bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐Term Survival of Intrastriatal Dopaminergic Grafts: Modulation of Acetylcholine Release by Graft‐Derived Dopamine
Authors: Jackisch Rolf , Duschek Markus , Neufang Brigitte , Rensing Hauke , Hertting Georg , Herman Jean‐Paul
Published: October 2006

Abstract: The nigrostriatal dopaminergic system of rats was unilaterally lesioned with 6‐hydroxydopamine. Part of the animals was grafted 2 weeks later with fetal dopaminergic cells on the lesioned side; untreated rats of the same strain served as controls. Both 3 and 12–14 months after surgery the striatal dopamine (DA) content and the in vivo rotational response following injection of d‐amphetamine showed significant changes in grafted as compared to lesioned animals. At 12–14 months after transplantation, the electrically evoked release of tritiated DA and acetylcholine (ACh) in slices (preincubated with [3H]DA or [3H]choline, respectively) of striata of intact, lesioned, or grafted animals was also investigated. Electrical field stimulation of striatal slices of the lesioned side did not evoke any significant [3H]DA overflow, whereas a marked [3H]DA release was observed in slices of grafted and control striata. Moreover, both DL‐amphetamine (3 μM) and nomifensine (10μM) strongly enhanced basal 3H outflow in these slices. Electrically evoked [3H]ACh release was significantly reduced in slices from all striatal tissues by 0.01 μM apomorphine. In slices from denervated striata a clearcut hypersensitivity for this action of apomorphine was present, indicating super sensitivity of DA receptors on cholinergic terminals; this hypersensitivity was significantly reduced in graft‐bearing striata. Furthermore, because this hypersensitivity was unchanged in slices of lesioned striata under stimulation conditions (four pulses/100 Hz) avoiding inhibition by endogenously released DA, it is concluded that lesion‐induced DA receptor super sensitivity is caused by an increase in receptor density or efficacy rather than by a decreased competition between endogenous and exogenous agonists. Both reuptake blockade of DA with nomifensine (10 μM) and release of endogenous DA by DL‐amphetamine (3 μM) potently reduced [3H]ACh release only in control and grafted but not in lesioned tissue. In experiments using potassium‐evoked [3H]ACh release, tetrodotoxin had no effect on the inhibitory activity of amphetamine and nomifensine, indicating that the DA receptors involved in their indirect inhibitory action are located directly on the cholinergic terminals.
Hypoxia Increases the Cyclic AMP Content of the Cat Carotid Body In Vitro
Authors: Delpiano M. A. , Acker H.
Published: October 2006

Abstract: The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 ± 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 ± 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 μM) nor by propranolol plus haloperidol (27 μM). Inhibition of the cyclic nucleotide phosphodiesterase with 1‐methyl‐3‐isobutylxanthine (0.8 μM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hyppxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N‐(cis‐2‐phenylcyclopentyl)azacyclotridecan‐2‐imine hydrochloride (MDL 12330A; 20–1,000 μM) under zero‐Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2+‐calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50–200 μM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the Po2‐sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone‐receptor interactions.
Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes
Authors: Poli A. , Lucchi R. , Vibio M. , Barnabei O.
Published: October 2006

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K+ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R‐phenylisopropyladenosine. All these effects were reversed by the selective adenosine A1 receptor antagonist 8‐cyclo‐pentyltheophylline. In the same synaptosomal preparation, K+ (30 mM) strongly stimulated the release of the preloaded [3H]adenosine in a partially Ca2+‐dependent and tetrodotoxin (TTX)‐sensitive manner. Moreover, in the same experimental conditions, both l‐glutamate and l‐aspartate enhanced the release of [3H]adenosine derivatives ([3H]ADD). The gluta‐mate‐evoked release was dose dependent and appeared to be Ca2+ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d‐glutamate and d‐aspartate were able to stimulate [3H]ADD release. In contrast, the specific glutamate agonists N‐methyl‐d‐aspartate, kainate, and quisqualate failed to stimulate [3H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg2+ and l‐glutamate‐evoked release was not inhibited by adding the specific antagonists 2‐amino‐5‐phosphonovaleric acid or 6–7‐dinitroquinoxaline‐2, 3‐dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ‐anunobutyric acid stimulated [3H]ADD release in a dose‐related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.
Rapid Isolation of Rat Brain Nuclei on Percoll Gradients
Authors: Vakakis Nicholas , Hearn Milton T. W. , Veitch Barry , Austin Lawrence
Published: October 2006

