bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats
Authors: Nakahara Ichiro , Kikuchi Haruhiko , Taki Waro , Nishi Syogo , Kito Makoto , Yonekawa Yasuhiro , Goto Yasunobu , Ogata Nobuyoshi
Published: October 2006

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1‐ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acid chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time‐dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.
Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord
Authors: Fone Kevin C. F. , Johnson Janel V. , Putland Anthony P. , Bennett Geoffrey W.
Published: October 2006

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed‐volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15–50 mM) produced a dose‐related increase in basal NA release that was calcium dependent The potassium‐evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective α2‐adrenoceptor antagonist idazoxan (10−6‐10−4M) or (b) the thyrotrophin‐releasing hormone (TRH) analogue RX 77368 (pGlu‐His‐3,3′‐dimethyl ProNH2; 10−5 and 10−4M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 μM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10−7M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10−4M) significantly attenuated the potassium‐or RX 77368‐induced release of NA, whereas substance P (3 × 10−5 and 1 × 10−4M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.
Processing of Proenkephalin in Adrenal Chromaffin Cells
Authors: Wilson Steven P.
Published: October 2006

The processing of proenkephalin was studied using [35S]methionine pulse‐chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin‐derived peptides were extracted from the cells and separated by reverse‐phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met‐enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met‐enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radio‐labeling and a 6‐h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co‐storage of catecholamines.
Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay
Authors: Kawashima Koichiro , Hayakawa Toru , Kashima Yuko , Suzuki Takeshi , Fujimoto Kazuko , Oohata Hisayo
Published: October 2006

A vertical‐type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholincsterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 ± 0.6 fmol/min, n = 16). Tetrodotoxin (1 μM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1–1 μM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 μM) to the perfusion fluid produced about a 100‐fold increase in the amount of ACh collected. In the presence of physostig mine, topical administration of atropine and pirenzepine (0.01–1 μM) through a dialysis probe produced a further three‐to fourfold increase in ACh output, whereas a slight increase was produced by AF‐DX 116 at the highest concentration (1 μM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M, muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.
Regional Heterogeneity of L‐Glutamate and L‐Aspartate High‐Affinity Uptake Systems in the Rat CNS
Authors: Fletcher Elizabeth J. , Johnston Graham A. R.
Published: October 2006

The high‐affinity uptake of L‐[3H]glutamate and L‐[3H]aspartate into synaptosomes prepared from rat cerebral cortical, hippocampal, and cerebellar tissue was reduced by a number of structural analogues of L‐glutamate and L‐aspartate. thero‐3‐Hydroxy‐L‐aspartic acid was a more potent inhibitor of L‐glutamate uptake than of L‐aspartate uptake in the cerebral cortex, but not in the hippocampus or cerebellum. A similar pattern of selectivity was observed for cis‐l‐aminocyclobutane‐1,3‐dicarboxylic acid. Dihydrokainate was also more potent against L‐glutamate than against L‐aspartate in the cerebral cortex, but in the hippocampus, it was more potent against L‐aspartate than against L‐glutamate. By contrast, L‐α‐aminoadipate was significantly more potent in the cerebellum than in the cerebral cortex and hippocampus as an antagonist of both L‐glutamate and L‐aspartate. These results support other evidence that there is regional heterogeneity in acidic amino acid uptake sites and that the amino acids L‐glutamate and L‐aspartate may be taken up by a number of transport systems with overlapping substrate specificity but different inhibitor profiles.
Stereospecificity of High‐and Low‐Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes
Authors: Ferguson S. S. G. , Diksic M. , Collier B.
Published: October 2006

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)‐α‐methylcholine and S(+)‐β‐methylcholine were significantly better inhibitors of the high‐affinity choline transport system than were their enantiomers. Although both enantiomers of α‐and of β‐methylcholine inhibited [3H]choline transport, only R(+)‐α‐methylcholine and S(+)‐β‐methylcholine could be transported by the high‐affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high‐affinity transporter are dearly different. In addition to high‐affinity choline transport, Na+‐independent low‐affinity transport was measured. This process transported R(+)‐α‐methylcholine, but not S(‐)‐α‐methylcholine; however, it showed no stereoselectivity for the enantiomers of β‐methylcholine. Thus, high‐and low‐affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.
Identification of Serotonin Receptors Recognized by Anti‐Idiotypic Antibodies
Authors: Tamir Hadassah , Liu Kuo‐peing , Hsiung Shu‐chi , Yu Peiying , Kirchgessner Annette L. , Gershon Michael D.
Published: October 2006

