bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Metabolism and Toxicity of l‐Methyl‐4‐Phenyl‐l,2,3,6‐Tetrahydropyridine in Cultures of Embryonic Rat Mesencephalon
Authors: Mytilineou Catherine , Friedman Linda
Published: October 2006

Abstract: The metabolism of l‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was studied in dissociated cell cultures prepared from embryonic rat mesencephalon. MPTP was added to the feeding medium and at the end of the incubation period MPTP was separated from the water‐soluble metabolite by ether extraction. Our results demonstrate that, in cultures, MPTP is oxidized predominantly by monoamine oxidase B, since deprenyl but not clorgyline had an inhibitory effect on its metabolism. The metabolite of MPTP diffuses freely in the feeding medium (99% of the total) and its concentration increases with time. The concentration of the metabolite can be increased by increasing the number of cells plated into the tissue culture dish and the toxicity to dopamine neurons depends on the amount of metabolite accumulated in the feeding medium. The metabolism of MPTP is reduced by decreasing the number of glial cells present in the cultures.
Muscarinic Agonists Evoke Neurotransmitter Release: Possible Roles for Phosphatidyl Inositol Bisphosphate Breakdown Products in Neuromodulation
Authors: Diamant Sophia , Lev‐Ari Itzhak , Uzielli Illana , Atlass Daphne
Published: October 2006

Abstract: Carbachol (CCh), a muscarinic agonist that elicits the formation of inositol trisphosphate (IP3) and diacylglyc‐erol (DG), induces a calcium‐dependent [3H]norepi‐nephrine ([3H]NE) release [IC50= (2.7 ± 0.5) × 10‐4M] in rat brain slices. Similarly, other muscarinic agonists evoke [3H]NE release which is specifically inhibited by muscarinic antagonists such as 3‐quinuclidinyl benzilate, atropine, and N‐methyl‐4‐piperidyl benzilate. The atropine‐sensitive evoked release is effectively inhibited by neomycin (IC50= 50 μM), a phospholipase C inhibitor that interferes with IP3‐dependent cellular processes. In addition, polymyxin B, a rather selective inhibitor of protein kinase C (PK‐C), abolishes the agonist‐mediated release with a half‐maximal effective concentration of 0.53 μM(750 ng/ml). These results have a significant implication for the mechanism by which agonists generating IP3 and DG act as inducers of neurotransmitter release in the CNS. However, since both neomycin and polymyxin B act also as N‐calcium‐channel blockers, other possible mechanisms are discussed. The CCh‐induced release suggests that in the CNS an agonist‐receptor interaction leads to a calcium‐dependent neurotransmitter release, most likely via promoting the IP3/DG as second messengers followed by activation of PK‐C.
Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester‐Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity
Authors: Malhotra Ravindra K. , Bhave Sanjiv V. , Wakade Taruna D. , Wakade Arun R.
Published: October 2006

Abstract: The effects of phorbol esters [phorbol 12,13‐dibutyrate (PDB), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and phorbol 13‐acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10‐day‐old chick embryo were cultured in serum‐free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]‐norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]‐norepinephrine over the nonstimulation period. Stimulation‐evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+‐free Krebs solution containing 1 mM EGTA completely blocked stimulation‐evoked release of [3H]‐norepinephrine. Stimulation‐evoked release of [3H]‐norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two‐to fourfold in the presence of PDB or TPA. PDB at 1–100 nM produced a concentration‐dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the paniculate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the paniculate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the paniculate fraction by six‐ to eightfold. Phorbol 13‐acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.
Neuronotrophic Factors Released by C6 Glioma Cells
Authors: Westermann R. , Hardung M. , Meyer D. K. , Ehrhard P. , Otten U. , Unsicker K.
Published: October 2006

Abstract: Glial cells have been shown previously to release factors that promote survival of central and peripheral neurons [neuronotrophic factors (NTFs)]. We have investigated the release of NTFs by C6 cells, a rat glioma cell line, under different modes of conditioning. Media conditioned in the presence or absence of serum [C6 cell conditioned media (C6CMs)] were analyzed using biological, biochemical, and immunological assays. We report that (a) nuclear and cytoskeletal proteins were not present in C6CMs, indicating that C6CM proteins result from release by C6 cells rather than from cell death; (b) C6CM contained 1–3 μg protein/ml, corresponding to a secretion rate of about 0.5 pg protein per cell and day; (c) C6CM contained the neurite‐promoting factor laminin and low amounts of nerve growth factor; (d) the presence of fetal calf serum in the culture medium was essential for synthesis and release of NTFs; and (e) our C6CM contained at least three NTFs differing by their temporal secretory patterns and three NTFs differing by biochemical properties, indicating that C6 cells produce and secrete six different NTFs. Within these, nerve growth factor seems to be the only established NTF.
Formation of a New Biogenic Aldehyde Adduct by Incubation of Tryptamine with Rat Brain Tissue
Authors: Susilo Rudy , Damm Henning , Rommelspacher Hans
Published: October 2006

