bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of Various Carbohydrate Moieties Including β‐Galactose and N‐Acetylglucosamine Residues on Solubilized Porcine Brain Neurokinin‐1/Substance P Receptors
Authors: Liu Ya Fang , Quirion Remi
Published: October 2006

Neurokinin‐1 (NK‐1)/substance P (SP) receptors were solubilized using 10 mM 3‐[(cholamidopropyl)‐dimethylammonio]‐1‐propanesulfate from porcine striatal membranes (solubilization yield, 80%). In solubilized preparations, [3H]SP apparently bound to a single class of high‐affinity sites (KD= 0.82 ± 0.13 nM) as in membrane ho‐mogenates. The ligand selectivity pattern observed in both membrane and solubilized receptor preparations indicated that [Sar9, Met(O2)11]SP = SP ± senktide = [Nle10]neurokinin A4–10.This suggests the selective labeling of the NK‐1 receptor class in both assays. Solubilized receptors were retained on agarose‐coupled lectins that bind N‐acetylglucosamine‐galactose and β‐galactose (Ricinus communis I and Ricinus communis II), mannose (concanavalin A and lentil), and N‐ acetylglucosamine (wheat germ agglutinin) but not on lectins binding fucose (Lotus A) and N‐acetylgalactosamine (Dolichos biflorus A). Thus, it appears that porcine brain NK‐1/SP receptors are enriched with various carbohydrate moieties, β‐galactose and N‐acetylglucosamine‐galactose residues being especially abundant. This situation is rather different from that in various other members of the rhodopsin seven‐trans‐membrane receptor superfamily.
Effect of Unilateral Perinatal Hypoxic‐Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High‐Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study
Authors: Kostic Vladimir , Przedborski Serge , Jackson‐Lewis Veraice , Cadet Jean Lud , Burke Robert E.
Published: October 2006

The binding characteristics and distribution of M1and M2 muscarinic cholinergic receptors and high‐affinity choline uptake sites were studied in the striatum of the rat at 3–4 and 9–12 weeks of age after exposure to unilateral perinatal hypoxic‐ischemic brain injury. High‐affinity choline uptake sites were labeled with [3H]hemicholinium‐3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AFDX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine‐labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium‐3‐labeled high‐affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF‐DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium‐3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that Striatal cholinergic systems are resistant to hypoxic‐ischemic injury.
Cyclic AMP Modulates Differentially the Release of Dopamine Induced by Hypoxia and Other Stimuli and Increases Dopamine Synthesis in the Rabbit Carotid Body
Authors: Pérez‐García M. T. , Almaraz L. , González C.
Published: October 2006

We have investigated the effects of different treatments that increase cyclic AMP levels on the in vitro synthesis and release of catecholamines in the rabbit carotid body. We also measured the rate of 45Ca2+ efflux from previously loaded carotid bodies under different conditions. Forskolin produced a dose‐dependent increase in the release of [3H]dopamine elicited by a hypoxic stimulus of medium intensity (Po2= 33 mm Hg) without altering basal [3H]dopamine release (100% O2‐equilibrated medium). At a concentration of 5 × 10‐‐6M, forskolin increased the release of [3H]dopamine induced by hypoxic stimuli of different intensities; the increase was maximal (498%) at the lowest intensity of hypoxic stimuli (Po2= 66 mm Hg), averaged 260% for hypoxic stimuli of intermediate intensity and 2 × 10‐‐4M cyanide, and was 150% under anoxia. Dibutyryl cyclic AMP (2 mM) and 3‐isobutyl‐1‐methylxanthine (0.5 mM) mimicked forskolin effects under hypoxic stimulation. Forskolin (5 × 10‐‐6M) also increased (180%) the release of [3H]dopamine induced by 20% CO2/pH 6.6, 2.5 × 10‐‐4M dinitrophenol, and 3 × 10‐‐5M ionomycin. Forskolin and 3‐isobutyl‐1‐methylxanthine were without effect on the release of [3H]dopamine elicited by 30 mM extracellular K+. Forskolin (5 × 10‐‐6M) augmented significantly the rate of 45Ca2+ efflux induced by hypoxic stimuli (Po2 of 33 and 66 mm Hg) and 2 × 10‐‐4M cyanide and showed a tendency to increase (20%) the 45Ca2+ efflux induced by dinitrophenol and low pH and to decrease (21%) the efflux induced by 30 mM K+ without altering the rate of efflux under basal conditions. Finally, forskolin (10‐‐5M) produced a 40% increase in the rate of [3H]dopamine synthesis from [3H]tyrosine. From the present and previously published observations, two physiological roles emerge for cyclic AMP in the carotid body: (a) The release of dopamine elicited by natural stimuli (low Po2 and low pH/high Pco2) is positively modulated by the associated increase in cAMP levels. In the case of the hypoxic stimulus, cyclic AMP may act on the O2‐sensing machinery and the exocytotic process, whereas in the case of the acidic stimuli, it may affect only the exocytotic process. (b) The activation of dopamine synthesis observed during hypoxic stimulation may be produced. at least in part, by the concomitant increase in level of cyclic AMP. via a cyclic AMP‐dependent phosphorylation of tyrosine hydroxylase.
GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3‐/GM2‐Synthase Activity Ratio Throughout Development
Authors: Daniotti J. L. , Lancia C. A. , Rösner H. , Maccioni H. J. F.
Published: October 2006

