bluuebunny/crossref_metadata_2025 huggingface dataset:
BASAL, DOPAMINE‐ AND OCTOPAMINE‐STIMULATED ADENYLATE CYCLASE ACTIVITY IN THE BRAIN OF THE MOTH, MAMESTRA CONFIGURATA, DURING ITS METAMORPHOSIS1
Authors: Bodnaryk Robert P.
Published: October 2006

—The specific activities of basal, dopamine‐ and octopamine‐stimulated adenylate cyclase in the brain of the moth, Mamestra configurata, increased by an order of magnitude or more during the transition from pupal to adult brain. Increases in adenylate cyclase activity outpaced increases in brain weight and protein content by a wide margin, especially during the last 10 days of metamorphosis when most of the change in enzyme activity occurred.

The greatly enhanced adenylate cyclase activity appears to reflect increased demands placed on the adult brain as an integrative centre, especially in the integration of sensory input from the eyes and antennae. Growth of the optic lobes during brain development contributed a substantial proportion of basal (33%), dopamine‐sensitive (21%) and octopamine‐sensitive (37%) adenylate cyclase activity to the adult brain. High levels of octopamine and the presence of active octopamine‐sensitive adenylate cyclase suggest a cyclic AMP‐mediated physiological function for octopamine in the optic lobes of the insect brain, probably as a neurotransmitter or neuromodulator.
Transport and Turnover of Acetylcholinesterase and Choline Acetyltransferase in Rat Sciatic Nerve and Skeletal Muscle
Authors: Wooten G. Frederick , Cheng Chuan‐Huan
Published: October 2006

We studied the axonal transport and turnover of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities in rat sciatic nerve and skeletal muscle. AChE activity accumulated proximal to a sciatic nerve ligation rapidly and linearly for 4‐6 h, and ChAT accumulated much more slowly, but linearly, for at least 4 days. In the nerve segment distal to a ligation, AChE activity accumulated rapidly and linearly for 3‐4 h, while no change was found for ChAT for up to 48 h. Only 13% of total AChE activity in rat sciatic nerve moved in the rapidly transported phase. The absolute rate of rapid orthograde axonal transport of AChE activity was estimated to be approximately 390 mm per day and for retrograde transport approximately 230 mm per day. ChAT transport occurred only in the orthograde direction and at a rate of approximately 1‐2 mm per day. It is suggested that previous investigations of turnover of AChE and ChAT activities in skeletal muscle over‐estimated the true rate of turnover. We estimate that the theoretical minimal rate of turnover for total AChE and ChAT activities in skeletal muscle were 200 days and 950 days, respectively.
Regulation of Muscarinic Receptor‐Mediated Cyclic GMP Synthesis by Cultured Mouse Neuroblastoma Cells
Authors: El‐Fakahany Esam , Richelson Elliott
Published: October 2006

Mouse neuroblastoma clone N1E‐115 has muscarinic acetylcholine receptors that mediate cyclic GMP synthesis. This receptor‐mediated response is not significantly higher than background until the cells have been maintained in the stationary phase for at least 1 week. The basis of the influence of time in culture on the cyclic GMP response was investigated. The relative amount of cyclic GMP synthesized by intact cells was measured by radioactively labeling the GTP pool with [3H]guanine, incubating cells with agonists, and then chromatographically isolating [3H]cyclic GMP. Carbamylcholine‐, ionophore X‐537A‐, and sodium azide‐induced cyclic GMP formation increased with time in culture to a maximum of 13‐, 9‐, and 2.5‐fold above basal, respectively. There was no change in the number or the apparent affinity of the muscarinic receptors as measured by [3H]quinuclidinyl benzylate ([3H]QNB) binding. In addition, there was no change in the apparent affinity of the receptors for agonist as measured by the ability of carbamylcholine to displace the specific binding of [3H]QNB. Guanylate cyclase activity per milligram protein and per cell in‐creased six‐ and sevenfold, respectively, from day 0 to day 22. However, this increase in guanylate cyclase appeared to precede the marked increase in sensitivity of the cells to agonists. These data suggest that, in addition to guanylate cyclase and muscarinic receptors, there is another factor which is responsible for the development of this muscarinic receptor‐mediated response.
Asparagine as Precursor for Transmitter Aspartate in Corticostriatal Fibres
Authors: Reubi J. C. , Toggenburger G. , Cuénod M.
Published: October 2006

