bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Sodium and Sulphydryl Groups on [3H]Sulpiride Binding Sites in Rat Striatal Membranes
Authors: Freedman S. B. , Poat Judith A. , Woodruff G. N.
Published: October 2006

Abstract: The effect of thiol reagents on the specific binding of the atypical neuroleptic, sulpiride, to rat striatal membranes was examined. Pretreatment of membranes with N‐ethylmaleimide (NEM), but not with iodoacetamide or dithiothreitol (DTT), diminishes [3H]sulpiride binding. The effect is dependent on time, temperature, and the concentration of NEM. The reaction proceeds with pseudo‐first‐order rate kinetics, indicating the involvement of a single essential SH group in the binding of [3H]sulpiride to striatal membranes sites. Both sodium and sulpiride, but not nucleotides, protect the [3H]sulpiride sites from NEM inactivation in a dose‐dependent fashion. As sodium is essential for [3H]sulpiride binding it is possible that in the presence of sodium and sulpiride a conformational change takes place that makes the essential SH group less accessible t o alkylation.
Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla
Authors: Holz Ronald W. , Senter Ruth A. , Frye Roy A.
Published: October 2006

Abstract: Carbachol or elevated K+ stimulated 45Ca2+ uptake into chromaffin cells two‐ to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca2+ uptake and catecholamine secretion induced by the mixed nicotinic‐muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca2+ uptake occurred within 15 s of stimulation by carbachol or elevated K+ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K+ was similar to that of Ca2+ uptake. There was a close correlation between Ca2+ uptake and catecholamine secretion at various concentrations of Ca2+. The concentration dependencies for inhibition of both processes by Mg2+ or Cd2+ were similar. Ca2+ uptake saturated with increasing Ca2+ concentrations, with an apparent Km for both carbachol‐induced and elevated K+‐induced Ca2+ uptake of approximately 2 mM. The Ca2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca2+ entry and a presumed increase in cytosolic Ca2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca2+‐ dependent processes associated with catecholamine secretion. Ca2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage‐sensitive Ca2+ channels.
Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain
Authors: Kimelberg H. K. , Stieg P. E. , Mazurkiewicz J. E.
Published: October 2006

Abstract: Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein‐positive, astrocytic monolayer. Smaller, process‐bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2′,3′‐dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5‐to 5‐fold less than that found for cerebral homogenates from perfused 3‐week‐old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10−8M, confirming that both rat brain and the astrocyte cultures possess the high‐activity type II enzyme. CA‐II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA‐II.
Concentration Gradients of Free and Total γ‐Aminobutyric Acid and Homocarnosine in Human CSF: Comparison of Suboccipital and Lumbar Sampling
Authors: Grove Jeffrey , Schechter Paul J. , Hanke N. F. J. , Smet Yves de , Agid Yves , Tell Guy , Koch‐Weser Jan
Published: October 2006

Abstract: Concentrations of free and total γ‐aminobutyric acid (GABA) and homocarnosine were determined in sequential aliquots of the first 30 ml of CSF obtained by lumbar puncture in five patients. Rostrocaudal gradients were calculated and compared to gradients estimated by determining concentrations of these substances in CSF obtained by simultaneous suboccipital and lumbar punctures in four more patients. In the lumbar fractions study, rostrocaudal mean gradients of 0.36, 36, and 21 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were calculated. In the suboccipital/lumbar study, gradients of 0.33, 30, and 24 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were estimated. These results indicate that valid comparison of CSF concentrations of these substances is restricted to similar fractions and suggest that in CSF the substances originate largely from brain rather than from peripheral sources.
Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells
Authors: Bremer Eric G. , Sapirstein Victor S. , Savage Tom , McCluer Robert H.
Published: October 2006

Abstract: The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain GM3, GM2, GM1, GD3, and GD1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GaINAc(β1→4) Gal(β1→4)Glc(β1→1)Cer (GgOse3Cer), and GaINAc(β1→3)Gal(α1→4) Gal‐(β1→4)Glc(β1→1)Cer (GbOse3Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mm‐EGTA showed a two‐ to threefold increase in GM3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mm‐EGTA plus 0.4 mm‐EDTA a similar increase in GM3 was observed but this change was now accompanied by decreases in GM2, GM1 GgOse3Cer, and GbOse4Cer. The EGTA‐EDTA effects were reversed when growth was in the presence of Ca2+ sufficient to bind all chelator. Mn2+ replacement reversed the chelator effects differentially; GM2 and GM1 levels were the most sensitive to increases in Mn2+ concentration; GgOse3Cer and GbOse4Cer were also sensitive, whereas GM3 was the least affected. These results suggest calcium serves an important regulatory role on GM3 levels and that manganese concentration may regulate the levels of galactosamine‐containing glycolipids in mouse NB41A neuroblastoma cells.
Homocholine and Short‐Chain N‐Alkyl Choline Analogues as Substrates for Torpedo Choline Acetyltransferase
Authors: Luqmani Y. A. , Richardson P. J.
Published: October 2006