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a gangliosidc of the lactosylceramidc series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N‐acetylneuraminic acid (Neu NAc)] to adult (30% of total gangliosidic Neu NAc). GD3‐synthase specific activity increased about 1.5‐fold from birth to day 7 and essentially plateaued thereafter. The GD3‐/GM2‐synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GDI a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other “b” pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3‐/GM2‐synthase activity ratio throughout development of the tissue.
Endogenous ADP‐Ribosylation in Brain: Initial Characterization of Substrate Proteins
Authors: Duman Ronald S. , Terwilliger Rose Z. , Nestler Eric J.
Published: October 2006

Cholera and pertussis toxin‐mediated ADP‐ribo‐sylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP‐ribosylation of G proteins in brain. The present study demonstrates endogenous ADP‐ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and SO kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The 42‐ and 45‐kDa proteins comigrated precisely with the major cholera toxin‐labeled bands. Furthermore, the endogenous ADP‐ribosylated and cholera toxin‐ADP‐ribosylated bands yielded identical 32p‐labeled peptide fragments by one‐dimensional peptide mapping, indicating that they are probably the same proteins, presumably the α‐subunits of Gs. In contrast, peptide maps of the 50‐kDa protein, which migrated close to a 48‐kDa cholera toxin‐labeled band, demonstrated that this protein is distinct from the toxin‐labeled band and from Gsα. Levels of endogenous ADP‐ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP‐ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP‐ribosylation activity in brain have so far been unsuccessful with one exception. Sodium nitroprusside, which increases the formation of nitric oxide, dramatically stimulated the endogenous ADP‐ribosylation of a 36‐kDa protein, as reported previously by others, and, under certain conditions, also stimulated ADP‐ribosylation of the 42‐, 45‐, and 50‐kDa proteins, although to a lesser extent than the 36‐kDa protein. These findings demonstrate that GSα and additional, as yet unidentified, proteins are ADP‐ribosylated in brain in the absence of bacterial toxins and suggest that such endogenous covalent modification may regulate the functional activity of these proteins in vivo.
Intranigral Iron Injection Induces Behavioral and Biochemical “Parkinsonism” in Rats
Authors: Ben‐Shachar D. , Youdim M. B. H.
Published: October 2006

Elevated iron concentrations in the substantia nigra (SN) pars compacta have been implicated in the development of idiopathic Parkinson's disease. Because, as a transitional metal, iron promotes free radical formation, the role of iron in the degeneration of the nigrostriatal dopamine neurons in Parkinson's disease has received much attention. This study further investigates the cytotoxic effects of iron in the SN. Various concentrations of FeCl3 (1, 5, and 50 μg of Fe3+ in 5 μl) were unilaterally injected into the SN of adult rats. The two lower doses of iron had no effect on striatal dopamine levels or on the behavioral responses of the rats. However, injection of 50 μg of Fe3+ resulted in a substantial selective decrease of striaial dopamine (95%), 3,4‐dihydroxyphenylacetic acid (82%), and homo‐vanillic acid (45%), without any change in norepinephrine concentration. Dopamine‐related behavioral responses, such as spontaneous movements in a novel space and rearing, were significantly impaired, whereas amphetamine administration induced ipsilatcral rotation in the iron‐treated rats. The present study indicates that the nigrostriatal dopamine neurons are susceptible to the presence of ionic iron and thus supports the assumption that iron initiates dopaminergic neu‐rodegeneration in Parkinson's disease.
A One‐Step Procedure for Isolation of Poly(A)+ mRNA from Isolated Brain Capillaries and Endothelial Cells in Culture
Authors: Boado Ruben J. , Pardridge William M.
Published: October 2006