Abstract: Tryptamine was degraded by incubation with rat brain homogenate to an unknown product. The reaction was stimulated by the nonionic detergents Triton X‐100 and Lubrol PX and less by the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio] 1 ‐propanesulfonate (CHAPS). The same results were obtained with pig brain and bovine brain. The monoamine oxidase inhibitor pargyline inhibited the reaction strongly, indicating the partici pation of the enzyme on the reaction. Addition of 17,000 g supernatant from rat brain homogenate increased the for mation effectively whereas phospholipids or chloroform/methanol (7:3) extract from the 17,000 g supernatant showed only little or no effect. Chromatographic and electrophoretic properties as well as the chemical reaction of the product with specific reagents suggest that the compound consists of an indole part and an amino acid part. The product could be identified by fast atom bombardment mass spectrometry and by comparison with the synthetic substance (4R)‐2‐(3‐indolylmethyl)‐1,3‐thiazolidine‐4‐carboxylic acid. It is formed by the enzymatic oxidation of tryptamine producing indole‐3‐acetaldehyde which sponta neously cyclizes with free l‐cysteine from the tissue. The results suggest that the reaction of biogenic aldehydes with brain macromolecules may proceed via an analogous reaction.
Subcellular Distribution of UDP‐Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia
Authors: Sato Carmen , Black Joel A. , Yu Robert K.
Published: October 2006

Abstract: Oligodendrocytes isolated from 18–19‐day‐old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32–0.8 (F1), 0.8–1.05 (F2), and 1.05–1.3 M (F3). The distribution of UDP‐galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid‐containing ceramides (NFA‐CgalT activity) or 2‐hydroxy fatty acid‐containing ceramides (HFA‐CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA‐CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA‐CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.
Synthetic Sialyl Compounds as Well as Natural Gangliosides Induce Neuritogenesis in a Mouse Neuroblastoma Cell Line (Neuro2a)
Authors: Tsuji Shuichi , Yamashita Tatsuro , Tanaka Makoto , Nagai Yoshitaka
Published: October 2006

Amphipathic compounds containing N‐acetylneuraminic acid (sialic acid) [for example, D‐N‐acetylneuraminyl‐(α2‐1)‐2S, 3R,4E‐2‐N‐tetracosanoyl sphingenine, sialyl alkyl glycerol ethers, and sialyl cholesterols] induced neuritogenesis in a neuroblastoma cell line (Neuro2a). The sialic acid in the hydrophilic moiety of the compounds is specifically required for neuritogenesis. The requirement for molecular specificity of the hydrophobic moiety, however, is rather low. Regarding the hydrophobic moiety, no preference for cholesterol, alkyl glycerol ether, or ceramide residues was observed as to their neuritogenic activity. Sialyl compounds with α‐ketosidic sialyl linkages were more active than the corresponding β‐anomers. These sialyl compounds induced the growth of only one neurite, but a long one, from the cell body. This type of neuritogenesis is completely different from that induced by compounds capable of elevating the concentration of intracellular cyclic AMP, which induced the appearance of many neurites from a single cell body. Besides this morphological change, the active sialyl compounds also caused a change in the carbohydrate composition of the cell surface. Sialyl compound treatment drastically increased the concentration of peanut agglutinin binding sites.
K+‐Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C
Authors: Chowdhury Muhammad , Fillenz Marianne
Published: October 2006

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na+ increased synthesis rate; this was blocked in Ca2+‐free medium and by addition of the Ca2+/calmodulin inhibitor N‐6‐aminohexyl‐5‐chloro‐1‐naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12‐O‐tetradecanoylphorboI‐13‐acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K+‐stimulated synthesis was unchanged in Ca2+‐free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K+ was additive with that of 8‐Br cyclic AMP and of Na+ removal; the combined effect of 50 mM K+ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K+ is mediated by protein kinase C.
Biosynthesis of Neutral Glucocerebroside Homologues in the Absence of Myelin Assembly After Nerve Transection
Authors: Yao Jeffrey K. , Poduslo Joseph F.
Published: October 2006