Abstract: The kinetic parameters, Km and Vmax, for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the Km for choline was similar in both cases (0.51 and 0.42 mm), the crude enzyme showed a three‐ to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N‐alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 mm. There was an increase in both Km and Vmax for all substrates; choline, N,N,N‐dimethylmonoethylaminoethanol, ‐monomethyldiethylaminoethanol and ‐dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N‐triethylaminoethanol and ‐dimethylmonopropylaminoethanol and homocholine were much less affected. However, in all cases, the kinetic parameter Vmax/Km remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 mm had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes.
Kinetics of Homovanillie Acid and Determination of Its Production Rate in Humans
Authors: Elchisak M. A. , Polinsky R. J. , Ebert M. H. , Kopin I. J.
Published: October 2006

Abstract: Homovanillie acid (HVA) labeled with either two or five deuterium atoms (d2‐HVA or d5‐HVA) was used to label the peripheral body pool of endogenous HVA (d0‐HVA) in control humans and in neurological patients. Either d2‐ or d5‐HVA was rapidly injected intravenously and concentrations of d2‐ or d5‐ and d0‐HVA in sequential samples of blood plasma were determined by gas chromatography‐mass spectrometry (GC‐MS). Parameters describing the distribution and elimination of HVA, as well as its pool size, turnover, and synthesis rate were then calculated. Results indicate that the decline of plasma d2‐ or d5‐HVA with time was multiexponential in five out of six subjects. Basal levels of endogenous HVA averaged 14.4 ± 1.3 ng/ml in the control patients and 8.69 ± 2.4 ng/ml in the neurological patients. The biological half‐life averaged 71.3 ± 8.0 min in the control subjects and 91.3 ± 15 min in the neurological patients. The apparent volume of distribution of HVA in the body was 31–100 liters. Plasma clearance was 243–679 ml/min. The size of the peripheral body pool, calculated from plasma kinetic parameters, was 392–673 μg. The HVA rate of production, calculated for the whole body, was 166–323 μg/h. This technique can be used to determine accurately the rate of HVA production by the whole body over short time intervals. Since sample size was very limited (n= 3 per group) and effects of variables such as age and sex on these data have not been excluded, more thorough investigations are needed to document any differences between normal controls and neurological patients.
Developmental Changes in the Folate‐Dependent Enzymes of De Novo Purine Biosynthesis in Rat Brain
Authors: Domínguez Joaquín , Ordóñez Luis A.
Published: October 2006

Abstract: The activities of the two folate‐dependent enzymes in the de nova purine biosynthetic pathway (e.g., glycinamide ribonucleotide transformylase and aminoimidazolecarboxamide ribonucleotide transformylase), have been evaluated as a function of age in crude extracts from rat brain, liver, kidney, and spleen. The activities of the enzymes in brain are similar to those found in liver and kidney. In all tissues the activity of both enzymes was higher during early development, more than nine times above adult levels. In the CNS the enzymatic activities are apparently related to the periods of increased nucleic acid synthesis, with different activities being found in different regions during development. Our findings lend strong support to the suggestion that folic acid‐dependent metabolism plays an important role during early development of the brain.
Testosterone 5α‐Reductase in Spinal Cord of Xenopus laevis
Authors: Jurman M. E. , Erulkar S. D. , Krieger N. R.
Published: October 2006

Abstract: Testosterone 5α‐reductase, the enzyme that converts testosterone to 5α‐dihydrotestosterone, is present in the spinal cord of Xenopus laevis. In adult males the enzymatic activity is optimal at pH 7.4 and 27°C; the apparent Km is 2.0 × 10−5m and the Vmax is 10.0 pmol/mg protein/h. Enzymatic activity was assayed in segments of the spinal cord in each of four groups: control untreated males, females, castrated males, and sexually active clasping males. Striking differences in both the amount of dihydrotestosterone produced with time and in the pattern of its distribution were seen in spinal cords of clasping males compared with those of the other groups. The differences are greatest in the basal medulla and rostral segments of the spinal cord. Neurons in these segments innervate the muscles primarily involved in clasping.
Alterations of Nuclear Thyroid Hormone Receptors in Cerebral Cortex In Vivo
Authors: Thrall Cary L. , Yanagihara Takehiko
Published: October 2006