Anti‐idiotypic antibodies were generated by immunizing rabbits with affinity‐purified antibodies to serotonin (5‐hydroxytryptamine; 5‐HT). Anti‐5‐HT activity was removed from the resulting antisera by chromatography through a 5‐HT affinity column. The anti‐idiotypic antibodies were demonstrated by enzyme‐linked immunosorbent assay to bind to affinity‐purified whole anti‐5‐HT antibodies and their Fab fragments. Anti‐idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5‐HT were coupled, competed with 5‐HT (covalently bound to protein) for the binding sites on anti‐5‐HT antibodies and serotonin binding protein. The anti‐idiotypic antibodies antagonized the binding of [3H]5‐HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti‐ idiotypic antibodies also blocked the binding of the 5‐HT1B‐ selective ligand (‐)‐[125I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti‐idiotypic antibodies failed to inhibit binding of the 5‐ HT1A‐selective ligand 8‐hydroxy‐2‐(di‐n)‐[3H]propylamino)‐ tetralin ([3H]8‐OH‐DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti‐idiotypic antibodies may recognize some 5‐HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti‐idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5‐ HT1C or 5‐HT2 receptor or with a genomic clone encoding the 5‐HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5‐ HT1C and 5‐HT2 receptors, but not on the surfaces of HeLa cells that expressed 5‐HT1A receptors. Immunostaining of cells by the anti‐idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5‐HT1C receptors was blocked by mesulergine (but not ketanserin, 8‐OH‐DPAT. or spiperone), whereas that of cells expressing 5‐HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8‐OH‐DPAT). The anti‐ idiotypic antibodies failed to inhibit the uptake of [3H]5‐HT by serotonergic neurons. It is concluded that the anti‐idiotypic antibodies generated with anti‐5‐HT serum recognize the 5‐ HT1B, 5‐HTlC, and 5‐HT2 receptor subtypes; however, neither 5‐HT1A receptors nor 5‐HT uptake sites appear to react with these antibodies.
Binding of Botulinum and Tetanus Neurotoxins to Ganglioside GT1b and Derivatives Thereof
Authors: Schengrund Cara‐Lynne , DasGupta Bibhuti R. , Ringler Nancy J.
Published: October 2006

The ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b‐coated plastic wells was investigated and compared with the binding characteristics of the parent ∼ 150‐kDa protein. Although the ∼50‐kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b‐coated wells was exhibited by the ∼ 50‐kDa carboxy‐terminal half of the ∼ 100‐kDa heavy chain of BTxA; the amino‐terminal half of the heavy chain lacked the ability to bind. Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy‐terminal half of its heavy chain. Binding of BTxA and the C‐terminal half of the heavy chain was optimal in buffers of low ionic strength (μ≤ 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at μ≤ 0.10. TTx and the ∼ 50‐kDa C‐terminal half of its ∼ 100‐kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA. Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b. Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by ∼60 and ∼90%, respectively. Determination of the IC50 values for GT1b derivatives (molar concentration needed to reduce toxin binding to GT1b by 50%) indicated that (a) the carboxyl moiety of the sialosyl residues is recognized by BTxA and TTx, (b) the lipid segment of GT1b enhances BTxA and TTx binding, and (c) the N‐acetyl groups and C(8)‐C(9) on the sialosyl residues are not essential components of the binding site for BTxA and TTx.
Depolarization and Agonist‐Stimulated Changes in Inositol 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex
Authors: Challiss R. A. John , Nahorski Stefan R.
Published: October 2006

