrast to the myelin fraction, which contains 0.88 μg ganglioside NeuNAc per mg dry weight, This axolemma‐enriched fraction contains all the major brain‐type gangliosides, while myelin contains mostly ganglioside GMI. The lipid composition of these fractions is compared with that of other neuronal and axolemmal preparations. The origin and function of the axolemmal glycolipid are discussed.
Multiple Molecular Forms of Acetylcholinesterase in Rat Vagus Nerve, Smooth Muscle, and Heart
Authors: Skau K. A. , Brimijoin S.
Published: October 2006

Extracts of rat tissues were subjected to ultracentrifugation on linear density gradients of sucrose (5–20%) and fractions of these gradients were analyzed for acetylcholinesterase (AChE) activity. Most of the AChE activity in extracts of vagus nerve was found to sediment at 10S. However, 16S AChE, previously thought to be confined to somatic motor nerves and skeletal muscle, was observed to accumulate rapidly at a nerve ligation. A search for this rapidly sedimenting form of AChE was carried out in tissues receiving vagal innervation. Significant amounts of 16S AChE were detected in the heart, especially in the right atrium, and in several regions of the gut. It was concluded that 16S AChE is distributed more widely than has previously been recognized.
Influence of Synchronized Sleep on the Biosynthesis of RNA in Two Nuclear Classes Isolated from Rabbit Cerebral Cortex
Authors: Giuditta Antonio , Rutigliano Bruno , Vitale‐Neugebauer Anna
Published: October 2006

Abstract: The biosynthesis of RNA during sleep has been studied in two purified nuclear fractions separated from rabbit cerebral cortex after subarachnoidal injection of radioactive orotate. The biochemical parameters have been referred to the percent EEG synchronization recorded during the period of incorporation (1 hr). The content of radioactive RNA per nucleus increases significantly with percent synchronization in the fraction of large nuclei (of neuronal and astroglial origin). While sedimentation and electrophoretic analyses of this RNA are consistent with the hypothesis of an enhanced turnover of rRNA during wakefulness, the accumulation of labelled RNA which is observed during sleep may be due to a modified turnover of nuclear heterogeneous RNA. On the other hand, in the fraction of small nuclei (mostly of oligodendroglial origin) the content of radioactive RNA per nucleus and the pattern of sedimentation of labelled RNA show no dependence on the electrical state of the cortex. These data indicate that in the cerebral cortex the sleepwakefulness transition is accompanied by a differential cellular response in RNA turnover.
Effects of Denervation and Simple Disuse on Rates of Oxidation and on Activities of Four Mitochondrial Enzymes in Type I Muscle
Authors: Nemeth Patti M. , Meyer Dan , Kark R. A. Pieter
Published: October 2006

Abstract: To differentiate the effect of muscle contractile activity from that of motor nerve on oxidative processes in type I muscle, oxidative processes were studied in muscle after immobilization and after denervation. The two processes led to similar atrophy of muscle weight and of the mean diameter of muscle fibers. Disuse of soleus muscle (type I) did not affect rates of oxidation of 14C‐labeled substrates although these were reduced by disuse of the vastus lateralis (type II). Disuse of the soleus did not affect activities of several mitochondrial enzymes assayed by histochemical or biochemical methods. However, denervation of the soleus did lead to a fall in metabolic rates and enzyme activities. The activity of 3‐hydroxybutyrate dehydrogenase fell more than did the activities of succinic dehydrogenase, lipoamide dehydrogenase, or cytochrome–c oxidase in both homogenates and in mitochondrial fractions. These results suggest nerve may regulate mitochondrial enzymes in type I muscle. The mechanism appears to be different from that which regulates oxidative processes in type II muscle.
Elevation of Substance P‐like Immunoreactivity in Rat Central Nervous System by Protease Inhibitors
Authors: Hanson Glen R. , Lovenberg Walter
Published: October 2006