Abstract: Investigation was conducted under in vivo conditions in the adult male rat to determine the basic characteristics of the nuclear thyroid hormone receptors in the cerebral cortex. Equilibrium with cortical nuclei of an intravenous dose of triiodothyronine (T3) occurred 3 h after injection and showed a t1/2 of 1 h for dissociation. Saturation of receptors occurred at 0.5–0.6 ng/mg DNA. The endogenous level of binding in the normal rat was 0.07–0.1 ng/mg DNA, representing a 15% occupancy of total receptors at a serum concentration of 66 ng/dl. These characteristics were then examined under several pathophysiological conditions. In the hypothyroid rat, an apparent 37% in‐crease in total binding sites occurred. Under either fasting conditions or insulin or glucagon administration declines in serum T3 were noted, and the endogenous binding also decreased in parallel. Only glucagon produced a significant reduction in total binding sites. Under the hypoxic condition produced by maintenance under a 7% oxygen atmosphere, a slight increase in apparent total binding sites was found with no change in endogenous binding level. Severe narcosis resulted in no effects on T3 binding parameters. These results demonstrate specific alterations of thyroid hormone receptors that may be important physiologically.
Phosphatidylserine Enhancement of [3H]γ‐Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes
Authors: Chweh A. Y. , Leslie S. W.
Published: October 2006

Abstract: [3H]γ‐Aminobutyric acid ([3H]GABA) binding to purified lipids was examined in an organic solvent‐aqueous partition system. In addition, the [3H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium‐dependent [3H]GABA uptake into synaptosomes isolated from rat whole brains. [3H]GABA was found to bind to all of the lipids studied in the organic solvent‐aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [3H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [3H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μm). These results suggest that PS may play a role in the sodium‐dependent GABA reuptake process in the presynaptic nerve end.
On the Use of Microwave Radiation Energy for Brain Tissue Fixation
Authors: Schneider David R. , Felt Barbara T. , Goldman Harold
Published: October 2006

Abstract: Focused microwave irradiation (MWR) is an increasingly accepted method of sacrifice of laboratory animals such as the mouse or rat. By fixing the brain within a fraction of a second with heat inactivation, the investigation of fast neurochemical events may be obtained. Even though the technique is widely utilized, its application is inconsistent. This report illustrates some of the requirements necessary for the proper application of MWR for the sacrifice of animals, particularly those related to the length of time MWR is applied and the efficiency with which generated MWR power is coupled to the brain tissue. Studies were performed on the mouse, using either a 2.5 KW or 6.3 KW generator with a focused, closed system waveguide at time intervals of 350 or 500 ms or 1.4 s. During each of these intervals MWR was varied so that core brain temperatures for all groups were held between 83 and 95°C. In contrast with reported studies that used full animal restraint, all animals were minimally restrained for less than 1 s before sacrifice. Tissue content of cyclic AMP, an index of neuronal activity grossly affected by subtle changes in the activity of adenylate cyclase and/or phosphodiesterases, was monitored. No differences in tissue cyclic AMP content in any of 12 brain regions were detected after MWR, either at 350 or 500 ms. A substantial increase in cyclic AMP content occurred in 8 of 12 brain regions examined following microwave irradiation for 1.4 s. On the basis of these experiments, accurate determination of cyclic AMP in rodent brain requires that the maximum time interval of MWR exposure should not exceed 500 ms.
Sympathetic Regulation of Circadian Rhythm of Serotonin N‐Acetyltransferase Activity in Pineal Gland of Infant Rat
Authors: Deguchi Takeo
Published: October 2006

Abstract: The circadian rhythms of serotonin N‐acetyltransferase activity in the pineal glands of infant and adult rats were compared. The nighttime increase of N‐acetyltransferase activity in the pineals of infant rats was blocked by removal of superior cervical ganglion or by pretreatment with reserpine, l‐propranolol, and cycloheximide. Injection of isoproterenol to infant rats markedly elevated pineal N‐acetyltransferase activity. When the pineal glands of infant rats were organ‐cultured, N‐acetyltransferase activity spontaneously increased 7–12 h after the rats were killed. When infant rats were previously denervated or pretreated with reserpine and their pineals were cultured, this spontaneous elevation of N‐acetyltransferase activity was abolished, indicating that the transient increase of the enzyme activity in organ culture was due to a liberation of catecholamine from degenerating nerve endings. Additional illumination until midnight prevented the nighttime increase of N‐acetyltransferase activity in intact infant rats but not in blinded infant rats. These observations are taken to indicate that N‐acetyltransferase rhythm in immature rat pineals is regulated by the sympathetic nerves in the same manner as in adult rat pineals, that the immature rat pineal does not contain a time‐keeping system, and that there is no extraretinal light perception in infant rats as far as N‐acetyltransferase rhythm is concerned.
Mg2+‐ or Ca2+‐Activated ATPase in Squid Giant Fiber Axoplasm
Authors: Shecket Gordon , Lasek Raymond J.
Published: October 2006

Abstract: A divalent cation‐activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of the enzyme with those of myosin and Na+–K+‐ATPase suggests that it differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.
Cellular Level of Purine Compounds in Ischemic Gerbil Brain by High Performance Liquid Chromatography
Authors: Morimoto Kazuyoshi , Tagawa Kunio , Hayakawa Torn , Watanabe Fusao , Mogami Heitaro
Published: October 2006