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and myo‐inositol 1,3,4,5‐tetrakis‐phosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values forcarbachol. Omission of buffer Ca2+ ([Ca2+]e 10–20 μM) reduced basal lns (1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K+‐evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L‐type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3‐kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.
Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin‐Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells
Authors: Purkiss John , Murrin Richard A. , Owen P. Jane , Boarder Michael R.
Published: October 2006

The role of lipid‐bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal Chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor‐linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase(DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of Chromaffin cells prelabelled (24 h) with 32P1 leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When Chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [γ‐32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.
Ca2+‐Independent Release of Glutamate During In Vitro Anoxia in Isolated Nerve Terminals
Authors: Rubio Inmaculada , Torres Magdalena , Miras‐Portugal María Teresa , Sánchez‐Prieto Jose
Published: October 2006

The effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extrasynaptosomal concentration of glutamate ([Glu]cxt) under aerobic conditions was 2.3 μM and increased to 4.9 μM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]cxt The accumulation of glutamate in the external medium during anoxia was Ca2+ in dependent and insensitive to a significant reduction of the Ca2+‐dependent release of the amino acid. These results indicate that a Ca2+‐independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.
Solubilized Rat Brain Adenosine Receptors Have Two High‐Affinity Binding Sites for l, 3‐Dipropyl‐8‐Cyclopentylxanthine
Authors: Oliveira Júlia C. , Sebastião A. M. , Ribeiro J. A.
Published: October 2006

The specific binding of L‐N6‐[3H]phenylisopropyladenosine (L‐[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L‐[3H]PIA (0.5–12.0 nA/) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 ± 0.03 and 0.66 ± 0.05 pmol/mg of protein. In the presence of GTP, the binding of L‐[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 ± 0.09 and 0.93 ± 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L‐[3H]PIA binding by 1, 3‐dipropyl‐8‐cydopentylxanthine (DPCPX) (0.1–300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 ± 0.02 and 0.74 ± 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.
Neuronal Survival Factor from Bovine Brain Is Identical to Neuron‐Specific Enolase
Authors: Takei Nobuyuki , Kondo Jun , Nagaike Kazuhiro , Ohsawa Keiko , Kato Kanefusa , Kohsaka Shinichi
Published: October 2006

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high‐speed centrif‐ugation was used as the starting material. At the step of DE‐52 ion‐exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti‐F14 antiserum recognized mainly a 30‐kDa protein in F14 and anti‐F23 antiserum recognized mainly a 44‐kDa protein in F23. After sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of F23, the 44‐kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron‐specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti‐NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose‐dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.
Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain
Authors: Raisman‐Vozari Rita , Hirsch Etienne , Javoy‐Agid France , Vassort Cecile , Savasta Marc , Feuerstein Claude , Thibault Jean , Agid Yves
Published: October 2006

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S‐labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high‐sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer‐assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S‐labelled antibody, the detection of TH could be performed at the cellular level.
Amitriptyline‐Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity
Authors: Wong Ka L. , Bruch Richard C. , Farbman Albert I.
Published: October 2006

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3‐isobutyl‐l‐methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline‐mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin‐stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP‐dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline‐mediated inhibition of neurite outgrowth.
Resolution of Biphasic Binding of the Opioid Antagonist Naltrexone in Brain Membranes
Authors: Remraers Ann E. , Medzihradsky Fedor
Published: October 2006

In synaptosomal membranes from rat brain cortex, in the presence of 150 mM NaC1, the opioid antagonist [3H] naltrexone bound to two populations of receptor sites with affinities of 0.27 and 4.3 nM, respectively. Guanosine‐5′‐(3‐thiotriphosphate) had little modulating effect and did not alter the biphasic nature of ligand binding. On the other hand, receptor‐selective opioids differentially inhibited the two binding components of [3H] naltrexone. As shown by nonlinear least‐squares analysis, the μ opioids Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol or sufentanil abolished high‐affinity [3H] naltrexone binding, whereas the δ‐selective ligands [D‐ Pen2, D‐Pen5] enkephalin, ICI 174, 864, and oxymorphindole inhibited and eventually eliminated the low‐affinity component in a concentration‐dependent manner. These results indicate that, in contrast to the guanine nucleotide‐sensitive biphasic binding of opioid‐alkaloid agonists, the heterogeneity of naltrexone binding in brain membranes reflects ligand interaction with different opioid‐receptor types.
Glutamic Acid Decarboxylase Activity of the Preoptic Area and Hypothalamus Is Influenced by the Serotonergic System
Authors: Munaro N. I. , Morello H. , Taleisnik S.
Published: October 2006