The role of asparagine as precursor for the neurotransmitter aspartate was investigated in rat striatum in vitro.14C‐asparagine incubated with striatal slices is converted to a great extent to 14C‐aspartate which is released in a calcium‐dependent manner by high KCl. Furthermore, a frontoparietal cortex ablation of two weeks produces a decrease of more than 70% in the striatal release of newly synthetized 14C‐aspartate, whereas the striatal GABA release is unaffected. This suggests that asparagine is a possible pre‐cursor in vitro for transmitter aspartate in the striatum. This reaction is dependent on intact corticostriatal fibres.
Lysosomal Acid Hydrolases in Developing Huml Brain Regions
Authors: Sinha L. , Sinha A. K.
Published: October 2006

β‐D‐Glucosidase, β‐D‐glucosaminidase, acid phosphatase, and β‐D‐galactosidase were monitored in the human foetal brain at different gestaional periods. Glucosidase specific activity in all brain regions exhibits two peaks, at 8 g and 32 g foetal weights. Acid phosphatase exhibits very high specific activity in all brain regions at 5 g, but the cerebellar activity forms a peak at 220 g foetal weight, the midbrain at 135 g, and the spinal activity at 760 g. β‐D‐Glucosaminidase has a peak at 220 g and 660 g in the midbrain, and β‐D‐galactosidase specific activity is highest in the cortex and cerebellum in late gestation (neuronal differentiation phase). The midbrain medulla and the spinal cord show peak activity at 8 g and 220 g foetal weight. The results suggest an inter‐ and intraregional heterogeneity of acquisition for these enzymes in human brain ontogeny.
Preparation of Neurofilament Protein from Guinea Pig Peripheral Nerve and Spinal Cord
Authors: Shecket Gordon , Lasek Raymond J.
Published: October 2006

Abstract: A simple and rapid method for preparation of enriched neurofilament protein from mammalian peripheral nerve or spinal cord is described. Tissue extracts from guinea pig nerve or spinal cord are fractionated by ammonium sulfate fractionation, chromatography on Sepharose 4B, and precipitation with ethanol. Molecular exclusion chromatography on Sepharose 4B, in which the neurofilament protein elutes quantitatively in the exclusion volume of the column, with little contamination by other proteins, is found to be a highly effective purification step. The protein is found to precipitate in ammonium sulfate fractions over a wide range of salt concentration, from 20 to 80% saturation. It is found to be quantitatively precipitated in 40% v/v ethanol‐water. The preparative method described yields 0.25 mg of neurofilament protein per gram of nerve or spinal cord, with a purity of approximately 50%. The three principal neurofilament polypeptides, which have molecular weights by SDS‐polyacrylamide gel electrophoresis of 200K, 145K, and 68K, are found to be present in the preparation in a molar ratio of 1:2:6. A variant form of neurofilament protein occurring in approximately 20% of Hartley strain guinea pigs is described, which has the polypeptide composition: 200K, 192K, 145K, 68K.
Production and Release of Acetylcholinesterase by a Primary Cell Culture of Bovine Adrenal Medullary Chromaffin Cells
Authors: Mizobe Fumio , Livett Bruce G.
Published: October 2006

Abstract: Bovine adrenal chromaffin cells were isolated and maintained as primary culture monolayers. Total acetylcholinesterase (AChE) activity in the cells increased during the culture period, and AChE activity appeared in the culture medium. We have examined the role of the AChE synthesized by the cells on ACh‐evoked release of catecholamine from the cells. A progressive decrease in the efficacy of ACh (5 × 10‐5m) to evoke release of [3H] norepinephrine from day 3–15 cultures suggests that exogenously applied ACh is hydrolyzed by the nascent AChE synthesized by the cells. These findings provide evidence that chromaffin cells produce AChE and release it into their immediate environment.
Calcium and the Muscarinic Synaptosomal Phospholipid Labeling Effect
Authors: Fisher Stephen K. , Agranoff Bernard W.
Published: October 2006

Abstract: The role of calcium in the muscarinic phospholipid labeling effect in synaptosomes has been investigated. In the absence of added calcium, acetylcholine doubled phosphatidate labeling and increased phosphatidy linositol labeling 40% in synaptosomes when incubated in a medium that contained [32P]orthophosphate. Inclusion of calcium or the omission of magnesium resulted in a marked elevation of acetylcholine‐stimulated phosphatidylinositol labeling (70–80%) while phosphatidate stimulation was unaltered. Calciumchelating agents, EGTA and EDTA, reduced the stimulated labeling of both phosphatidate and phosphatidylinositol, but this inhibition could be reversed by calcium addition. The calcium ionophore A23187, which promotes entry of calcium into cells, selectively increased labeling of both phosphatidate and phosphatidyl‐inositol. This effect, unlike acetylcholine‐stimulated labeling, was not blocked by the addition of atropine. The calcium dependency of the acetylcholine stimulation, on the one hand, and the insensitivity of the ionophore to a muscarinic antagonist, on the other, argue strongly that the acetylcholine‐receptor interaction regulates calcium mobilization and that the latter is linked to the stimulated labeling of phosphatidate and phosphatidylinositol.
Feedback Inhibition of Tyrosine Hydroxylase from Five Regions of Rat Brain by 2‐Hydroxyestradiol and Dihy droxy pheny lalanine
Authors: Lloyd Tom , Ebersole Barbara Jones
Published: October 2006