The study of the regulation of low‐abundance blood‐brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single‐step method for isolation of polyfA)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo‐de‐oxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was—15‐19 #g/g of brain or choroid plexus, 14‐17 μg per batch of isolated capillaries in a single bovine forebrain (190 g), and 6‐12 μg/107 ECL cells. Northern blot analysis showed characteristic and undegraded 2.1‐and 1.7‐kb actin transcripts in brain capillaries and a 2.1‐kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up‐regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two‐step method.
Heterogeneity of N‐Methyl‐D‐Aspartate Receptors Regulating the Release of Dopamine and Acetylcholine from Striatal Slices
Authors: Cai Ning‐sheng , Kiss Béla , Erdö Sándor L.
Published: October 2006

In an attempt to examine some functional characteristics of the N‐methyl‐D‐aspartate (NMDA) receptor complex, the NMDA‐evoked effluxes of endogenous dopamine (DA) and [3H]acetylcholine ([3H]ACh) were simultaneously examined in a rat Striatal slice preparation. NMDA induced release of both DA and ACh in a concentration‐dependent, Ca2+‐, Mg2+‐, and tetrodotoxin‐sensitive manner. These release responses were remarkably reduced by long‐term pre‐treatment with a low concentration of NMDA. an indication of the desensitization of the NMDA receptor. Glycine was potent in reversing the desensitization‐related reduction of DA release but failed to reverse the diminution of ACh release in the same slices. Our results indicate that the NMDA receptors regulating the release of DA and ACh are different with respect to their glycine modulatory site. This finding is consistent with a functional heterogeneity of the NMDA receptor complex in the rat striatum.
Calcium Effects on the Solubilization of Membrane‐Bound Histidine Decarboxylase in the Rat Brain
Authors: Toledo A. , Rodriguez R. , Sabriá J. , Rodriguez J. , Blanco I.
Published: October 2006

Abstract: In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane‐bound form. A major part (82%) of the membrane‐bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane‐containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of ∼50% of membrane‐bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.
Dopamine Synthesis Precedes Dopamine Uptake in Embryonic Rat Mesencephalic Neurons
Authors: Fiszman Mónica L. , Zuddas Alessandro , Masana Mónica I. , Barker Jeffery L. , di Porzio Umberto
Published: October 2006

Abstract: We have measured [3H]dopamine ([3H]DA) uptake and tyrosine hydroxylase‐immunopositive immunostaining in cells acutely dissociated from the embryonic ventral mesencephalon (MSC). DA and its metabolites as well as catechol‐O‐methyltransferase (COMT) and monoamine oxidase (MAO) activities were determined in homogenates taken from the MSC and striatum (STR). In the embryonic ventral MSC measurable DA and tyrosine hydroxylase (TH) immunostaining were present as early as embryonic day (E) 12.5. At E14 the number of TH+ neurons was about 50% of the values at E18. In the MSC, DA concentration increased sharply at E16 and reached a plateau before birth that was 10‐fold lower than adult values. In the STR, DA was first detected at E16, suggesting that DA fibers reach the STR at this embryonic stage. High‐affinity DA uptake appeared in the MSC only at E16, concomitantly with the arrival of DA fibers in the STR, increased sharply between E16 and E18, and reached a plateau before birth. This uptake mechanism was not selective for catecholamine uptake inhibitors. Thus, DA synthesis in the MSC preceded the onset of high‐affinity uptake mechanism, which could be correlated to the beginning of striatal DA innervation. Measurable MAO and COMT activities were detected as early as E13 (MSC) and E15 (STR), but not DA metabolites, which appeared later. We conclude that the high‐affinity DA uptake mechanism in MSC DA neurons develops coincident with the arrival of DA fibers to the STR. The sharp increase of DA uptake between E16 and E18 is due only in part to an increase in the number of TH+ cells. These results support the hypothesis that in vivo the target STR neurons regulate the maturation of MSC DA cells.
Effects of Peptides of the Secretin‐Glucagon Family and Cyclic Nucleotides on Tyrosine Hydroxylase Activity in Sympathetic Nerve Endings
Authors: Schwarzschild Michael A. , Zigmond Richard E.
Published: October 2006