Abstract: Several substance P‐rich areas in rat CNS had increased levels of substance P‐like immunoreactivity following the intraventricular injection of the protease inhibitors SQ 20881, SQ 14225, and leupeptin. There were significant differences in response patterns from region to region, possibly on account of an interaction of anatomical, biochemical, or physiological variables. Although the compound SQ 14225 appeared to be the most potent of the inhibitors examined, it had no apparent effect on CNS substance P‐like immunoreactivity when administered peripherally.
Bidirectional Movement of γ‐Aminobutyric Acid in Rat Spinal Cord Slices
Authors: Moscowitz Joyce A. , Cutler R. W. P.
Published: October 2006

Abstract: The bidirectional movement of GABA (γ‐aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [14C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm. Elevated potassium (60 mm) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium‐dependent process. Ouabain (0.1 mm) more than doubled the unidirectional release of endogenous GABA in a calcium‐independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm) stimulated unidirectional release and inhibited unidirectional uptake of GABA.
Effects of Catechol Estrogens1 and Catecholamines on Hypothalamic and Corpus Striatal Tyrosine Hydroxylase Activity
Authors: Foreman M. M. , Porter J. C.
Published: October 2006

Abstract: The effects of catechol estrogens on tyrosine hydroxylase activity in hypothalamic and corpus striatal extracts were evaluated. When assayed in the presence of subsaturating concentrations of pterin cofactor, tyrosine hydroxylase activity was depressed by 2‐hydroxyestrone, 2‐hydroxyestradiol, Lnorepinephrine, or dopamine. However, estrone, 17β‐estradiol, 2‐ methoxyestrone, or 2‐methoxyestradiol had no consistent inhibitory effect on tyrosine hydroxylase activity under in vitro conditions. Moreover, a decrease in pterin binding affinity (elevated Km) in the presence of either catecholamines or 2‐hydroxyestrogens was found. These findings were suggestive of a competitive interaction between catechols and pterin. Catechol estrogens and catecholamines were shown to inhibit both membrane‐bound and soluble forms of tyrosine hydroxylase. The membrane‐bound form of tyrosine hydroxylase, however, was found to have a greater binding affinity (lower Kl) for 2hydroxyestradiol and norepinephrine than did the soluble form. The results of the present study are suggestive of a cytoplasmic effect of estrogen that may be mediated by 2‐hydroxyestrogen and terminated by O‐methylation.
Mechanisms of Cyclic AMP Regulation in Cerebral Anoxia and Their Relationship to Glycogenolysis
Authors: Gross Robert A. , Ferrendelli James A.
Published: October 2006

Abstract: Anoxia elevates levels of cyclic AMP and depresses levels of cyclic GMP in cerebral cortex of mice. Similar effects are also observed in other regions of the brain. Aminophylline inhibits accumulation of cyclic AMP about 50% in hippocampus and cerebellum, but not in cerebral cortex and striaturn; however, this effect requires high doses (250 mgikg). Pretreatment of animals with reserpine, which depletes brain stores of norepinephrine, dopamine, and serotonin, and also produces sedation and mild hypothermia, markedly inhibits accumulation of cyclic AMP in all regions of anoxic brain. Destruction of norepinephrine terminals by treatment of neonatal animals with 6‐OH‐ dopamine, which does not sedate or produce hypothermia, has an effect on cyclic AMP levels similar to that of reserpine. None of the above treatments modifies the effect of anoxia on cyclic GMP levels. These data indicate that norepinephrine is a major regulator of cyclic AMP levels in anoxic brain and that adenosine and, perhaps, other unidentified substances have lesser roles in this process. In contrast, biogenic amines and adenosine appear to have no effect on cyclic GMP regulation in anoxic brain. Reserpine slows the activation of phosphorylase and the utilization of ATP, and slightly attenuates the breakdown of glycogen caused by anoxia, but has no effect on the changes in glucose, lactate, or phosphocreatine. In contrast, 6‐OH‐dopamine has no effect on any of these anoxiainduced changes. It is concluded that the effect of reserpine on phosphorylase, glycogen, and ATP is most likely related to the hypothermic and sedative effect of the drug, and that either cyclic AMP is not responsible for initiating glycogenolysis in anoxic brain or only a small rise in cyclic AMP levels is necessary for this process.
Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentrations of Oxidized and Reduced Glutathione in the Rat
Authors: Rehncrona Stig , Folbergrova Jaroslava , Smith David S. , Siesjo Bo. K.
Published: October 2006