Abstract: Inhibition of tyrosine hydroxylase from five regions of rat brain by a model catecholamine, DOPA and a model catecholestrogen, 2‐hydroxyestradiol, was examined. Tyrosine hydroxylase preparations from amygdala, preoptic, hypothalamic, striatal and hippocampal regions were freed of small molecules by gel filtration before use. The feedback inhibition of tyrosine hydroxylase by the two model catechols was studied using a spectrum of reduced pterin cofactors, including tetrahydrobiopterin, 6‐methyl‐tetrahydropterin and 6, 7‐dimethyltetrahydropterin. Micromolar levels of either inhibitor produced marked inhibition of tyrosine hydroxylase from all regions when subsaturating levels of the endogenous cofactor, tetrahydrobiopterin, were used.
The Receptor‐Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat
Authors: Ikeno Takeyuki , Dickens Geneva , Lloyd Tom , Guroff Gordon
Published: October 2006

Abstract: The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine β‐hydroxylation is not increased. Preincubation experiments suggest that the carbachol‐induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.
Development of α‐Noradrenergic and Dopaminergic Receptor Systems Depends on Maturation of Their Presynaptic Nerve Terminals in the Rat Brain
Authors: Deskin R. , Seidler F. J. , Whitmore W. L. , Slotkin T. A.
Published: October 2006

Abstract: To study the relationship between ontogeny of rat brain catecholamine nerve terminals and the receptor systems for the catecholamine transmitters, the developmental patterns of synaptosomal uptake mechanisms were compared with those of α‐noradrenergic and dopaminergic receptor‐mediated effects. Uptakes of [3H]dopamine or [3H]norepinephrine into dopaminergic and noradrenergic nerve terminals were low during the 1st week postpartum and increased rapidly during the 2nd week. A similar pattern was obtained for ontogeny of dopaminergic receptor binding sites, as evaluated by [3H]domperidone binding. Stimulation of incorporation of 33Pi into brain phospholipids (elicited by intracisternal injection of dopamine), which is mediated by dopaminergic receptors, was shown to be highly correlated with the maturation of both receptor binding sites and presynaptic nerve terminal uptake. A similar result was seen with norepinephrine, in that the synaptosomal uptake mechanism and norepinephrine‐induced stimulation of 33Pi incorporation into phospholipids, an α‐noradrenergic effect, developed in a parallel fashion. To test the hypothesis that development of the receptor systems is linked to nerve terminal ontogeny, presynaptic nerve terminals were destroyed in neonates by intracisternal administration of 6‐hydroxydopamine. The lesions prevented the maturational increase in the number of dopamine receptor binding sites and produced a defect in development of the dopamine‐ and norepinephrine‐induced stimulation of 33Pi incorporation. The results suggest that ontogeny of both dopaminergic and α‐noradrenergic receptor systems depend upon development of the presynaptic nerve terminals containing the transmitters.
Identification of a Mitochondrial Phosphoprotein in Brain Synaptic Membrane Preparations
Authors: Magilen Gilbert , Gordon Adrienne , Au Alice , Diamond Ivan
Published: October 2006

Abstract: A 41,000‐dalton phosphoprotein in crude synaptosomal membrane fractions is characterized by its unique divalent and monovalent cation regulation. It is identified by two‐dimensional gel electrophoresis as the phosphoprotein whose phosphorylation is enhanced by repetitive electrical stimulation of hippocampal brain slices. After sucrose‐gradient ultracentrifugation, this phosphoprotein is found in the mitochondrial subfraction. This suggests that the electrically produced changes in the level of phosphorylation of the 41,000‐dalton polypeptide are probably effects on cellular energetics rather than on specialized neural membrane function.
Membrane‐Associated Cytoskeletal Proteins in Squid Giant Axons
Authors: Baumgold Jesse , Terakawa Susumu , Iwasa Kunihiko , Gainer Harold
Published: October 2006