The effect of the serotonergic system on glutamic acid decarboxylase (GAD) activity of the preoptic area and the hypothalamus was studied in female rats on the day of proestrus. A circadian rhythm of GAD activity was observed with higher values in rats killed at 1130 h than in rats killed at 1500 h. In rats bearing lesions of the median raphe nucleus (MRn), a nucleus that sends 5‐hydroxytryptamine nerve terminals to the areas under study decreased GAD activity. On the contrary, electrochemical stimulation of the MRn enhanced GAD activity in intact rats killed at 1500 h, but not in those killed at 1130 h, an effect that was prevented by the injection of the 5‐hydroxytryptamine antagonist, methysergide. Furthermore, the injection of 5‐hydroxytryptamine into the third ventricle, either in intact rats in the afternoon or in MRn‐lesioned rats in the morning, also increased GAD activity. The results of the present study suggest that activation of the serotonergic system increases GAD activity in the preoptic area and hypothalamus.
Vasoactive Intestinal Peptide Stimulates Catecholamine Biosynthesis in Isolated Adrenal Chromaffin Cells: Evidence for a Cyclic AMP‐Dependent Phosphorylation and Activation of Tyrosine Hydroxylase
Authors: Waymire J. C. , Craviso G. L. , Lichteig K. , Johnston J. P. , Baldwin C. , Zigmond R. E.
Published: October 2006

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel‐filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12‐O‐tetradecanoylphorbol 13‐acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP‐dependent phosphorylation of TH is responsible for these effects.
Changes in Extracellular Concentrations of Glutamate, Aspartate, Glycine, Dopamine, Serotonin, and Dopamine Metabolites After Transient Global Ischemia in the Rabbit Brain
Authors: Baker A. J. , Zornow M. H. , Scheller M. S. , Yaksh T. L. , Skilling S. R. , Smullin D. H. , Larson A. A. , Kuczenski R‐
Published: October 2006

Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmittiers (e.g., glycine and doparaine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1‐, 5‐, and 13‐fold and by 4‐, 9‐, and 31‐fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700‐fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4‐fold, but remained elevated throughout the 80‐min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann‐Whitney test). That glutamate and aspartate concentrations in the hippocampus co‐vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury. The observation that dopamine concentrations increased independently of ischemic duration indicates a maximal release with only S min of ischemia and suggests that dopamine's role in the incremental injury seen with increasing ischemic duration is limited to prolonged high concentrations rather than increasing peak levels as seen with glutamate and aspartate. The sustained elevation of glycine concentrations after ischemia may explain the ability of excitatory amino acids to produce delayed toxicity in the reperfusion phase, as glycine has been shown to facilitate glutamate's activity at the N‐ methyl‐D‐aspartate receptor.
Stimulation of γ‐[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur‐Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release
Authors: Dunlop John , Grieve Angus , Schousboe Arne , Griffiths Roger
Published: October 2006