Abstract: The influence of complete and pronounced incomplete cerebral ischemia on cortical concentrations of reduced (GSH) and oxidized (GSSG) glutathione was studied in lightly anaesthetized (70% N2 O) rats. GSH was extracted with HCl‐methanol‐perchloric acid and GSSG with trichloroacetic acid in the presence of N‐ethylmaleimide and measured fluorometrically, giving normal concentrations in cortical tissue of about 2 and 0.01 μmol.g−1 respectively. Reversible complete ischemia was induced by increasing the intracranial pressure to above the systolic blood pressure by infusing mock CSF into the cisterna magna. Reversible pronounced incomplete ischemia was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. Whether complete or incomplete, a 30‐min ischemic period caused a similar decrease in cortical GSH concentration (to about 90% of control) without any concomitant accumulation of GSSG in the tissue (or in CSF). Prolongation of the ischemic period (complete ischemia) to maximally 120 min caused an almost linear decrease of the tissue glutathione concentration to 45% of the preischemic value. During subsequent recirculation following a 30 min period of either complete or pronounced incomplete ischemia, there was a further decrease in cortical GSH concentrations without a reciprocal increase in GSSG concentrations. Lipid peroxidation (verified by determination of malondialdehyde production) induced in brain cortical tissue in vitro caused oxidation of tissue GSH with accumulation of GSSG. As the observed decrease in GSH during brain ischemia in vivo was not accompanied by any reciprocal increase in GSSG the results fail to support the hypothesis that peroxidative damage occurs during or following brain ischemia. The finding of an unchanged GSSG concentration does, however, not exclude the possibility of an increased turnover rate in the glutathione reductase reaction. It is concluded that the observed decrease in tissue GSH concentration mainly reflects a decrease in the glutathione pool size, due to an imbalance between breakdown and synthesis secondary to tissue energy failure.
Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions
Authors: Childers Steven R. , Snyder Solomon H.
Published: October 2006

Abstract: Guanine nucleotides differentiate binding of tritium‐labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [3H]‐labeled agonists by 20–60% and [3H]‐labeled antagonists by 0–20%. In the presence of 100 mM‐NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high‐affinity [3H]dihydromorphine sites by 52% and low‐affinity sites by 55%. Without sodium, GTP reduced high‐affinity [3H]‐naloxone sites by 36%; in the presence of 100 mM‐NaCl, GTP had no effect on either high‐ or low‐affinity [3H]naloxone sites. GTP increased the association rate of [3H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [3H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [3H]‐diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40‐ 120‐fold, with met‐ and leu‐enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [3H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [3H]‐labeled agonist binding by stimulating membrane‐bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.
Regulation by Cations of [3H]Spiroperidol Binding Associated with Dopamine Receptors of Rat Brain
Authors: Usdin Ted B. , Creese Ian , Snyder Solomon H.
Published: October 2006

Abstract: The effects of monovalent and divalent cations on binding of [3H]spiroperidol to dopamine receptors in rat corpus striatum were studied. Both monovalent and divalent cations as well as several chelating agents increase the number of [3H] spiroperidol binding sites. Manganese is most potent, enhancing binding at 1 μm concentration, while magnesium and calcium are at least two orders of magnitude less potent and the monovalent cations sodium, potassium and lithium are still weaker. Divalent cations enhance the potency of dopaminergic agonists in competing for [3H]spiroperidol binding, an effect which appears to be independent of the ionic augmentation of [3H]spiroperidol binding. Divalent cations decrease both the association and dissociation rates of [3H]spiroperidol binding to dopamine receptor sites.
Cerebral Cortical Glucose Utilization in the Conscious Rat: Evidence for a Circadian Rhythm
Authors: Crane P. D. , Braun L. D. , Cornford E. M. , Nyerges A. M. , Oldendorf W. H.
Published: October 2006