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF‐soluble and ‐insoluble forms. The KF‐insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.
Immunocytochemical Localization of (Na+,K+)‐ATPase in the Goldfish Optic Nerve
Authors: Schwartz Michal , Ernst Stephen A. , Siegel George J. , Agranoff Bernard W.
Published: October 2006

Antiserum to the catalytic subunit of goldfish brain (Na+,K+)‐ATPase has been employed at the electron microscopic level by means of the peroxidase‐antiperoxidase immunohistochemical method. In optic nerve, an‐tigenic sites are restricted to the nodes of Ranvier. No reaction product is detected in underlying internodal neurolemma. Outgrowing neurites for cultured retinal explants devoid of glial ensheathment exhibit a continuous distribution of the enzyme subunit. Antibodies against eel electroplax (Na+, K+)‐ATPase cross‐react with the goldfish brain enzyme and show a similar immunocytochemical distribution pattern.
Ontogeny of Adenosine Binding Sites in Rat Forebrain and Cerebellum
Authors: Marangos P. J. , Patel J. , Stivers J.
Published: October 2006

Abstract: The metabolically stable adenosine analogue N6‐cyclohexyl [3H]adenosine ([3H]CHA) was used to label adenosine receptors in rat fore‐brain and cerebellum during development. [3H]CHA binding develops rather slowly, with adult binding levels obtained at 24 days in cerebellum and later in the forebrain. Ontogenetic profiles in both areas are consistent with the onset of neuronal differentiation. High and low affinity sites appear to develop in parallel, since Scatchard analysis in forebrain tissue obtained from 5‐day‐old animals revealed both binding sites.
The Topographical Distribution of Alanine, Aspartate, γ‐Aminobutyric Acid, Glutamate, Glutamine, and Glycine in the Pigeon Optic Tectum and the Effect of Retinal Ablation
Authors: Fonnum Frode , Henke Hermann
Published: October 2006

Abstract: The concentrations of alanine, aspartate, γ‐aminobutyric acid, glutamine, glutamate, and glycine were measured in the pigeon optic nerve and in the individual tectal layers. Characteristic topographical distribution patterns were observed for the different amino acids. After unilateral retinal ablation, the concentration of aspartate and glutamate was decreased in the nerve and contralateral tectum. The reduction was restricted to the superficial part of the tectum, which receives a direct retinal input. The maximal loss was measured in the first two layers, where aspartate was reduced by 51% and glutamate by 75% in comparison with the ipsilateral side 4 weeks after ablation. The results favor a special role for aspartate and glutamate in pigeon retino‐tectal afferents.
γ‐Aminobutyric Acid and Benzodiazepine Binding Sites in Audiogenic Seizure‐Susceptible Mice
Authors: Horton R. W. , Prestwich S. A. , Meldrum B. S.
Published: October 2006

Abstract: The properties of γ‐aminobutyric acid recognition sites, benzodiazepine binding sites and the effect of exogeneous γ‐aminobutyric acid on benzodiazepine binding were determined in crude membrane fractions prepared from the brains of DBN/2 mice at ages before (8‐9 and 17‐18 days), during (22‐23 and 28‐29 days) and after (40‐43 days) the age of high susceptibility to audiogenic seizures. These have been compared with data from age‐ matched mice of a strain (TO) with lower audiogenic seizure susceptibility. The number of high‐affinity [3H]γ‐aminobutyric acid binding sites was lower at all ages in DBN/2 mice compared with TO mice, but the affinity was higher in DBN/2 mice. The number of low‐affinity [3H]y‐aminobutyric acid binding sites was lower at 8‐9 days and 40‐43 days in DBN/2 mice, but was not significantly different from TO mice at other ages. For [3H]flunitrazepam binding, the only difference found was a slight reduction in the number of binding sites at 28‐29 days of age in DBN/2 mice. γ‐Aminobutyric acid stimulation of [3H]‐flunitrazepam binding was not significantly different up to 22‐23 days of age, but was higher in DBN/2 mice at 28‐29 days and lower at 40‐43 days. Impairment of γ‐aminobutyric acid function is a possible permissive factor in the age‐dependent audiogenic seizure susceptibility in DBN/2 mice.
Glycerol Phosphate Dehydrogenase, Glucose‐6‐Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains
Authors: Cammer Wendy , Snyder D. Stephen , Zimmerman Thomas R. , Farooq Muhammad , Norton William T.
Published: October 2006