Abstract: The cellular level of ATP and related compounds in ischemic gerbil brain was investigated by high performance liquid chromatography (HPLC). Brain samples were obtained in situ following ligation of the common carotid artery. AMP and ADP peaks in the brain extracts in the ischemic group became much larger whereas the ATP peak decreased dramatically. The most striking finding was an extensive increase of adenosine: 50–100 fold. The levels of inosine and hypoxanthine also increased greatly in typical symptomatic gerbil.
Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography
Authors: Martini C. , Lucacchini A. , Ronca G. , Hrelia S. , Rossi C. A.
Published: October 2006

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200‐fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 mm‐chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS‐polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant (KD) is 8 nm for [3H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.
The Use of Inhibitors of GABA‐Transaminase for the Determination of GABA Turnover in Mouse Brain Regions: An Evaluation of Aminooxyacetic Acid and Gabaculine
Authors: Bernasconi R. , Maitre L. , Martin P. , Raschdorf F.
Published: October 2006

Abstract: The accumulation of γ‐aminobutyric acid (GABA) after inhibition of GABA‐T (4‐aminobutyrate: 2‐oxoglutamate aminotransferase, EC 2.6.1.19) by various doses of aminooxyacetic acid (AOAA) and gabaculine was studied in four different regions of the mouse brain. The dose‐response curve for GABA accumulation after treatment with AOAA was linear up to 10 mg/kg i.p., and then leveled off. The increase in GABA accumulation after gabaculine treatment was linear up to 100 mg/kg i.p. No further increase was observed with doses up to 300 mg/kg i.p. The selectivity of both GABA‐T inhibitors was assessed by measuring their effects on the content of free amino acids in mouse brain. Apart from the substantial increase in the GABA concentration, there were significant decreases in the content of glutamic acid, aspartic acid, alanine and glutamine, and an increase in ornithine content after administration of gabaculine. The same changes in amino acid content were observed after treatment with AOAA, but the level of lysine was also increased and the change in alanine level was biphasic. All these changes, however, were very small compared with the large increase in GABA level. A method for estimating the rate of the GABA turnover in vivo by measuring the initial rate of GABA accumulation after administration of AOAA or gabaculine is proposed, and the validity of the two techniques is discussed. The effect of diazepam on GABA levels and on the gabaculine‐induced accumulation of GABA was studied. The results obtained with diazepam show that this method can provide valuable insight into the effects of drugs on GABAergic mechanisms in vivo.
A Combined In Vivo/In Vitro Study of the Presynaptic Release of Adenosine Derivatives in the Hippocampus
Authors: Lee Kevin , Schubert Peter , Gribkoff Valentin , Sherman Brimmer , Lynch Gary
Published: October 2006

Abstract: To investigate the release of adenine compounds from defined neuronal pathways, we employed a hippocampal slice preparation in which a selective‐loading of the releasable pools was achieved in vivo with the aid of axonal transport. By injecting radioactive adenosine stereotaxically into the entorhinal cortex, the major afferent system to the dentate gyrus (the perforant path) was loaded within 20–36 h, at which time the rats were killed and hippocampal slices were prepared. The efflux of radioactive material, as recovered from the perfusate and measured in a scintillation counter, was found to be significantly increased in response to electrophysiologically controlled stimulation of the perforant path but not to stimulation of an alternative fiber tract, the fimbria. These findings provide supportive and more direct evidence for an activation‐coupled release of adenosine derivatives from presynaptic sites in the central nervous system.
Development of Ion Metabolism in Reaggregated Brain Cell Cultures
Authors: Marks Michael J. , Seeds Nicholas W.
Published: October 2006

Abstract: Mouse brain cell reaggregates have been used to study changes in sodium‐ and potassium‐dependent ouabain‐sensitive adenosine phos‐phohydrolase (Na+,K + ‐ATPase) activity and in 86Rb+ uptake and exit during development. Na+, K+‐ATPase activity in these cultures has two ouabain‐inhibitable components, both of which increased severalfold between day 3 and day 17 in culture. This increase, however, was less than that in developing brain. Little change in either total or extracellular water or in the equilibrium levels of Na+ and K+ occurred during development. The uptake of 86Rb+ measured a 10‐min incubation showed only a modest increase during culture, whereas the exit of 86Rb+ from reaggregates preloaded with the tracer increased approximately fourfold. The exit consisted of both K +‐independent and K+ ‐stimulated components and the K+ ‐stimulated fraction contributed most of the developmental change. When uptake rates were corrected for the contribution of the developmental changes in exit, these rates were found to increase as well. The 86Rb+ uptake correlated closely with the activity of the Na+, K+‐ATPase during development. The pattern of developmental changes in enzyme activity and 86Rb+ uptake and exit suggest that, while little change in the steady‐state levels of the ions occurred, the rates of ion movement increase markedly.
Enzymatic Detyrosination of Tubulin Tyrosinated in Rat Brain Slices and Extracts
Authors: Barra H. S. , Argaraña C. E. , Caputto R.
Published: October 2006