Abstract: The presence of a circadian rhythm of glucose utilization was demonstrated in vivo in rat cerebral cortex. The activity pattern of the rats, living in a controlled lighting regimen with lights on from 7 a.m. to 7 p. m., appeared to coincide with the rate of glucose consumption in the brain. The rate of utilization was measured at 3‐h intervals throughout the day and was found to fall from a maximum at 3 a.m. of 0.98 ± 0.13 μmol min−1 g−1 to a minimum of 0.70 ± 0.08 μmol min−1 g−1 at 3 p. m. Brain glucose also varied with time and its fluctuating level weakly correlated with its rate of utilization. Animals entrained on a 5‐h (4: 30‐9: 30 p. m.) feeding schedule had a similar circadian rhythm, with only a slight increase in amplitude. Reversal of the light cycle caused a disruption in the normal rhythm, but utilization still varied significantly with time of day. The results both indicate the potential error that can be encountered in experiments done at different times of the day and stress the need for awareness of time of day as a factor in measurements of alterations of metabolic rate in the brain.
Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry
Authors: Silbergeld Ellen K. , Hruska Robert E. , Miller Leonard P. , Eng Nancy
Published: October 2006

Abstract: Alterations in aspects of neurotransmission utilizing ‐γ‐aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K+‐stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium‐dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (Km) was increased and maximal velocity (Vmax) was reduced. Sodium‐independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.
Simultaneous Measurement of Cerebral Blood Flow and Unidirectional Movement of Substances Across the Blood‐Brain Barrier: Theory, Method, and Application to Leucine
Authors: Sage Jacob I. , Van Uitert Robert L. , Duffy Thomas E.
Published: October 2006

Abstract: The uptake of compounds by the brain depends upon cerebral blood flow. To determine the normal blood flow‐cerebral extraction relationship, a method for rapid, simultaneous measurement of cerebral blood flow and brain extraction was developed and applied to blood‐brain leucine transfer. Awake rats were injected intravenously with a mixture of n‐[14C]butanol and [3H]leucine. The quantities of indicators accumulated over the following 5–12 s in brain and in a sample of arterial blood withdrawn at a known rate were used to determine the flux of butanol and leucine into brain. Butanol extraction was assessed independently by measuring arterial and cerebral venous concentrations of the indicator after a bolus injection. Cerebral blood flow was equal to the ratio of butanol flux into brain to butanol extraction by brain; leucine extraction was then calculated as the ratio of leucine influx to cerebral blood flow. Leucine extraction by brain and cerebral blood flow were shown to be related exponentially. The maximum velocity of active leucine transport was virtually the same at flows of 150 and 400 ml/100 g/min. The present method is theoretically applicable to the measurement of the extraction of any compound from blood by brain. By measuring the noimal blood flow‐extraction relationship, one can differentiate changes in extraction secondary to altered flow from changes intrinsic to pathologic conditions with inconstant cerebral blood flow.
Differences in the Structure of A and B Forms of Human Monoamine Oxidase
Authors: Cawthon Richard M. , Pintar John E. , Haseltine Florence P. , Breakefield Xandra O.
Published: October 2006

Abstract: [3H]Pargyline‐labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). [3H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [3H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS‐PAGE of [3H]pargyline‐bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS‐solubilized, [3H]pargyline‐labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one‐dimensional peptide mapping in SDS gels, different patterns of [3H]pargyline‐labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.
Prediction of the Secondary Structure of Myelin Basic Protein
Authors: Martenson Russell E
Published: October 2006