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose‐6‐phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4‐fold during development, and at 120 days cells of this type had 1.4‐fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10‐day‐old rats were 1.4‐fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20‐, 60‐, and 120‐day‐old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin‐related membrane vesicles.
Calmodulin‐Sensitive and Calmodulin‐Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides
Authors: Treisman Glenn J. , Bagley Stuart , Gnegy Margaret E.
Published: October 2006

Abstract: The interaction between the Ca2+‐binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal paniculate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine‐5′‐(3,7‐imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 p.M GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100‐fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The cal‐modulin‐stimulatable component constituted 60% of the total activity. Our two‐component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP‐binding proteins. The finding that GTP was unable to activate the calmodulin‐sensitive component suggests that this component has either a different mode of binding to a GTP‐binding protein or inherently higher GTPase activity than has the calmodulin‐insensitive component. The results suggest there are two components of adenylate cyclase activity that can be differentiated by their sensitivities to calmodulin and guanyl nucleotides.
Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding
Authors: Gee Kelvin W. , Yamamura Henry I.
Published: October 2006

Abstract: Recently, it was proposed that β‐carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [3H]diazepam binding was lost in photolabeled membranes, specific [3H]propyl β‐carboline‐3‐carboxylate ([3H]PCC) binding was spared. In this study, the binding of the BZD antagonists [3H]PCC, [3H]Ro15 1788 and [3H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent KD and small reductions in the Bmax of 3H antagonist binding were observed. However, in the same membranes, up to 89% of specific [3H]FLU binding was lost. When [3H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [3H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[3H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β‐carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.
Dual Effects of Ascorbate on Serotonin and Spiperone Binding in Rat Cortical Membranes
Authors: Muakkassah‐Kelly S. F. , Andresen J. W. , Shih J. C. , Hochstein P.
Published: October 2006

Abstract: Ascorbate‐induced lipid peroxidation, as measured by malonyldialdehyde (MDA) production, caused irreversible decreases in Bmax of both [3H]5‐HT and [3H]spiperone binding. CaCl2 (4 mM) inhibited ascorbateinduced MDA formation at ascorbate concentrations >0.57 mM, but not at ≤0.57 mM. Under the standard assay conditions (5.7 mM ascorbate and 4 mM CaCl2), CaCl2 inhibited the MDA production caused by ascorbate by 88%, and the loss in [3H]5‐HT binding by 57%. Ascorbate still decreased [3H]5‐HT binding when lipid peroxidation was completely inhibited by EDTA. This additional effect of ascorbate was reversible after washing the membranes. Other reducing agents (dithiothreitol, glutathione, and metabisulfite) also decreased the binding of [3H]serotonin. In contrast, [3H]spiperone binding was not affected by ascorbate in the absence of lipid peroxidation or by other reducing agents. These experiments demonstrate that ascorbate has a dual and differential effect on serotonin binding sites. First, ascorbate‐induced lipid peroxidation irreversibly inactivates both [3H]5‐HT and [3H]spiperone binding. Second, independent of lipid peroxidation, there is a direct, reversible effect of ascorbate on [3H]serotonin but not on [3H]spiperone binding, which is probably due to the difference in the biochemical nature of the two serotonin binding sites.
Reduced Cortical Glutamine Concentrations in Electrically Kindled Rats
Authors: Leach M. J. , Miller A. A. , O'Donnell R. A. , Webster R. A.
Published: October 2006

Abstract: The effect of kindling rats (with an electrical stimulus applied daily to the frontal cortex) on the concentrations of taurine, aspartate, glutamate, glutamine, and γ‐aminobutyric acid has been investigated. When compared with control groups, cortical glutamine concentrations were significantly decreased in kindled rats by ∼20%. This decrease in glutamine directly correlated with the after‐discharge duration (r= 0.84, p = 0.005). The significance of this in relation to glutamate metabolism and kindling is discussed here.
Characterization of Tubulin in Mouse Brain Myelin
Authors: Néchaud Béatrice de , Wolff Annie , Jeantet Claude , Bourre Jean‐Marie
Published: October 2006

Abstract: Analysis of mouse brain myelin by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis showed that in the high‐molecular‐weight range it contained, besides the Wolfgram protein doublet, proteins comigrating with actin and with both subunits of tubulin. The occurrence of these α and β subunits was confirmed by peptide mapping in myelin analyzed by two‐dimensional electrophoresis. This tubulin did not arise from an artifactual binding of soluble brain tubulin to the myelin fraction: addition of exogenously labeled tubulin to brain homogenates proved that during myelin isolation by the procedure of Norton and Poduslo (1973) the contaminating tubulin was washed out. On the other hand, the distribution of tubulin isoforms in myelin was investigated by isoelectric focusing and compared with the distribution of the 21 isoforms listed for the whole brain soluble tubulin. It was shown that many isoforms were found in myelin (three isoforms for the α subunit and nine for the β subunit), and that some isoforms were represented both in myelin and in soluble tubulin, but in different relative proportions.
Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells
Authors: Nathanson Neil M.
Published: October 2006