The biosynthesis of myelin‐associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H‐labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endo‐neurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection. The biosynthesis of these GlcCe homologues, therefore, may represent the phenotypic expression of Schwann cells in the absence of myelin assembly and axonal contact.
Release of Proteins from the Surface of Bovine Central Nervous System Myelin by Salts and Phospholipases
Authors: Smith Ross , Braun Peter E.
Published: October 2006

Incubation of bovine CNS myelin with phospho‐lipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholi‐pase C from Clostridium perfringens, phosphatidylinositol‐specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9‐12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.
Neuronal Localization of Specific Angiotensin II Binding Sites in the Rat Inferior Olivary Nucleus
Authors: Walters D. E. , Speth R. C.
Published: October 2006

Adult male Sprague‐Dawley rats were treated with 3‐acetylpyridine, a neurotoxin selective for the inferior olivary nucleus. Following treatment, the rats exhibited deficits in locomotor behavior indicative of destruction in the inferior olivary nucleus. The rats were sacrificed 3 weeks later, and the binding of125I‐sarcosine1, isoleucine8angiotensin II to brain homogenates and slide‐mounted sections of brainstem was determined. Treatment with 3‐acetylpyridine significantly decreased specific125I‐sarco‐sine', isoleucine8angiotensin II binding in homogenates of posteroventral brainstem (containing the inferior olivary nucleus) by approximately 50%. Homogenates of dorsal brainstem and hypothalamus‐thalamus‐midbrain showed no significant changes in specific binding. Treatment with 3‐acetylpyridine did not significantly alter the radioligand binding affinity which was determined in the hypothalamus‐thalamus‐midbrain. Autoradiographic analysis of125I‐sarcosine1, isoleucine8angiotensin II binding in the brainstem sections indicated that specific angiotensin II binding sites in the inferior olivary nucleus were virtually eliminated by the 3‐acetylpyridine treatment. In addition, a comparatively small, but significant, decrease in specific125I‐sarcosine1, isoleucine8angiotensin II binding occurred in the solitary tract nucleus/dorsal vagal motor nucleus complex. These results indicate that specific angiotensin II binding sites in the inferior olivary nucleus occur exclusively on neuronal perikarya and/or dendrites.
Distribution of [3H]Dihydrotetrabenazine Binding in Bovine Striatal Subsynaptic Fractions: Enrichment of Higher Affinity Binding in a Synaptic Vesicle Fraction
Authors: Meshgin‐Azarian Sassan , Chang Warren , Cugier Debbie L. , Vincent Michael S. , Near Joseph A.
Published: October 2006

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3‐9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95‐400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.
Biochemical Mapping of Peptidyl‐Glycine α‐Amidation Activity in the Rat CNS
Authors: Meng Fan , Tsou Kang
Published: October 2006

Abstract: Peptidyl‐glycine α‐amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using d‐Tyr‐Phe‐Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (>30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10–30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1–10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no α‐amidation activity (<0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.
Prostaglandin Induces Ca2+ Influx and Cyclic GMP Formation in Mouse Neuroblastoma × Rat Glioma Hybrid NG108–15 Cells in Culture
Authors: Miwa Naoto , Sugino Hiroaki , Ueno Ryuji , Hayaishi Osamu
Published: October 2006

Abstract: Various prostaglandins (PGs) (10 nM‐30 μM) were added to NG108–15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2α, PGD2, and PGE2 (10 μM) transiently increased the cyclic GMP content 7.5‐, 3.9‐, and 3.1‐fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 μM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG‐induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose‐dependently inhibited the PG‐induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage‐dependent Ca2+ entry blockers, such as verapamil (10 μM), nifedipine (1 μM), and diltiazem (100 μM); however, the PG (10 μM)‐induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG‐induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage‐dependent Ca2+ channels may not be involved in the PG‐induced rise in Ca2+ content.
Phosphatidic Acid and Phosphoinositide Turnover in Myelin and Its Stimulation by Acetylcholine
Authors: Kahn David W. , Morell Pierre
Published: October 2006

Abstract: Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]‐glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phos‐phatidate (PA). Distribution of the 32P‐labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5‐fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol‐containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20–60% stimulation of [32P]‐phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C‐dependent cycle relative to de novo biosynthesis. We conclude that a significant fraction of both background and neurotransmitter‐enhanced phospholipid metabolism seen in whole brain tissue relates to myelin metabolism.
Chronic Exposure to Pertussis Toxin Alters Muscarinic Receptor‐Mediated Regulation of Cyclic AMP Metabolism in Neuroblastoma × Glioma NG108–15 Hybrid Cells
Authors: Westlind‐Danielsson Anita , Gillenius Pekka , Askelof Per , Bartfai Tamas
Published: October 2006