Abstract: An investigation into the probable secondary structure of the myelin basic protein was carried out by the application of three procedures currently in use to predict the secondary structures of proteins from knowledge of their amino acid sequences. In order to increase the accuracy of the predictions, the amino acid substitutions that occur in the basic protein from different species were incorporated into the predictive algorithms. It was possible to locate regions of probable α‐helix, β‐structure, β‐turn, and unordered conformation (coil) in the protein. One of the predictive methods introduces a bias into the algorithm to maximize or minimize the amounts of α‐helix and/or β‐structure present; this made it possible to assess how conditions such as pH and protein concentration or the presence of anionic amphiphilic molecules could influence the protein's secondary structure. The predictions made by the three methods were in reasonably good agreement with one another. They were consistent with experimental data, provided that the stabilizing or destabilizing effects of the environment were taken into account. According to the predictions, the extent of possible α‐helix and β‐structure formation in the protein is severely restricted by the low frequency and extensive scattering of hydrophobic residues, along with a high frequency and extensive scattering of residues that favor the formation of β‐turns and coils. Neither prolyl residues nor cationic residues per se are responsible for the low content of α‐helix predicted in the protein. The principal ordered conformation predicted is the β‐turn. Many of the predicted β‐turns overlap extensively, involving in some cases up to 10 residues. In some of these structures it is possible for the peptide backbone to oscillate in a sinusoidal manner, generating a flat, pleated sheetlike structure. Cationic residues located in these structures would appear to be ideally oriented for interaction with lipid phosphate groups located at the cytoplasmic surface of the myelin membrane. An analysis of possible and probable conformations that the triproline sequence could assume questions the popular notion that this sequence produces a hairpin turn in the basic protein.
Postmortem Changes of Amino Compounds in Human and Rat Brain
Authors: Perry Thomas L. , Hansen Shirley , Gandham Satnam S.
Published: October 2006

Abstract: Contents of 35 amino acids and related compounds were measured in whole rat brain, and in superficial areas of biopsied and autopsied human brain, after incubation for various intervals at temperatures simulating those likely to occur in cadavers under mortuary conditions. These data should aid interpretation of values for amino compounds determined in autopsied brain from patients with neurological or psychiatric disorders. The contents of glutamic acid, glutamine, taurine, phosphoethanolamine, cystathionine, and homocarnosine remain unchanged for long periods in human brain. Aspartic acid content is stable for 4 h after death, but thereafter rises rapidly. Glycine content rises rapidly, as do the contents of most amino acid components of proteins. Glutathione content drops rapidly in human brain after death. GABA content is stable for about 30 min, and rises to a maximum 2 to 3 h after death, after which it remains unchanged for at least 24 h. In rat brain, GABA content rises more rapidly, aspartate content rises more slowly, homocarnosine content decreases progressively, and glycerophosphoethanolamine content decreases more rapidly than in human brain.
Endogenous Substrates Utilized by Rat Brain in Severe Insulin‐Induced Hypoglycemia
Authors: Agardh Carl‐David , Chapman Astrid G. , Nilsson Bengt , Siesjö Bo K.
Published: October 2006

Abstract: Several previous studies have demonstrated that severe hypoglycemia is accompanied by consumption of endogenous brain substrates (glycolytic and citric acid cycle metabolites and free amino acids) and some have shown a loss of structural components as well, notably phospholipids. In the present study, on paralysed and artificially ventilated rats, we measured cerebral oxygen and glucose consumption during 30 min of hypoglycemic coma (defined as hypoglycemia of sufficient severity to cause cessation of spontaneous EEG activity) and calculated the non‐glucose oxygen consumption. In an attempt to estimate the missing substrate we measured tissue concentrations of phospholipids and RNA.

After 5 min of hypoglycemic coma, tissue phospholipid content decreased by about 8% with no further change during the subsequent 55 min. A similar reduction remained after 90 min of recovery, induced by glucose administration following 30 min of coma. Since no preferential loss of polyenoic fatty acids or of ethanolamine phosphoglycerides occurred, it is concluded that loss of phospholipids was due to phospholipase activity rather than to peroxidative degradation. The free fatty acid concentration increased sixfold after 5 min of coma and remained elevated during the course of hypoglycemia. A 9% reduction in tissue RNA content was observed after 30 min of hypoglycemia.

Calculations indicated that available endogenous carbohydrate and amino acid substrates were essentially consumed after 5 min of coma, and that other non‐glucose substrates must have accounted for approximately 50μmol·g−1 of oxygen (8.3 μmol·g−1 in terms of glucose equivalents) within the 5–30 min period. The 10% reduction in phospholipid‐bound fatty acids was more than sufficient (in four‐ to fivefold excess) to account for this oxygen consumption. However, since no further degradation occurred in the 5–30 min period, there is no simple, direct, quantitative relationship between oxygen consumption and cortical fatty acid oxidation during this interval. The possibility thus remains that unmeasured exogenous or endogenous substrates were utilized.