Abstract: The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N‐methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low‐affinity sites on intact cells with an affinity identical to that found for the low‐affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (> 30 s) high‐affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.
Dopaminergic Receptors in Bovine Retina and Their Interaction with Thyrotropin‐Releasing Hormone
Authors: Reading H. William
Published: October 2006

Abstract: A superfusion technique was employed to study the release of [3H]dopamine from isolated bovine retina. Only K+‐stimulated release was observed from both light‐ and dark‐adapted retina; release by other stimuli was from dark‐adapted retina only. Light‐evoked release of [3H]dopamine from dark‐adapted retina was blocked by thyrotropin‐releasing hormone (TRH), which has previously been identified as a retinal neuropeptide. TRH itself released small amounts of [3H]dopamine from dark‐adapted retina. These results are interpreted as indicating that TRH acts as a modulator of dopaminergic activity in retina through the agency of presynaptic autoreceptors. Evidence of the existence of a feedback inhibition system, probably mediated by dopaminergic autoreceptors, was found by the inclusion of sulpiride, a dopaminergic D2 receptor antagonist in the perfusate, which, in a stereoselective manner, enhanced spontaneous and light‐evoked release of [3H]dopamine. On the other hand, dopamine (1 μM) reduced these effects. TRH did not affect the high‐affinity uptake system for dopamine in retina; this, then, could not account for the effects on release. Radioligand binding showed a specific, saturable high‐affinity binding system for [3H]TRH, with an apparent KD of 2.2 nM and a Bmax of 23 fmol/mg protein in bovine retinal membranes. Displacement experiments showed that specific [3H]TRH binding was displaced in the nanomolar range by spiperone and in the micromolar range by dopamine, whereas l‐(‐)‐sulpiride was virtually inactive in displacing [3H]TRH. It was concluded that these results indicate the existence of at least three classes of dopaminergic receptor or binding site in retina, some of which can be classed as presynaptic autoreceptors which take part in a modulatory control mechanism for the release of dopamine from amacrine cells.
Presynaptic Distribution of the Cholinergic‐Specific Antigen Chol‐1 and 5′‐Nucleotidase in Rat Brain, as Determined by Complement‐Mediated Release of Neurotransmitters
Authors: Richardson Peter J.
Published: October 2006

Abstract: Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, γ‐aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4‐dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement‐mediated lysis. Three antisera were used: anti‐5′‐nucleotidase, anti‐Chol‐1, and anti‐rat cerebrum. Anti‐5′‐nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti‐Choi‐1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Antirat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5′‐nucleotidase was not present on any of the terminals studied, and that Chol‐1 is only present on cholinergic terminals.
Biogenic Amine‐Stimulated Cyclic Adenosine‐3′,5′‐Monophosphate Formation in the Rat Carotid Body
Authors: Mir Anis K. , Pallot David J. , Nahorski Stefan R.
Published: October 2006

Abstract: The subcutaneous injection of isoprenaline, salbutamol, histamine, and adrenaline to rats, which were subsequently killed by microwave irradiation, resulted in a rapid increase in the cyclic AMP content of the carotid body. On the other hand, noradrenaline, dopamine, adenosine, and 5‐hydroxytryptamine, at doses at least 100 times greater than that of isoprenaline, did not significantly alter the cyclic nucleotide content in vivo. The response to isoprenaline was dose related, with an ED50 of 15 μg · kg−1, and reached a peak level 1–1.5 min after injection. Incubation of intact carotid bodies with isoprenaline (10‐−5M)in vitro also resulted in a 10‐fold increase in cyclic AMP content. The in vivo response to isoprenaline could be blocked stereo‐selectively by propranolol, and ICI 118.551, a β2‐selective antagonist, blocks the isoprenaline‐elicited increase in cyclic AMP completely at a dose of 30 μg · kg−1; whereas betaxolol, a β1‐selective antagonist, was ineffective, even at a dose of 300 μg · kg−1. Hypoxia (5% oxygen in 95% N2) did not result in a significant increase in the cyclic AMP content, nor did it significantly alter the isoprenaline‐stimulated increase in the cyclic AMP content of the rat carotid body. These results suggest that some catecholamines may stimulate cyclic AMP formation by interacting with a β2‐adrenoceptor in the rat carotid body.
Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons
Authors: Webb Michael
Published: October 2006