Abstract: Tubulin was tyrosinated in slices and in extracts of brain of rats of 3, 25, and 120 days of age by successive incorporation of [14C]tyrosine and [3H]‐tyrosine, respectively. The release of the incorporated amino acid was measured by using tubulinyl‐tyrosine carboxypeptidase, carboxypeptidase A, and tubulin‐tyrosine ligase. With the carboxypeptidases no differences in either the rates or the extents of the release of tyrosine between these two differently labeled tubulins were found. Differences were found when the detyrosination was catalyzed by the ligase and these were attributed to a higher inactivation of tubulin labeled in slices than of that labeled in extracts.
Mass Fragmentographic Determination of Homovanillic Acid in Individual Dopaminergic Neuron Systems of Rat Brain: Application of Reaction Gas Chromatography
Authors: Matsumoto T. , Uchimura H. , Hirano M. , Kim J. S. , Nakahara T. , Tanaka K.
Published: October 2006

Abstract: Homovanillic acid (HVA) levels of 12 discrete rat brain areas were determined by a mass fragmentographic method using the reaction gas chromatographic technique. The use of reaction gas chromatography increased the sensitivity for determination of HVA. The sensitivity of this method allows measurement of HVA in small amounts of brain tissue. The HVA levels in polar, medial, and lateral fields of prefrontal cortex, anterior cingulate cortex, septum, amygdala, A12, A13, and A14 dopaminergic neurons were 0.417 ± 0.018 ng/mg protein, 0.689 ± 0.004, 0.753 ± 0.024, 0.496 ± 0.029, 1.311 ± 0.046, 0.555 ± 0.008, 1.949 ± 0.077, 1.109 ± 0.112, and 0.489 ± 0.019, respectively. The HVA levels in these areas are first reported in the present paper.
Studies on [3H]Diazepam and [3H]Ethyl‐β‐Carboline Carboxylate Binding to Rat Brain In Vivo. II. Effects of Electroconvulsive Shock
Authors: Nutt D. J. , Minchin M. C. W.
Published: October 2006

Abstract: In vivo specific binding of [3H]diazepam was not altered by a single electroconvulsive shock given 5, 30, or 60 min, or 24 h previously, nor 24 h after the last of 10 daily shocks. Similarly, in vivo [3H]ethyl‐β‐carboline carboxylate binding was not changed in the brains of animals that had been given a single electroconvulsive shock 30 min previously or a series of 10 daily shocks. Brain areas examined included cerebral cortex, hippocampus, cerebellum, and striatum. However, cortical binding of [3H]diazepam was increased by 32% in animals which were present in the same room while another was being injected and killed. This may represent a response to stress and/or anxiety.
Congenital Copper Deficiency: Copper Therapy and Dopamine‐β‐Hydroxylase Activity in the Mottled (Brindled) Mouse
Authors: Wenk Gary , Suzuki Kinuko
Published: October 2006

Abstract: The mottled (Mo) mouse is an animal model of the human congenital copper (Cu) deficiency disorder, Menkes' kinky hair syndrome. Intraperitoneal Cu chloride injections have been shown to produce clinical and morphological improvements in this mutant mouse. Cu injections (10 μg/g) on postnatal days 7 and 10 are shown to increase endogenous activity of the Cu‐dependent enzyme dopamine‐β‐hydroxylase in the brains of Mo mice.
Isolation of the Secretory Form of Soluble Acetylcholinesterase by Using Affinity Chromatography on Edrophonium‐Sepharose
Authors: Hodgson Anthony J. , Chubb Ian W.
Published: October 2006

Abstract: A single molecular from of soluble acetylcholinesterase was isolated from a variety of mammalian tissues by use of a novel affinity matrix. This matrix was synthesised by coupling the reversible cholinesterase inhibitor, edrophonium chloride, to epoxy‐activated Sepharose. This simple synthesis produced a matrix which was exceptionally stable and had the novel property of selectively binding only one molecular form of acetylcholinesterase. Soluble proteins from a variety of mammalian tissues, including brain, adrenal glands, cerebrospinal fluid, and blood, were separated by centrifugation. These contained combinations of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8), varying from a single form of acetylcholinesterase to multiple forms of both acetylcholinesterase and cholinesterase. The edrophonium‐Sepharose matrix bound only one form of acetylcholinesterase. This form of acetylcholinesterase corresponded in molecular size and electrophoretic mobility to the unique form found in cerebrospinal fluid, i.e. secretory acetylcholinesterase. Cholinesterase was not bound to the matrix.
Effect of Some Calcium Antagonists on Muscarinic Receptor‐Mediated Cyclic GMP Formation
Authors: El‐Fakahany Esam , Richelson Elliott
Published: October 2006