Abstract: A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000‐g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five‐step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five‐step Percoll gradients to yield subtractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in as‐trocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are ∼90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.
Glycine‐Like Modulation of N‐Methyl‐d‐Aspartate Receptors by a Monoclonal Antibody That Enhances Long‐Term Potentiation
Authors: Haring Rachel , Stanton Patric K. , Scheideler Mark A. , Moskal Joseph R.
Published: October 2006

Abstract: We have identified a monoclonal antibody, B6 B21, that significantly elevates long‐term potentiation when applied to CA1 pyramidal cell apical dendrites in rat hippocampal slices and characterized its binding to N‐methyl‐d‐aspartate‐receptor complexes using extensively washed hippocampal membranes. Five micrograms of affinity‐purified B6B21 per 100 μg of membranes gave a two‐to threefold elevation in N‐[1‐(2‐thienyl)cyclohexyl]‐3,4‐[3H]piperidine ([3H]TCP) binding. When [3H]TCP binding was stimulated by the combined addition of maximal concentrations of glutamate, glycine, and magnesium, B6B21 no longer stimulated [3H]TCP binding. Like glycine, B6B21 enhanced the effect of N‐methyl‐d‐aspartate and glutamate in stimulating [3H]TCP binding. Moreover, B6B21 reversed 7‐chlorokyn‐urenic acid inhibition of [3H]TCP binding, but it had no effect on the inhibition of [3H]TCP binding by d‐(−)‐2‐amino‐5‐phosphonovaleric acid. B6B21 increased the rate of association and dissociation of [3H]TCP, but had no effect on equilibrium binding. Glutamate, but not glycine, however, increased B6B21‐enhancement of [3H]TCP association and dissociation. B6B21 binding at strychnine‐insensitive glycine sites was confirmed by direct measurement of [3H]glycine binding. These results suggest that B6B21 binds directly to N‐methyl‐d‐aspartate receptors and displays properties similar to glycine.
Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1‐Opioid Receptors in Guinea Pig Hippocampus
Authors: Wagner John J. , Evans Christopher J. , Chavkin Charles
Published: October 2006

Abstract: Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a K1‐selective radioligand. Veratridine‐induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid pep‐tide‐containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous K ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1–8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin‐and proenkephalin‐derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of K1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that pro‐dynorphin‐derived opioids released under physiological conditions from the mossy fibers act at K1 receptors in the guinea pig dentate gyrus.
Rapid ATP Loss Caused by 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine in Mouse Brain
Authors: Chan Piu , DeLanney Louis E. , Irwin Ian , Langston J. William , Di Monte Donate
Published: October 2006

Abstract: The effects of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) on ATP levels in different areas of mouse brain were studied after rapid fixation of cerebral tissue in situ by microwave irradiation. ATP levels in the striatum, ventral mesencephalon, and cerebellum of untreated C57BL/6 mice killed by microwave irradiation were 2‐3 times greater than values measured in the brains of animals killed by cervical dislocation. In microwaved mice, administration of MPTP (40 mg/kg s.c.) caused a 10‐20% decrease in ATP concentrations as compared to control animals injected with saline. This decrease was relatively rapid and selective because it occurred in both the striatum and ventral mesencephalon, but not in the cerebellar and frontal cortex, at 30, 60, 120, and 240 min after MPTP exposure. Furthermore, ATP loss in the striatum was prevented by mazindol, a catecholamine uptake blocker, indicating a rather selective effect of MPTP on the ATP content of dopaminergic terminals. Results of this study are consistent with mitochondrial damage in the MPTP model of parkinsonism and provide the first direct experimental evidence in vivo that a decrease in ATP may play a role in MPTP‐induced neurotoxicity.
Sodium Fluoride Mimics the Effect of Prostaglandin E2 on Catecholamine Release from Bovine Adrenal Chromaffin Cells
Authors: Ito Seiji , Negishi Manabu , Mochizuki‐Oda Noriko , Yokohama Hiromitsu , Hayaishi Osamu
Published: October 2006