Chronic pertussis toxin treatment (5 days) of NG108–15 neuroblastoma × glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10−‐4M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5‐day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1‐stimulated cyclic AMP production. Chronic exposure (5 days) of NG108–15 cells to carbachol (10−‐5M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24‐h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.
Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers
Authors: Oláh Z. , Novak R. , Lengyel I. , Dux E. , Joó F.
Published: October 2006

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer‐aided x‐ray film densitometry. With the use of this method, Ca2+‐calmodulin (CAM)‐, Ca2+/phospholipid (PK C)‐, cyclic GMP (cGMP)‐, and cyclic AMP (cAMP)‐dependent protein kinases were detected. CAM‐dependent protein ki‐nase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP‐dependent protein kinase was much higher than that of the cAMP‐dependent one. Autophosphoryla‐tion of both the α‐ and β‐subunits of CAM‐dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothe‐lial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood‐brain barrier.
γ‐Aminobutyric Acid Agonist‐Induced Alterations in the Ultrastructure of Cultured Cerebellar Granule Cells Is Restricted to Early Development
Authors: Hansen Gert H. , Belhage Bo , Schousboe Arne , Meier Eddi
Published: October 2006

The effect of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol (THIP) on the ultrastructural composition of cultured cerebellar granule cells was investigated during development by quantitative electron microscopy (morphometric analysis). Granule cells were exposed to THIP (150 μM) for 6 h after 7 and 14 days, respectively, in culture. THIP treatment of 7‐day‐old cultures led to a statistically significant increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles, and coated vesicles, whereas no significant increase in the cytoplasmic density of these organelles was observed in 14‐day‐old cultures exposed to THIP for 6 h. These findings show that the effect of THIP on the ultrastructural composition of cultured cerebellar granule cells is restricted to early development.
Slow Penetration of Thyrotropin‐Releasing Hormone Across the Blood‐Brain Barrier of an In Situ Perfused Guinea Pig Brain
Authors: Zloković Berislav V. , Lipovac Milo N. , Begley David J. , Davson Hugh , Rakić Ljubiša
Published: October 2006

Transport of 3H‐labelled thyrotropin‐releasing hormone (TRH) across the blood‐brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 × 10‐−3 to 1.22 × 10‐−3 ml min−1 g−1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43–48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood‐brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained. l‐Prolinamide, the N‐terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood‐brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood‐brain barrier, the rate of transfer being some three times higher than that of d‐mannitol.
Noncholinergic Transmitter(s) Maintains Secretion of Catecholamines from Rat Adrenal Medulla for Several Hours of Continuous Stimulation of Splanchnic Neurons
Authors: Wakade Arun R.
Published: October 2006

Abstract: The effect of continuous stimulation of splanchnic nerves at1, 3, and 10 Hz on the secretion of catecholamines from the isolated rat adrenal gland was examined. Secretion evoked at 10 Hz declined over 60% in 1 h, and by the end of 4 h the secretion was only 10% of the initial value. The secretion evoked at 3 Hz was unchanged in the first hour, but showed a gradual decline in subsequent hours. In contrast, secretion evoked at 1 Hz was well maintained for several hours. Even after 6 h of continuous stimulation, the decline was only about 35%. Atropine plus hexamethonium reduced the secretion evoked at 10 Hz by over 80%, but that evoked at 1 Hz was reduced by about 35%; addition of naloxone reduced it to 75%. When the secretion declined to very low levels after continuous stimulation at 10 Hz for 100 min, a change in frequency to 3 Hz or 1 Hz caused a sharp rebound in the secretory response. Returning the frequency back to 10 Hz led to a sharp drop in the secretion, whereas reducing the frequency to 1 or 3 Hz once again increased the secretion. The rebound in the secretory response after switchover of frequencies was observed in the presence of atropine plus hexamethonium, but was abolished by naloxone. Extensive stimulations, which caused large amounts of catecholamine secretions at each frequency, were not associated with any loss in tissue catecholamine contents. The major conclusion is that secretion of catecholamines is maintained uninterrupted for several hours when splanchnic nerves are stimulated at low frequency. Under these conditions, the release of noncholinergic transmitter(s) from splanchnic nerves is mostly responsible for inducing the secretion of catecholamines.
Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla
Authors: Corda M. G. , Longoni B. , Cau A. , Paci S. , Salvadori S. , Laudani U. , Biggio G.
Published: October 2006