Abstract: Several calcium antagonists were screened for their effect on muscarinic acetylcholine receptor‐mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E‐115). Mn2+, Ni2+, and verapamil rapidly antagonized the response noncompetitively, with the following order of potency: verapamil > Mn2+ > Ni2+. The effects of Mn2+ and Ni2+, but not those of verapamil, were largely reversed by increasing extracellular calcium concentration. Additional effects of these agents included displacement of [3H]quinuclidinyl benzilate binding by verapamil and elevation of cyclic GMP levels by Mn2+ and Ni2+ in the absence of agonists. These results are in support of the hypothesis that receptor‐mediated cyclic GMP formation by these cells is dependent upon entry of calcium into the cell and demonstrate the complexity of the effects of calcium antagonists.
Local Cerebral Glucose Utilization in Two Models of B12 Deficiency
Authors: Hakim Antoine M. , Cooper Bernard A. , Rosenblatt David S. , Pappius Hanna M.
Published: October 2006

Local cerebral glucose utilization (LCGU), as measured by the 2‐deoxy‐D‐[l‐14C]glucose technique, reflects local cerebral functional activity. In an effort to elucidate mechanisms of the encephalopathy associated with deficiency of vitamin B12, LCGU was determined in two recently described models of effective B12 deficiency: exposure of rats to subanesthetic doses of nitrous oxide (N2O) and/or administration of 1‐amino‐cyclopentane‐1‐carboxylic acid (cycloleucine). Our results show that exposure of adult rats to N2O depresses LCGU selectively in cortical, auditory, and limbic structures, in association with a depression in whole‐brain activities of the vitamin B12‐dependent methyltetrahydrofolate‐homocysteine methyl‐trans‐ferase (EC 2.1.1.13, methionine synthetase). Cycloleucine has no discernible effect on LCGU in the adult rat and does not change the cerebral activity of methionine synthetase.
Effects of Dopaminergic Drugs on Cerebellar Prostaglandin Concentrations
Authors: Millia C. , Nicoletti F. , Grasso A. A. , Patti F. , Condorelli D. F. , Rapisarda E. , Rampello L. , Costa G. , Scapagnini U.
Published: October 2006

Abstract: Previous data indicate that the injection of dopaminergic drugs induces changes in cerebellar 3′,5′‐guanosine monophosphate (cGMP) content. Accordingly, we have investigated the effects of haloperidol, sulpiride, or apomorphine on cerebellar prostaglandin (PG) concentration, a parameter related to cGMP content. Results obtained show that dopamine receptor blocking agents, such as haloperidol and sulpiride, significantly decrease cerebellar PGE2 and PGF2α concentrations, while opposite changes are induced by apomorphine, a dopamine receptor agonist.
Relation of Cholesterol to Oligodendroglial Differentiation in C‐6 Glial Cells
Authors: Volpe Joseph J. , Obert Kathleen A.
Published: October 2006

Abstract: The relation of cellular cholesterol content to a biochemical expression of oligodendroglial differentiation was studied in cultured C‐6 glial cells. Induction of the oligodendroglial marker enzyme 2′: 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) was determined after alteration of the sterol content of cellular membranes by exposure to compactin, a specific inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and cholesterol synthesis. The sterol content and as a consequence, the sterol/phospholipid molar ratio of C‐6 glial cells were decreased by treating the cells, in 10% lipoprotein‐poor serum, with various concentrations of compactin for 24 h. The degrees of sterol depletion thus produced were maintained for 48 h after removal of the compactin if the cells were maintained in serum‐free medium, the culture conditions necessary for induction of CNP in untreated cells. Forty‐eight hours after removal of serum, no induction of CNP occurred in cells previously treated with 0.5 μg/ml of compactin, whereas untreated cells exhibited a three‐ to fourfold increase in CNP activity. Intermediate degrees of sterol depletion resulted in intermediate degrees of inhibition of the CNP induction. Moreover, the morphological expressions of glial differentiation observed in the untreated cells did not occur in the sterol‐depleted cells. That the effect of compactin on the induction of CNP relates to depletion of sterol was indicated by the finding that when low‐density lipoprotein was added to the compactin‐treated cells, the induction of CNP, the morphological expressions of differentiation and the sterol/phospholipid molar ratios were preserved. The degree of sterol depletion that totally prevented the induction of CNP had no effect on (Na++ K+)‐activated ATPase activity, total protein synthesis and cell viability. The data define a critical role for sterol in oligodendroglial differentiation in this model system.
Inhibition of Rabbit Brain Prolyl Endopeptidase by N‐Benzyloxycarbonyl‐Prolyl‐Prolinal, a Transition State Aldehyde Inhibitor
Authors: Wilk Sherwin , Orlowski Marian
Published: October 2006

Abstract: Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin‐releasing hormone, and luteinizing hormone‐releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl‐prolyl‐proline, with a Ki of 50 μM, prompted the synthesis of benzyloxycar‐bonyl‐prolyl‐prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl‐prolyl‐prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.
Alternative Pathways of Glucose Utilization in Brain: Changes in the Pattern of Glucose Utilization and of the Response of the Pentose Phosphate Pathway to 5‐Hydroxytryptamine During Aging
Authors: Zubairu Salmata , Hothersall John S. , El‐Hassan Asia , McLean P. , Greenbaum A. Leslie
Published: October 2006