Abstract: We have reported recently that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+‐ATPase, synergistically induced a gradual secretion of catecholamines from the cells. Here we examined the involvement of a GTP‐binding protein(s) in PGE receptor‐induced responses by using NaF. In the presence of Ca2+ in the medium, NaF stimulated the formation of all three inositol phosphates, i.e., inositol monophosphate, bisphosphate, and trisphosphate, linearly over 30 min in a dose‐dependent manner (15–30 mM). This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. NaF also induced catecholamine release from chromaffin cells, and the dependency of stimulation of the release on NaF concentration was well correlated with those of NaF‐enhanced inositol phosphate formation and increase in cytosolic free Ca2+. Although the effect of NaF on PGE2‐induced catecholamine release in the presence of ouabain was additive at concentrations below 20 mM, there was no additive effect at 25 mM NaF. Furthermore, the time course of catecholamine release stimulated by 20 mM NaF in the presence of ouabain was quite similar to that by 1 μM PGE2, and both stimulations were markedly inhibited by amiloride, with half‐maximal inhibition at 10 μM. Pretreatment of the cells with pertussis toxin did not prevent, but rather enhanced, PGE2‐induced catecholamine release over the range of concentrations examined. These results demonstrate that NaF mimics the effect of PGE2 on catecholamine release from chromaffin cells and suggest that PGE2‐evoked catecholamine release may be mediated by the stimulation of phosphoinositide metabolism through a putative GTP‐binding protein insensitive to pertussis toxin.
Characterization of Two Neuroblastoma Cell Lines Expressing Recombinant Nerve Growth Factor Receptors
Authors: Reddy Usha Rani , Venkatakrishnan Gita , Roy Amit K. , Chen Jie , Hardy Mattie , Mavilio Fulvio , Rovera Giovanni , Pleasure David , Ross Alonzo H.
Published: October 2006

Abstract: In earlier studies, a 75,000‐dalton glycoprotein (gp75) has been identified as a component of both low‐ and high‐affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF‐induced up‐regulation of fos oncogene expression, and did not efficiently internalize NGF. LANS is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high‐affinity NGFRs but no detectable low‐affinity sites. The transformant line LAN5/NGFR expressed both high‐ and low‐affinity NGFRs, displayed NGF‐induced up‐regulation of fos oncogene, and efficiently internalized NGF. The number of high‐affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high‐affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high‐affinity NGFRs, the putative second factor may also limit NGF responsiveness.
Stimulation of Neurite Outgrowth in Neuroblastoma Cells by Neuraminidase: Putative Role of GM1 Ganglioside in Differentiation
Authors: Wu Gusheng , Ledeen Robert W.
Published: October 2006

Abstract: Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro‐2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neur‐aminidase‐stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti‐GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A‐103 line, previously characterized as “neurite minus,” did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.
Presynaptic Changes in Long‐Term Potentiation: Elevated Synaptosomal Calcium Concentration and Basal Phosphoinositide Turnover in Dentate Gyrus
Authors: Lynch M. A. , Voss K. L.
Published: October 2006