Abstract: In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled “in vitro” with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of γ‐ [3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration‐dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the CI‐ ionophore, t‐[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl‐ into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl‐ uptake into rat brain membrane vesicles. Finally, intravenous administration of the β‐carboline inverse agonist 6,γ‐dimethoxy‐4‐ethyl‐;β‐carboline‐3‐carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pen‐tylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA‐coupled chloride ionophore in the fish brain as compared with the mammalian brain.
Acrylamide‐Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve
Authors: Harry G. J. , Goodrum J. F. , Bouldin T. W. , Toews A. D. , Morell P.
Published: October 2006

Abstract The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5‐mm segments determined. In both control and acrylamide‐treated animals, outflow profiles of [35S]methionine‐labeled proteins showed a well defined crest which moved down the nerve at a rate of ± 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine‐labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide‐treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide‐treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide‐induced increase in the demand for axonally transported material.
α2‐Adrenoceptors Mediate Inhibition of Cyclic AMP Production in the Spinal Cord After Stimulation of Cyclic AMP with Forskolin but Not After Stimulation with Capsaicin or Vasoactive Intestinal Peptide
Authors: Uhlén Staffan , Wikberg Jarl E. S.
Published: October 2006

Abstract: In slices obtained from the ventral and the dorsal guinea pig spinal cord both forskolin and vasoactive intestinal peptide (VIP) caused a dose‐dependent stimulation of the production of cyclic AMP. By contrast capsaicin stimulated cyclic AMP formation only in the dorsal cord; no effect was observed in the ventral cord. The (α2‐adrenergic agonist UK‐14,304 dose‐dependently inhibited the production of cyclic AMP in both the dorsal and ventral aspects of the cord when the formation of cyclic AMP had been stimulated with 3 μM forskolin, the maximal inhibition amounting to 25–32%. Also the basal (i.e., unstimulated) production of cyclic AMP was inhibited, the inhibition amounting to about 16–18%. However, after stimulation of cyclic AMP formation in the dorsal cord with capsaicin, UK‐14,304 was virtually ineffective in inhibiting the accumulation of cyclic AMP. Also, when the formation of cyclic AMP was stimulated with VIP, UK‐14,304 was virtually ineffective in inhibiting the formation of cyclic AMP both in the ventral and the dorsal parts of the cord. When cyclic AMP production had been stimulated with forskolin the ability of UK‐14,304 to inhibit the formation of cyclic AMP was not attenuated by capsaicin, either in the ventral or in the dorsal cord. The results are discussed with the notion that cyclic AMP inhibitory spinal cord α2‐adrenoceptors are located on cells accessible to stimulation of cyclic AMP with forskolin but not with capsaicin or VIP.
Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats
Authors: Lowery Joan M. , Berti‐Mattera Liliana N. , Zhu Xi , Peterson Richard G. , Eichberg Joseph
Published: October 2006

Abstract: Sciatic nerve from streptozotocin‐induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol‐4,5‐bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [γ‐32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P] orthophosphate for 2 h, the specific activity of PIP2 was 50–120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P‐labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a “nonmyelin” fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin‐enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.
Cloning of a Putative Neuronal Nicotinic Acetylcholine Receptor Subunit
Authors: Isenberg Keith E. , Meyer Grant E.
Published: October 2006

Abstract: A cDNA clone was isolated from a rat superior cervical ganglion cDNA library with an oligonucleotide that hybridized to muscle‐like nicotinic acetylcholine receptor (nAChR) subunit cDNA. The deduced amino acid sequence possesses characteristics expected of a nAChR subunit that does not bind acetylcholine, in addition to distinctive features such as unique cysteine residues and N‐linked glycosylation sites.
Tyrosination State of Tubulin and the Activity of Tubulin:Tyrosine Ligase and Tubulin Carboxypeptidase in the Developing Retina of the Chick
Authors: Arregui Carlos , Barra Héctor S.
Published: October 2006