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO4 oxidation/NaB 3H4 reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO4 or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO4 oxidation, about 80% of the cell‐associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO4/NaB 3H4 in time course studies of cultures up to 8 days in vitro. Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.
A Sensitive Radiometric Assay for Tryptophan Hydroxylase Applicable to Crude Extracts
Authors: Beevers S. Jane , Knowles Richard G. , Pogson Christopher I.
Published: October 2006

Abstract: We describe here a simple and convenient method for assay of tryptophan 5‐monooxygenase (hydroxylase), applicable to enzyme in all states of purification. It is based on the enzyme‐catalysed formation of 5‐hydroxy‐[4‐3H]tryptophanfrom [5‐3H]tryptophan, and the subsequent acid‐dependent quantitative release of 3H as 3H2O; unreacted substrate is removed with activated charcoal. The assay is linear with respect to both protein concentration and time, and gives results similar to those in a standard fluorimetric assay.
Acetylcholine Releases ATP from Varicosities Isolated from Guinea Pig Myenteric Plexus
Authors: White Thomas D. , Al‐Humayyd Mohammed
Published: October 2006

The effects of cholinergic agonists and antagonists on the release of ATP from isolated myenteric varicosities were studied using a firefly luciferin‐luciferase technique. In a previous study, acetylcholine and nicotine released ATP from isolated myenteric varicosities, whereas the muscarinic agonist bethanechol did not. In the present study, release of ATP by acetylcholine was shown to be Ca2+dependent. d‐Tubocurarine competitively antagonized the release of ATP by either acetylcholine or nicotine. Maximal release of ATP by acetylcholine (10‐3M) was approximately 24% that observed with the depolarizing drug veratridine (5 x 105M), suggesting either that not all of the varicosities capable of releasing ATP possess nicotinic receptors or that acetyl‐choline does not depolarize the varicosities to the degree that veratridine does. Tetrodotoxin slightly but significantly reduced ATP release induced by acetylcholine or nicotine, indicating some involvement of Na+ channels in the release process. Finally, 6‐hydroxydopamine pre‐treatment produced a 48% reduction in the acetylcholine‐evoked release of ATP, suggesting that much, but possibly not all, of the ATP release occurs from noradrenergic varicosities present in the preparation.
Determination of Some Molecular Parameters of Tyrosine Hydroxylase From Rat Adrenal, Rat Striatum, and Human Pheochromocytoma
Authors: Rosenberg Robert C. , Lovenberg Walter
Published: October 2006

The molecular parameters of tyrosine hydroxylase (EC 1.14.16.2) from rat adrenal, rat striatum, and human pheochromocytoma were determined by combined gel filtration and sucrose gradient ultracentrifugation. The enzyme from rat adrenal has a calculated molecular weight of 228,000, a Stokes radius of 60.9 Å, a sedimentation coefficient of 9.10S, and a frictional ratio of 1.39. The enzyme from rat striatum has a calculated molecular weight of 210,000, a Stokes radius of 54.3 Å, a sedimentation coefficient of 9.38S, and a frictional ratio of 1.28. Tyrosine hydroxylase from human pheochromocytoma tissue has a calculated molecular weight of 255,000, a Stokes radius of 68.2 Å, a sedimentation coefficient of 9.08S, and a frictional ratio of 1.50. These results indicate that the tyrosine hydroxylases from central and peripheral tissue in the rat are quite similar although the human enzyme appears to be significantly larger.
The Benzodiazepine Receptor in Rat Brain and Its Interaction with Ethyl /3‐Carboline‐3‐Carboxylate
Authors: Martin I. L. , Doble A.
Published: October 2006

[3H]Ethyl β‐carboline‐3‐carboxylate ([3H]β‐CCE) binds to a homogeneous population of recognition sites in rat whole brain membranes with high affinity. The [3H]β‐CCE binding is completely displaceable by low concentrations of a number of benzodiazepines with similar potencies found when using a 3H‐benzodiazepine as the ligand. This suggests that the recognition sites for β‐CCE and the benzodiazepines are identical or that they are involved in a close interaction. The binding of [3H]β‐CCE does not obey simple mass‐action kinetics. [3H]Flunitrazepam dissociation from its receptor population is biphasic, and different methods of initiation of this dissociation indicate that cooperative interactions take place within the receptor population. We conclude that the benzodiazepine receptor is a single entity that can exist in two conformations, the equilibrium between which may be controlled by some as yet unidentified factor.
Studies on Subcellular Fractions Which Are Involved in Myelin Membrane Assembly: Isolation from Developing Mouse Brain and Characterization by Enzyme Markers, Electron Microscopy, and Electrophoresis
Authors: Pereyra Pedro M. , Braun Peter E.
Published: October 2006