Abstract: The oxidation of differentially labelled glucose, pyruvate and glutamate in brain slices from rats aged 20 days to 26 months has been studied and the partition of the glucose used into the glycolytic‐tricarboxylic acid cycle pathway, the pentose phosphate pathway and the glutamate‐GABA shunt has been calculated. Over the time range 4 to 26 months, there is an approximately 20% decrease in the production of CO2 via the glycolytic‐tricarboxylic acid cycle route, as there is in the rate of glucose phosphorylation. The glutamate‐GABA pathway falls by about 50% over this same time span. The broad activity of the pentose phosphate pathway falls rapidly and cannot be detected in the brains of rats aged 18 months or more, whereas the fully stimulated pathway, i.e. in the presence of the artificial electron acceptor phen‐azine methosulphate, declines only marginally over this period, falling sharply only after 23 months. The pentose phosphate pathway is stimulated by the presence of 5‐hy‐droxytryptamine and this stimulation appears to increase with age.
Temperature Dependence of Catecholamine Secretion from Cultured Bovine Chromaffin Cells
Authors: Kao Lung‐Sen , Westhead Edward W.
Published: October 2006

Abstract: Secretion of both epinephrine and norepinephrine by cultured chromaffin cells was studied at temperatures ranging from 0°C to 37°C. The percentage of epinephrine secreted was always lower than that of norepinephrine when the cells were stimulated with either acetylcholine or high K+ at any temperature. When the cells were stimulated with acetylcholine or carbachol the percentage of catecholamine secreted at 10 min increased with temperature from 4°C to 24°C and then decreased from 24°C to 37°C. Potassium‐stimulated cells secreted increasing amounts of catecholamine as the temperature was increased to 37°C. We found, however, that the initial rates of secretion increased continuously as temperature increased throughout the range for both carbachol‐and K+‐stimulated cells. The temperature maximum of acetylcholine‐stimulated secretion is caused by a faster shut‐off of secretion at higher temperature. The Arrhenius plots of initial rates show an inflection point at approximately 17°C for carbachol‐stimulated cells. The plot for K+‐stimulated cells is a straight line over the entire temperature range. The transition could be caused by a conformational change in the cholinergic receptor/ion channel molecule.
Substance P Protects Against Desensitization of the Nicotinic Response in Isolated Adrenal Chromaffin Cells
Authors: Boksa P. , Livett B. G.
Published: October 2006

Abstract: Substance P, a peptide endogenous to the splanchnic nerve, is known to inhibit the acetylcholineand nicotine‐induced release of catecholamines from isolated adrenal chromaffin cells. In the present study the effect of substance P on desensitization of catecholamine release from these cells was examined. Substance P (10−5M) completely protected against desensitization of catecholamine release produced by acetylcholine at 37°C or 23°C and by nicotine at 23°C; substance P also afforded appreciable protection against nicotine‐induced desensitization at 37°C. The peptide had no effect on K+‐induced desensitization of catecholamine release. Like substance P, d‐tubocurarine also prevented nicotinic desensitization. Substance P prevented both of two components of nicotinic desensitization, i.e. the Ca2+‐dependent component and the Ca2+‐independent, depletion‐independent component of desensitization. Substance P had little effect on subsequent catecholamine uptake, indicating that substance P's protection against desensitization is a result of facilitation of catecholamine release rather than inhibition of catecholamine reuptake. Nicotine‐induced catecholamine release and nicotinic desensitization of catecholamine release were Na+‐independent, although substance P's inhibition of nicotine‐induced catecholamine release was reduced by extracellular Na+. These in vitro studies suggest a similar role for substance P in vivo: substance P's protection against nicotinic desensitization may ensure a maintained output of adrenal catecholamines during stress, when the splanchnic nerve releases large amounts of acetylcholine.
Kinetics of Drug‐Induced Changes in Dopamine and Serotonin Metabolite Concentrations in the CSF of the Rat
Authors: Mignot E. , Laude D. , Elghozi J. L.
Published: October 2006