Abstract: The tyrosination state of tubulin and the enzymes involved in the tubulin tyrosination/detyrosination cycle—tubulin:tyrosine ligase and tubulin carboxypeptidase—were determined in chick retina during development. The amount of tyrosinable (tyrosinated plus detyrosinated) tubulin increased ∼110% from embryonic day 7 to 14. Then it decreased, and by day 19 it was similar to the value on day 7. This result did not change after hatching, at least up to day 20. The proportion of tyrosinated and detyrosinated tubulin significantly changed with the development of the animal. At embryonic day 7, these tubulin species were at a proportion of 70 and 30%, respectively, and after hatching, the values inverted, to 30 and 70%, respectively. This change did not correlate with the activity of the ligase relative to that of the carboxypeptidase, as measured in vitro. This observation suggested that a change in the turnover rate of microtubules, in the proportion of assembled and nonassembled tubulin pools, or in both had occurred. Coincident with the last possibility, the proportion of assembled tubulin was found to increase during the development of the animal. This finding suggests that the tyrosination state of tubulin may be determined, at least in part, by the assembly state.
Release of γ‐[3H]Aminobutyric Acid from Rat Olfactory Bulb and Substantia Nigra: Differential Modulation by Glutamic Acid
Authors: Jaffe E. H. , Vaello M. L.
Published: October 2006

Abstract: We have studied the glutamate modulation of γ‐[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N‐Methyl‐D‐aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2‐amino‐4‐phosphonobutyric acid and the N‐methyl‐D‐aspartate receptor antagonist 2‐amino‐5‐phosphonovaleric acid significantly inhibited the K+‐glutamate‐ and the kainate‐induced [3H]GABA release. Mg2+ (5 mM), which blocks the N‐methyl‐D‐aspartate receptors, significantly inhibited the K+‐glutamate‐induced but not the kainic acid‐induced [3H]GABA release. The K+‐glutamate‐stimulated release, but not the K+‐stimulated [3H]GABA release, was strongly inhibited by Na+‐free solutions or by 300 nM tetrodotoxin. Apparently the glutamateinduced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb. It is possible that in the external plexiform layer of the olfactory bulb there is a mixed population of voltage‐dependent excitatory amino acid receptors, capable of modulating GABA release through an interneuron. In the substantia nigra glutamate does not modulate GABA release.
Is the Augmentation of K+‐Evoked Intrasynaptosomal Ca2+ Concentration Due to the Influx of Ca2+ in Rat Brain Synaptosomes?
Authors: Okada Mitsuko , Mine Kazunori , Iwasaki Katsunori , Fujiwara Michihiro
Published: October 2006

Abstract: Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+‐channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin‐2‐loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 μM EGTA or 100 μM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 μM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration‐dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+‐evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.
Possible Involvement of Polysialogangliosides in Nerve Sprouting and Cell Contact Formation: An Ultracytochemical In Vitro Study
Authors: Seybold Volker , Rösner Harald , Greis Christian , Beck Eva , Rahmann Hinrich
Published: October 2006

Abstract: In Cichlid fish (Oreochromis mossambicus) primary cell cultures from whole brain and optic tectum, the differentiation‐dependent distribution of polysialogangliosides on the outer cell surface has been followed on an ultrastructural level. For this, a two‐step labeling technique with the monoclonal mouse antibody Q211, recognizing a polysialoganglioside‐associated epitope, followed by a secondary IgM antibody, coupled to colloidal gold sols as an electron‐dense marker, has been used. The gold grains are not uniformly distributed over the whole cell surface, but rather are clearly arranged clusters. In cells from freshly hatched larvae, both cell bodies and nerve fibers strongly exhibit the polysialoganglioside epitope on their surface. With progressing development, neuronal cell labeling is more and more restricted to nerve fibers and especially to cellular adhesion zones, including synaptic terminals, thus suggesting a functional involvement of polysialogangliosides in nerve sprouting and initiation of both cell‐to‐extracellular matrix and cell‐to‐cell contacts.
A Kinetic Analysis of k‐Opioid Agonist Binding Using the Selective Radioligand [3H]U69593
Authors: Smith J. A. M. , Hunter J. C. , Hill R. G. , Hughes J.
Published: October 2006

Abstract: The interaction of the nonselective opioid ligand [3H]bremazocine and of the k‐opioid [3H]U69593 with the k‐receptor was investigated in guinea‐pig cortical membranes. Each radioligand bound to a single population of high‐affinity sites, although [3H]U69593 apparently recognised only 70% of those sites labelled by [3H]bremazocine. Naloxone and the selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both [3H]bremazocine and [3H]U69593. Detailed analysis of the binding of [3H]U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl‐5′‐imidodiphosphate (GppNHp) inhibited binding of [3H]U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of [3H]U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high‐affinity complex between the k‐receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower‐affinity state of the receptor.