Abstract: In this report, two changes that occur in the presynaptic terminal following induction of long‐term potentiation in the dentate gyrus are examined, and the results demonstrate that the same changes are stimulated by the putative retrograde messenger arachidonic acid. First, there is an increase in the concentration of intracellular calcium in synaptosomes prepared from potentiated tissue compared with control tissue. This effect on intracellular calcium concentration was mimicked in control tissue by treatment of synaptosomes with either arachidonic acid or inositol 1,4,5‐trisphosphate in a dose‐dependent but nonadditive manner. Second, there is an increase in phosphoinositide turnover in synaptosomes prepared from potentiated tissue compared with control tissue, and this change can also be mimicked in control tissue by exposure of synaptosomes to arachidonic acid. These findings are consistent with the hypothesis that the increase in glutamate release associated with long‐term potentiation may be stimulated by arachidonic acid, as a result of an increase in intrasynaptosomal calcium concentration, perhaps occurring as a result of arachidonate‐stimulated phosphoinositide metabolism.
Thyroid Hormone Causes Sexually Distinct Neurochemical and Morphological Alterations in Rat Septal‐Diagonal Band Neurons
Authors: Westlind‐Danielsson Anita , Gould Elizabeth , McEwen Bruce S.
Published: October 2006

Abstract: Sex differences were investigated in cholinergic neurons of the septal‐diagonal band region of adult rats subjected to neonatal treatment with 3,3′,5‐triiodo‐L‐thyronine (T3). Neonatal hyperthyroidism resulted in a 44% increase in specific activity of choline acetyltransferase (ChAT; EC 2.3.1.6) in adult male rat septal‐diagonal band region, whereas no change in ChAT activity could be detected in either dorsal or ventral hippocampus. An increase in muscarinic cholinergic receptors, as measured by [3H]quinu‐clidinyl benzilate ([3H]QNB) binding, was discovered in both septum‐diagonal band and dorsal hippocampus of the T3‐treated male rats. Immunohistochemistry in the septal‐diagonal band region indicated a more intense staining in the neonatally T3‐treated adult male rats than in controls, with larger and more abundant ChAT‐positive and nerve growth factor receptor (NGF‐R)‐positive varicosities. ChAT immunocytochemistry showed a substantial decrease in cell body area in the medial septum and in the vertical limb of the diagonal band of T3‐treated male rats, while cell density increased twofold. Female littermates subjected to the same treatment showed no changes in any of the biochemical or immunohistochemical cholinergic markers. Only in the medial septum was morphology significantly altered in the female T3‐treated rats in that ChAT‐positive cell body area increased. These results indicate a marked sexual variation in the septal‐diagonal band region with respect to the sensitivity of postnatally developing cholinergic neurons to the actions of excess thyroid hormone.
Electroconvulsive Treatment Induces a Rapid and Transient Increase in Tyrosine Phosphorylatin of a 40‐Kilodalton Protein Associated with Microtubule‐Associated Protein 2 Kinase Activity
Authors: Stratton Kathleen R. , Worley Paul F. , Litz Julie S. , Parsons Sarah J. , Huganir Richard L. , Baraban Jay M.
Published: October 2006

Abstract: Recent studies have identified protein tyrosine phosphorylation as a major intracellular signaling pathway. However, little is known about regulation of this signaling pathway in neuronal systems. To help identify changes in levels of protein tyrosine phosphorylation in brain, we have utilized specific anti‐phosphotyrosine antibodies to detect phosphotyrosine‐containing proteins by immunoblotting techniques. We have found that electroconvulsive treatment induces a selective increase in tyrosine phosphorylation of a soluble 40‐kDa protein. The rise is rapid and transient, reaching maximal levels at 1–2 min and returning to basal levels by 8 min. The phosphotyrosine‐containing 40‐kDa protein is most prominent in hippocampus, smaller in neocortex, and not detected in brainstem or cerebellum. A phosphotyrosine‐containing 42‐kDa protein present in several cell types has recently been identified as a serine/threonine phosphotransferase, referred to as microtubule‐associated protein 2 kinase. Comparison of the levels of tyrosine phosphorylation of the 40‐kDa protein and microtubule‐associated protein 2 kinase activity during column chromatography of hippocampal extracts demonstrates that the phosphotyrosine‐containing 40‐kDa protein and microtubule‐associated protein 2 co‐purify. Moreover, the tyrosine phosphorylation of the 40‐kDa protein and microtubule‐associated protein 2 kinase activity are increased to a similar extent following electroconvulsive treatment. These findings suggest that the phosphotyrosine‐containing 40‐kDa protein identified in brain is closely related to microtubule‐associated protein 2 kinase.
Neurotensin Decreases the Affinity of Dopamine D2 Agonist Binding by a G Protein‐Independent Mechanism
Authors: von Euler Gabriel , van der Ploeg Ingeborg , Fredholm Bertil B. , Fuxe Kjell
Published: October 2006