Abstract: Cerebrospinal fluid (CSF) was removed at a constant flow rate of 1 μl/min from the third ventricle of anesthetized rats. Every 15 min, CSF dihydroxyphenyl‐acetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations were determined by direct injection of CSF into a liquid chromatographic system coupled with electrochemical detection. Mean CSF concentrations of DOPAC, HVA, and 5‐HIAA were 1.29 μM, 0.88 μM, and 2.00 μM, respectively. In order to determine the turnover rates of dopamine (DA) and serotonin, experiments using monoamine oxidase (MAO) inhibition were performed. Tranylcypromine (20 mg/kg i.p.) induced a sharp exponential decrease of CSF DOPAC, HVA, and 5‐HIAA, with respective half‐lives of 15.60 min, 16.91 min, and 77.23 min. Their respective turnover rates were 3.74, 2.22, and 1.18 nmol ml−1. h−1. m‐Hydroxybenzylhydrazine (NSD‐1015, 100 mg/kg i.p.) and monofluoromethyl‐DOPA (100 mg/kg i.p.), two decarboxylase inhibitors, induced a slow exponential decrease of all three CSF metabolites, α‐Methyl‐p‐tyrosine (250 mg/kg i.p.) also induced a slow exponential decrease of DOPAC and HVA. These decreases of CSF DOPAC and HVA induced by DA synthesis inhibitors may reflect the turnover of DA in vivo. Haloperidol (0.5 mg/kg i.p.) considerably enhanced CSF DOPAC and HVA without affecting 5‐HIAA, confirming that dopaminergic receptors modulate DA neurotrans‐mission in vivo. Haloperidol administered 1.5 h after NSD‐1015 did not increase DOPAC and HVA, in contrast to reserpine (5 mg/kg i.p.) injected under the same conditions. These findings indicate that the effect of haloperidol on DA metabolites may involve newly synthesized DA, but not the DA storage pool.
Phospholipid and Ganglioside Composition in Rat Brain After Chronic Intake of Ethanol
Authors: Vrbaški Snežana R. , Gruji‐Injac Bojana , Ristić M.
Published: October 2006

Abstract: A total of 18 60‐day‐old male Wistar rats were divided into three groups of six animals each. One group was fed a basal diet containing high levels of protein, fat, carbohydrate, vitamins, and minerals and separately a solution of 25% sucrose‐32% ethyl alcohol (wt/vol). A second group was offered water as the only drinking fluid and a similar solid diet, except that carbohydrate replaced ethanol isocalorically. A third group was maintained on the basal diet ad libitum. All groups of animals were killed in a sober state after 6 months of chronic ethanol treatment and lipid analyses were performed on brain homog‐enates. Chronic treatment of the animals with ethanol produces statistically significant modification of the phospholipid and ganglioside patterns in rat brain. A statistically significant decrease of the total phospholipid content and of some of the investigated fractions, i.e., phos‐phatidylcholine and phosphatidylserine, as well as an increase of phosphatidylinositol were observed. Chronic alcohol consumption was associated with a statistically significant increase in the total amount of ganglioside in rat brain. An increase in most of the investigated ganglioside fractions was indicated but the difference was statistically significant only for trisialoganglioside GT1b. The amount of disialoganglioside GD1a in these brains was decreased after chronic intake of ethanol.
Formation and Metabolism of Dopamine in Nine Areas of the Rat Brain: Modifications by Haloperidol
Authors: Westerink Ben H. C. , Bosker Fokko J. , Wirix Els
Published: October 2006

Abstract: The rate of removal of 3,4‐dihydroxyphenyla‐cetic acid (DOPAC) in nine rat brain areas (striatum, nucleus accumbens, tuberculum olfactorium, hypothal‐amus, lateral hippocampus, occipital cortex, brain stem, cerebellum, and retina) was calculated from its exponential decline after monoamine oxidase inhibition by par‐gyline. The experiments were carried out with rats pre‐treated with either saline or haloperidol. It appeared that the efficiency with which DOPAC was removed from the brain (expressed by the fractional rate constant k) varied considerably throughout the brain. Haloperidol dramatically decreased the k values, and in addition these effects differed widely in the various brain areas. Similarly to DOPAC, haloperidol had a pronounced retarding effect on the efflux of homovanillic acid (HVA) from the brain. These findings strongly suggest that great care should be taken when drug‐induced alterations in DOPAC and HVA concentrations are interpreted as changes in dopami‐nergic activity. The dopamine (DA) concentrations were measured in the same experiments, but it appeared that the pargyline‐induced rise in DA was of limited use for the estimation of the synthesis rate of the amine. We calculated the rate of catecholamine synthesis in the nine brain areas from the rise of 3,4‐dihydroxyphenylalanine (DOPA) during decarboxylase inhibition. In saline‐ as well as in haloperidol‐pretreated rats it was found that the total catecholamine synthesis rate in the typical dopa‐minergic areas (striatum, nucleus accumbens, and tuberculum olfactorium) was of the same order of magnitude as the DOPAC rate of removal. This confirms that DOPAC formation is quantitatively the main route of degradation in these brain areas. If it is assumed that DOPAC is also the main metabolite in other brain areas, it is possible to calculate from the DOPA accumulation rate and the DOPAC removal rate which part of the catecholamine synthesis is destined for formation of noradrenaline and which part is used for DA and subsequently metabolized to DOPAC. Finally we calculated the ratios between the rate of removal of DOPAC and the DA content for the nine brain areas. The results indicate that DA is metabolized much faster in the lateral hippocampus and cerebellum than in the other brain areas studied.