In primary cultures of mouse cerebral cortex neurons, sulphur‐containing excitatory amino acids (SAAs; namely, L‐cysteine sulphinate, L‐cysteate, L‐homocysteine sulphinate, L‐homocysteate, S‐sulphocysteine) at concentrations ranging from 0.1 μM to 1 mM evoked a saturable release of ‐γ‐[3H] aminobutyric acid ([3H] GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50), 100–150 nM) in releasing [3H] GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H] tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA‐stimulated release of [3H] GABA was shown to comprise two distinct components, calcium‐dependent and calcium‐independent, which occur after activation of N‐methyl‐B‐aspartate (NMDA) and non‐NMDA receptors. Thus, all SAA‐evoked responses were antagonized by the selective, competitive NMDA‐receptor antagonist, 3‐[(±)‐2‐carboxypiperazin‐4‐yl] propyl‐1‐phosphonic acid (IC50range, >50 μM) and the non‐NMDA‐receptor antagonist, 6, 7‐dinitroquinoxalinedione (IC50 range, 5–50 nM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA‐evoked responses without having any effect on basal levels of [3H] GABA efflux, a result consistent with an involvement of NMDA‐receptor activation. Calcium‐independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 pMexcitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 fiMSKF‐89976‐A, a non‐ transportable inhibitor of theGABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are dearly consistent with a receptor‐mediated, depolarization‐induced reversal of the GABA carrier.
Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site
Authors: Lomneth Rich , Martin Thomas F. J. , DasGupta Bibhuti R.
Published: October 2006

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described “cell cracking” procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell “ghosts” which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca2+‐activated [3H] nuorepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150‐kDa NT and the 50‐kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca2+‐activated [3H] norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100‐kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by “cell cracking.” The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]‐ norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.
D1 and D2 Dopamine Receptors in Human Substantia Nigra: Localization and the Effect of Aging
Authors: De Keyser Jacques , De Backer Jean‐Paul , Vauquelin Georges , Ebinger Guy
Published: October 2006

Abstract: D1 and D2 receptor densities in human substantia nigra were examined by use of the specific binding of, respectively, [3H]SCH 23390 [R(+)‐7‐chloro‐8‐hydroxy‐3‐[3H]methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine] and [3H]spiperone. A unilateral loss of striato‐ and pallidonigral pathways by an infarction (n = 4) had no effect on the ipsilateral nigral D2 receptors, but reduced the ipsilateral nigral D1 receptors by 48–60% compared with the intact side. These data suggest that a substantial fraction of D1 receptors in human substantia nigra is located on terminals of striato‐and/or pallidonigral neurons, whereas D2 receptors are confined to intrinsic nigral cells. We also examined the effect of aging on the D1 and D2 receptors in substantia nigra obtained from 25 postmortem human brains (age range 19–88 years). The densities of both receptor types were not affected by the aging process. Since nigrostriatal dopaminergic neurons degenerate with aging, these results suggest either that the nigral D2 receptors are up‐regulated in response to a progressive depletion of dopamine in the substantia nigra or that, in contrast to the rat, they are not located on dopaminergic neurons.
Altered Protein Tyrosine Phosphorylation in Alzheimer's Disease
Authors: Shapiro I. P. , Masliah E. , Saitoh T.
Published: October 2006

Abstract: The activity of protein tyrosine kinases was determined in extracts from Alzheimer's disease brains and age‐and postmortem time‐matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p < 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine‐positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.
Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA‐N‐2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover
Authors: Sandmann J. , Wurtman R. J.
Published: October 2006

Abstract: We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA‐N‐2, by measuring the formation of 3H‐inositol phosphates (3H‐IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)‐mediated formation of 3H‐IP in a time‐ and dose‐dependent manner (EC50= 40‐55 μM). In addition, in the presence of ethanol (170‐300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three‐ to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50= 50 μM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA‐N‐2 cells with the phorbol ester phorbol 12‐myristate 13‐acetate (PMA, 0.1 μM; 10 min) increased [3H]PEt levels by up to 10‐fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 μM) or by pretreatment for 24 h with 0.1 μM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 μM). In summary, these results suggest that, in LA‐N‐2 neuroblastoma cells, mAChR are coupled both to phosphoinositide‐specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.
Fatty Acid Oxidation and Ketogenesis by Astrocytes in Primary Culture
Authors: Auestad Nancy , Korsak Rose A. , Morrow Jack W. , Edmond John
Published: October 2006