Abstract: To examine whether GTP‐binding proteins (G proteins) mediate the ability of neurotensin to lower the affinity of dopamine D2 agonist binding, the modulation by neurotensin in vitro of N‐[3H]propylnorapotnorphine ([3H]NPA) binding was investigated following pretreatment with pertussis toxin and N‐ethylmaleimide in rat neostriatal membranes. Preincubation with N‐ethylmaleimide (100 μM) markedly inhibited pertussis toxin‐induced back‐ADP ribosylation of three proteins with apparent molecular masses of 41, 40, and 39 kDa, respectively. This inhibition was prevented by adding dithiothreitol (250 μM) during the preincubation. N‐Ethylmaleimide increased the KD (180 ± 30%) and decreased the Bmax (−31 ± 9%) of [3H]NPA binding sites but did not affect the binding properties of the selective D2 antagonist [3H]raclopride. N‐Ethylmaleimide pretreatment did not affect the neurotensin (3 nM)‐induced increase in the KD of [3H]NPA binding sites. Pertussin toxin treatment in vivo and in vitro was similarly ineffective. In conclusion, the present study indicates that neurotensin modulation of D2 agonist binding in neostriatal membranes is not mediated by G proteins.
Age‐Related Differences in Synaptosomal Peroxidative Damage and Membrane Properties
Authors: Viani Paola , Cervato Giovanna , Fiorilli Amelia , Cestaro Benvenuto
Published: October 2006

Abstract: Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+,K+‐ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age‐related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age‐dependent decrease in membrane fluidity, a reduction of Na+,K+‐ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K+‐ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to aging processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.
Recovery of Postischemic Brain Metabolism and Function Following Treatment with a Free Radical Scavenger and Platelet‐Activating Factor Antagonists
Authors: Gilboe D. D. , Kintner D. , Fitzpatric J. H. , Emoto S. E. , Esanu A. , Braquet P. G. , Bazan N. G.
Published: October 2006

Abstract: We have studied the metabolic and functional effects of two new platelet‐activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14‐min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high‐energy phosphates, carbohydrate content, and thiobarbituric acid‐reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14‐min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726‐treated ischemic brains than in DMSO‐treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO‐treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger. The PAF antagonists act at a different level, possibly restoring normal mitochondrial function or preventing some of the adverse effects of increased excitatory amino acid and/or intracellular Ca2+ levels. This is the only drug combination currently known to promote such complete short‐term metabolic and functional recovery during postischemic reoxygenation.
Subcellular Localization, Partial Purification, and Characterization of a Dynorphin Processing Endopeptidase from Bovine Pituitary
Authors: Devi Lakshmi , Gupta Prem , Fricker Lloyd D.
Published: October 2006

Abstract: An enzyme capable of cleaving dynorphin B‐29 to dynorphin B‐13 is present in bovine pituitary, with 40‐ to 50‐fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide‐containing secretory vesicles. The enzyme has been purified 2,800‐fold from whole bovine pituitaries using ion‐exchange and gel filtration chromatography. Purified dynorphin‐converting enzyme has a neutral pH optimum, and is substantially inhibited by the thiol‐protease inhibitor p‐chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B‐29 at Arg14, producing both dynorphin B‐14 and dynorphin B‐13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A‐17 at the single Arg cleavage site, generating both dynorphin A‐8 and A‐9 in a 7: 1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B‐29 and dynorphin A‐17, and possibly other peptides, at single Arg residues.
Quantitative Autoradiography Reveals Different Angiotensin II Receptor Subtypes in Selected Rat Brain Nuclei
Authors: Tsutsumi Keisuke , Saavedra Juan M.
Published: October 2006