Abstract: An extensive scheme for the subcellular fractionation of myelinating mouse brain is presented. Several centrifugation procedures for the separation of membranes involved in myelinogenesis are critically appraised, and guidelines for selection of centrifugation conditions are given. Characteristics of subcellular fractions are presented in the form of electron micrographs; also presented are distribution of RNA and protein; electrophoretic profiles of membrane proteins, and verification of the myelin‐specific basic proteins, proteolipid protein, and glycoprotein by the immuno‐electroblot technique; and the distribution of eight marker enzyme activities. Myelin‐related membranes were found to differ both qualitatively and quantitatively in their complement of myelin‐specific proteins. These myelin‐containing fractions appear to represent different stages of myeli‐nation that coexist in developing mouse brain. These results provide the fundamental methodologies and background information for kinetic radioisotope analysis of intracellular events in the assembly of myelin presented in a companion article.
Effects of Calcium on the Binding of Phencyclidine to Acetylcholine Receptor‐Rich Membrane Fragments from Torpedo californica Electroplaque
Authors: Oswald Robert E.
Published: October 2006

Abstract: The influence of calcium on the binding of phencyclidine (PCP) to acetylcholine (ACh) receptor‐rich membrane fragments was investigated. Calcium decreased the equilibrium affinity for PCP in the presence, but not in the absence, of the cholinergic agonist carbamylcholine. The effect of calcium was rapidly reversible by EGTA, indicating that it was not attributable to a calcium‐activated protease or a phospholipase. Following detergent solubilization of the nicotinic ACh receptor, the calcium effect on PCP remained, suggesting that calcium may interact directly with the receptor to exert its effect. Other divalent cations (Mn2+, La2+ Co2+, Mg2+) had similar effects. A correlate of “desensitization” of the ACh receptor can be observed using PCP binding, and a two‐step “desensitization” process can be observed. Calcium seemed to increase the amplitude of a rapid component of receptor “desensitization.” The results presented in this paper suggest that calcium may play a role in the modulation of the nicotinic ACh receptor.
Biochemical Evidence of Selective Nerve Cell Changes in the Normal Ageing Human and Rat Brain
Authors: Allen S. J. , Benton J. S. , Goodhardt M. J. , Haan E. A. , Sims N. R. , Smith C. C. T. , Spillane J. A. , Bowen D. M. , Davison A. N.
Published: October 2006

Abstract: Atrophy with ageing of human whole brain, entire temporal lobe, and caudate nucleus was assessed in autopsy specimens, by biochemical techniques. Only the caudate nucleus showed changes. Markers for several neurotransmitter systems were also examined for changes with age. In neocortex and temporal lobe of human brain, small decreases were detected in markers of cholinergic nerve terminals, whereas a large decrease (79%) occurred in the caudate nucleus. Findings were similar in striatum from 3–33‐month‐old rats. No change occurred in binding of [3H]quinuclidinyl benzilate by human samples. Markers of serotonergic terminals were also unchanged in human and rat brain. By contrast, binding of [3H]lysergic acid diethylamide and [3H]serotonin was decreased (32–81%) in human neocortex and temporal lobe, but not in caudate nucleus. A 43% loss of a marker of γ‐aminobutyrate terminals occurred in human neocortex, while [3H]muscimol binding increased (179%). No changes were detected in markers of catecholamine synapses in temporal lobe or rat striatum. Hence, with human ageing there appears to be a loss of markers of γ‐aminobutyrate neurones intrinsic to neocortex and acetylcholine cells intrinsic to the caudate nucleus, as well as a change in postsynaptic serotonin receptors in neocortex. These losses are accompanied by relative preservation of markers of ascending projections from basal forebrain and brain stem.
Degradation of Neurofilament Proteins by Purified Human Brain Cathepsin D
Authors: Nixon Ralph A. , Marotta Charles A.
Published: October 2006

Abstract: Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one‐or two‐dimensional sodium dodecyl sulfate‐polyacrylámide gel electrophoresis (SDS‐PAGE). Degradation was appreciable at enzyme activity‐to‐substrate protein ratios that were two‐to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP breakdown products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two‐dimensional SDS‐PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno‐cross‐reactive fragments generated from NFPs by CD comigrated on two‐dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskel‐etal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.