Abstract: The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D‐(–)‐3‐hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2‐day‐old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3–0.5 mM) was 50–90 nmol C2 units h−1 mg of protein−1. A similar rate was obtained using radiolabeled tracer methodology with [1‐14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1‐14C]‐ and [7‐14C]octanoate and [1‐14C]‐, [13‐14C]‐, and [15‐14C]palmitate indicated that a substantial proportion of the ω‐terminal fourcarbon unit of these fatty acids bypassed the β‐ketothiolase step of the β‐oxidation pathway and the 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1‐14C‐labeled fatty acids, obligated to pass through the acetyl‐CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (ω‐1)‐labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (ω‐3)‐labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3‐oxo‐acid‐CoA transferase (EC 2.8.3.5) or acetoacetyl‐CoA deacylase (EC 3.1.2.11) or both on acetoacetyl‐CoA and not by the action of the mitochondrial HMG‐CoA cycle involving HMG‐CoA lyase (EC 4.1.3.4), which was readily detected, and HMG‐CoA synthase (EC 4.1.3.5), which was barely measurable.
Characterization of the Release of Cholecystokinin‐8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters
Authors: Verhage Matthijs , Ghijsen Wim E. J. M. , Nicholls David G. , Wiegant Victor M.
Published: October 2006

Abstract: In the present study, the release of the neuropeptide cholecystokinin‐8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK‐like immunoreactivity (CCK‐LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2+‐dependent manner upon chemical depolarization, addition of 4‐aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK‐LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK‐LI is still present inside the synaptosomes. A correlation exists between the release of CCK‐LI and the elevations of [Ca]i. The release of CCK‐LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2+‐dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+. Furthermore, the relationship between the volume average [Ca]i (measured with fura‐2) and the extent of release is more or less linear for the release of CCK‐LI, whereas this relationship is clearly nonlinear for the release of endogenous glutamate and γ‐aminobutyric acid in similar preparations. We hypothesize that these differences between the neuropeptide CCK‐8 and classical transmitters are caused by three differences: (a) vesicle type; (b) Ca2+ sensitivity of the release mechanism; and (c) release site.
A Role for Acetylcholine‐Nicotinic Receptor Interactions in the Selective Increase of Rat Skeletal Muscle G4 Acetylcholinesterase Following Short‐Term Denervation
Authors: Hodges‐Savola Cheryl A. , Fernandez Hugo L.
Published: October 2006

Abstract: The present work addresses the effects of shortterm denervation on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in anterior gracilis muscles from adult male Sprague‐Dawley rats. It examines possible relationships between AChE isoform changes and other denervation phenomena, and evaluates the importance of acetylcholine (ACh)‐nicotinic receptor interactions in selectively modulating the activity of G4 AChE. Results confirm that denervation causes a specific, transient increase in G4 AChE and show that: most of the increment can be explained by the hydrophobic species of this isoenzyme; changes in AChE isoforms markedly precede the onset of spontaneous electromechanical activity (fibrillation), as well as acetylcholine receptor (AChR) proliferation; and the G4 AChE response is eliminated when AChRs are blocked by α‐bungarotoxin treatment performed before but not after (24 h) denervation. These data point to the absence of direct causal relationships between the G4 AChE increment and fibrillation, AChR proliferation, or changes in the release of this isoform from denervated muscle. In turn, they suggest the participation of AChR activation in triggering the G4 AChE response and emphasize the possible role of ACh‐AChR interactions in modulating the production of this isoenzyme in not only denervated but also innervated fast‐twitch muscles.
Structure‐Function Relationships of Endothelins, Sarafotoxins, and Their Receptor Subtypes
Authors: Sokolovsky Mordechai
Published: October 2006

Abstract: In brain synaptic membranes not extensively washed, (+)‐5‐[3H]methyl‐10,1 l‐dihydro‐5H‐dibenzo[a, d]‐cyclohepten‐5,10‐imine ([3H]MK‐801) binding was markedly inhibited in a concentration‐dependent manner (at concentrations above 1 μM) by several compounds having antagonistic activity at the Ca2+‐binding protein calmodulin. Scatchard analysis revealed that N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 μM. In membranes extensively washed and treated with a low concentration of Triton X‐100, L‐glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK‐801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W‐7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]‐MK‐801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W‐7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N‐methyl‐D‐aspartate‐sensitive subclass of excitatory amino acid receptors in rat brain.