Abstract: Heterogeneity of rat brain angiotensin II receptors was revealed by quantitative autoradiography after incubation with 125I‐Sar1‐angiotensin II and displacement with the angiotensin II antagonists CGP 42112 A and DuP‐753 and by receptor sensitivity to dithiothreitol. Receptors in areas involved in cardiovascular and fluid control—the subfornical organ, nucleus of the solitary tract, paraventricular nucleus, and area postrema—are displaced by DuP‐753 with an IC50 of 1 × 10−7M, are sensitive to 5 mM dithiothreitol, and thus are angiotensin II type‐1. Receptors in the inferior olive are displaced by CGP 42112 A (IC50, 1 × 10−9M) but not by DuP‐753 in concentrations up to 10−4M, are insensitive to 5 mM dithiothreitol, and thus are angiotensin II type‐2.
Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat
Authors: Sims Neil R.
Published: October 2006

Abstract: Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal‐lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19–45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal‐lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal‐lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal‐lateral striatum at each of the time points studied, but showed reductions (19–22%) during ischemia and after 24 h of recirculation in the paramedian neocortex. The results from this study provide evidence that reductions in the function of free mitochondria, apparently involving restriction of electron flow through complex I of the electron transport chain, develop selectively in an ischemia‐susceptible region at times coincident with initial histological evidence of neuronal damage.
Secretion of Catecholamines from Individual Adrenal Medullary Chromaffin Cells
Authors: Leszczyszyn David J. , Jankowski Jeffrey A. , Viveros O. Humberto , Diliberto Emanuel J. , Near Joseph A. , Wightman R. Mark
Published: October 2006

Abstract: Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon‐fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cationexchange polymer. Catecholamine secretion is induced by nicotine (10–500 μM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotineinduced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.
Alterations in Cortical [3H]Kainate and α‐[3H]Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy
Authors: Maddison Jill E. , Watson Wendy E. J. , Dodd Peter R. , Johnston Graham A. R.
Published: October 2006

Abstract: Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L‐[3H]Glutamate, (+)‐[3H]‐5‐methyl‐10, 11 ‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate ([3H]MK‐801), [3H]kainate, and α‐[3H]‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L‐[3H]‐glutamate or [3H]MK‐801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low‐affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non‐N‐methyl‐D‐aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.
Calcium Dependency of Muscarinic and Nicotinic Agonist‐Induced ATP and Catecholamine Secretion from Porcine Adrenal Chromaffin Cells
Authors: Xu Yanping , Duarte Emilia P. , Forsberg Erik J.
Published: October 2006

Abstract: The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine‐induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage‐dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration‐induced depolarization and presumably also intracellular Ca2+ mobilization.
Muscarinic Cholinergic Receptor‐Mediated Phosphoinositide Metabolism in Peripheral Nerve
Authors: Day Nancy S. , Berti‐Mattera Liliana N. , Eichberg Joseph
Published: October 2006

Abstract: Few receptor‐mediated phenomena have been detected in peripheral nerve. In this study, th'e ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo‐[3H]inositoI and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositoI trisphosphate ([3H]IP3) accounting for 7–8% and 1–2% of the total, respectively. In the presence of 100 γM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 γM carbamylcholine and stimulated IP generation was abolished by 1 γM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF‐DX 116 {11‐[[2‐[(diethylamino)methyl]‐1‐piperidinyl]acetyl]‐5,11 ‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one} (5.7), and 4‐diphenylacetoxy‐N‐methylpiperidinemethiodide (4‐DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin‐rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP‐ribosylated ∼40‐kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor‐mediated signal transduction via the phosphoinositide cycle.
Neuronal and Glial Marker Proteins in the Evaluation of the Protective Action of MK 801
Authors: Haglid K. G. , Wang S. , Hamberger A. , Lehmann A. , Moller C. J.
Published: October 2006

Abstract: A quantitative dot immunobinding procedure was used to quantify glial [the S‐100 protein and the glial fibrillary acidic (GFA) protein] and neuronal (the 68‐ and 200‐kDa neurofilament polypeptides, neuron‐specific enolase, and neuronal cell adhesion molecule) markers. A single intraperitoneal administration of 10 mg/kg of MK 801 blocked the increase of glial parameters and the decrease in content of neuronal marker proteins that occurred as the response to an N‐methyl‐D‐aspartate (NMDA) lesion in the rat hippocampus. The degradation products of GFA protein and the 68‐kDa neurofilament polypeptide that were induced by the NMDA lesion did not appear after MK 801 treatment. This study shows that brain‐specific proteins are a set of precise tools for the evaluation of neuroprotective effects of antagonists to excitatory amino acids.
Peptide Subunits of γ‐Aminobutyric AcidA/Benzodiazepine Receptors from Bovine Cerebral Cortex
Authors: Park Dongeun , de Blas Angel L.
Published: October 2006

Abstract: The γ‐aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single β subunit [57,000 Mr band in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE)] is composed of at least four different peptides of 54,000–57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the β subunit‐specific monoclonal antibody 62‐3G1. Peptides in the range of 54,000–57,000 Mr were photoaffinity‐labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity‐labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62‐3G1 nor photoaffinity‐labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS‐PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity‐labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity‐labeled by [3H]flunitrazepam did not react with antiserum A.
Lack of the Ganglioside Molecular Species Containing the C20‐Long‐Chain Bases in Human, Rat, Mouse, Rabbit, Cat, Dog, and Chicken Brains During Prenatal Life
Authors: Palestini Paola , Sonnino Sandro , Tettamanti Guido
Published: October 2006

Abstract: The long‐chain base composition of the total gangliosides from fetal brain of human, rat, mouse, rabbit, cat, dog, and chicken has been investigated during prenatal life. All the analyzed ganglioside mixtures contained the C18:1‐ and C18:0‐long‐chain bases, the latter covering 2–5% of the total long‐chain base content. The C20‐long‐chain bases, which are components of the total ganglioside mixture from adult animals, were absent.
Incorporation of Very‐Long‐Chain Fatty Acids into Sphingolipids of Cultured Neural Cells
Authors: Saito Mariko , Saito Mitsuo
Published: October 2006

We examined effects of exogenous very‐long‐chain fatty acids on lipids of cultured chick neurons and astrocytes. When chick neurons were incubated in chemically defined medium containing 10 μMnervonic acid (C24:1) for 7 days, it was found that a major fatty acid moiety of gangliosides and sphingomyelin was nervonic acid itself, which was not normally detected in the sphingolipid fraction. This alteration in the fatty acid composition apparently occurred in each ganglioside species. Under these experimental conditions, nervonic acid was not found in the glycerophospholipid fraction, and the amounts of triacylglycerol and free nervonic acid increased. Addition of behenic acid (C22:0) or erucic acid (C22:l) also induced changes in the fatty acid composition of gangliosides. When chick astrocytes were incubated in the presence of 10 μM nervonic acid for 7 days, no significant change was observed in the fatty acid composition of gangliosides. These studies indicate that the manipulation of the fatty acid moiety of sphingolipids in cultured neurons is possible.
Epidermal Growth Factor Enhances Striatal Dopaminergic Parameters in the 1‐Methyl‐4‐Phenyl‐l, 2, 3, 6‐Tetrahydropyridine‐Treated Mouse
Authors: Hadjiconstantinou Maria , Fitkin James G. , Dalia Asad , Neff Norton H.
Published: October 2006

Intracerebroventricular infusion of epidermal growth factor (EOF) into mice with 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP)‐induced degeneration of dopaminergic nigrostriatal neurons partially enhanced the content of dopamine (DA) and 3, 4‐dihydroxyphenylacetic acid as well as the activity of tyrosine hydroxylase in the striatum. EGF also enhanced these parameters in control, unlesioned animals. Neurotrophic activity also was observed in embryonic mesencephalic cultures, where EGF enhanced DA uptake after a lesion with the neurotoxic metabolite of MPTP, 1‐methyl‐4‐phenylpyridinium ion. Our in vivo and in vitro studies suggest that EGF may be a neurotrophic factor for dopaminergic neurons, or may act indirectly by inducing the release of a dopaminergic trophic factor from other cells.
Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor‐β
Authors: Kaplan Ruth , Zaheer Asgar , Jaye Michael , Lim Ramon
Published: October 2006

Glia maturation factor‐β, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor‐β, we screened a human brainstem cDNA library in δll. A 0.7‐kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor‐β, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.
Transport of Asparagine by Rat Brain Synaptosomes: An Approach to Evaluate Glutamine Accumulation
Authors: Erecińska Maria , Zaleska Malgorzata M. , Chiu Linda , Nelson David
Published: October 2006

Isolated rat brain synaptosomes accumulated L‐asparagine with a Km value of 348 μM and a Kmax value of 3.7 nmol/mg of protein/min at 28°C. Uptake of L‐asparagine was inhibited by the presence of L‐glutamine, whereas transport of L‐glutamine was blocked by L‐asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas α‐(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L‐asparagine had a substantial sodium‐dependent component, whereas that of the D‐stereoisomer was almost unaffected by the presence or absence of the cation. L‐Asparaginc was accumulated to a maximal gradient, [L‐Asn]i/[L‐Asn]o, of 20–30, and this value was reduced to 5–6 by withdrawal of sodium or addition of high [KG]. A plot of log [Na+]o/{Na+]i against the log [L‐Asn]i/[L‐Asn]o had a slope close to 1, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L‐asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L‐asparagine and L‐glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [GIn]i maintained by the brain in vivo.
Activation and Inactivation of Oxytocin and Vasopressin Release from Isolated Nerve Endings (Neurosecretosomes) of the Rat Neurohypophysis
Authors: Payza Kemal , Russell James T.
Published: October 2006

Neurosecretory terminals (neurosecretosomes, NSS) were isolated from rat neurohypophyses. High [K+]oor veratridine stimulated secretion of vasopressin and oxytocin by up to ∼ 100‐fold. Stimulated secretion was dependent on calcium and temperature, and could be elicited from NSS maintained in culture for 4 days. After overnight culture of the NSS, secretion was still inhibited by calcium channel blockers (cobalt, dihydropyridines, ω‐conotoxin, D 600) and K opiates (dynorphin and U50488). Ionomycin evoked dose and calcium‐dependent hormone release, with a Hill coefficient for calcium of 1.74. High [K+]o enhanced the 5 μMionomycin‐induced secretion, apparently through calcium entry rather than depolarization, as the increase in secretion was abolished by 100 μM D 600. During prolonged depolarization the hormone secretion peaked within 2 min, then declined to near basal levels. Depolarization for 25 min without calcium neither activated secretion nor prevented subsequent secretion on readdition of calcium, suggesting that the decline in secretion was not due to membrane depolarization. Indeed, the rates of decline in secretion were similar for different levels of depolarization (0.070 ± 0.003 and 0.081 ± 0.003 min−1 for 25 and 45 mM [K+]o, respectively). Four minutes after the onset of continuous depolarization (45 mM[K+]o) in the presence of calcium, the declining secretion was still dependent on voltage‐activated calcium influx through channels sensitive to D 600 and nitrendipine. The results presented here suggest that the decline in secretion during prolonged depolarizing stimuli may be due to exhaustion, inactivation, or desensitization of a calcium‐triggered event.