Abstract: Previous studies have shown that certain peptides of the secretin‐glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8‐Bromoadenosine 3′,5′‐cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8‐Bromoguanosine 3′,5′‐cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3‐isobutyl‐1‐methylxanthine and/or Ro 20–1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13‐dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3′,5′‐cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.
Vitamin C Deficiency Facilitates 5‐S‐Cysteinyldopamine Formation in Guinea Pig Striatum
Authors: Fornstedt Bodil , Carlsson Arvid
Published: October 2006

Abstract: Being a catechol, dopamine (DA) is easily autoxidized in solution to a semiquinone and then further to a quinone. These quinones and by‐products, as reduced forms of oxygen, are all cytotoxic. By quantifying quinone metabolites, such as 5‐S‐cysteinyl adducts of DA, 3,4‐dihydroxy‐phenylalanine (DOPA), and 3,4‐dihydroxyphenylacetic acid (DOPAC), an indirect measure of catechol autoxidation is available. Ascorbic acid (AA) has an important role as an antioxidant in the organism. A group of guinea pigs (Dunkin‐Hartley) received an AA‐free diet for 37 days, whereas a control group was fed an AA‐containing diet (1,400 mg/kg of pellets). To one group of AA‐deprived animals a single dose of AA (500 mg/kg, i.p.) was administered 2 h before death, whereas another group received two doses 9 and 24 h before death. The striatal levels of 5‐S‐cysteinyl adducts, DA, noradrenaline, and DOPAC and the cerebellar and the limbic levels of AA were determined. A significant increase in 5‐S‐cysteinyl‐DA content was found in the striatum of AA‐deficient animals (143 ± 12% of control values). A further increase was found 2 h after an AA injection (177 ± 16% of control values), which was significant compared with both controls and AA‐deficient animals. An elevation in 5‐S‐cysteinyl‐DA content was still observed following two AA injections during a 24‐h period (153 ± 7% of control values). The 5‐S‐cysteinyl‐DOPAC content increased significantly (134 ± 14% of control values) in the AA‐deficient animals given AA acutely (2 h), both compared with controls and with the AA‐deficient group. DA levels increased nonsignificantly to 113% 2 h following an AA injection and significantly to 117% following two doses of AA within 24 h of AA injection. No other changes compared with normal animals were detected for any catechol examined. In guinea pigs given a scorbutogenic diet the AA levels decreased to 11–12% in the cerebellum and in the limbic system. Following a single dose of AA the brain levels increased to 19–23%, and in animals given AA 9 and 24 h before death AA levels reached 61–72% of control values in the cerebellum and in the limbic system. Thus, the present study indicates an increased autoxidation rate of DA in the striatum of AA‐depleted guinea pigs and a prooxidant acute effect of AA administration to these animals.
Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells
Authors: Horwitz Joel
Published: October 2006

Abstract: In PC12 pheochromocytoma cells whose phospholipids had been prelabeled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1–2 min, before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin‐stimulated cells; bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.
Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells
Authors: Mochizuki‐Oda Noriko , Mori Kensaku , Negishi Manabu , Ito Seiji
Published: October 2006

Abstract: We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2‐induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2‐induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole‐cell voltage‐clamp recordings, an increase in inward current was observed over a 6–7‐min period following bath application of PGE2 (± 10 μM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2+‐free medium, but it was not inhibited by Mn2+, a blocker of voltage‐dependent Ca2+ channels. In cell‐attached patch‐clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2‐induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside‐out patch‐clamp configuration, inositol 1,4,5‐trisphosphate (2 μM) added to the cytoplasmic side activated the Ca2+‐channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage‐independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5‐trisphosphate.
Brain Adenosine and Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of the Adenosine Deaminase Inhibitor Deoxycoformycin
Authors: Phillis J. W. , Walter G. A. , Simpson R. E.
Published: October 2006

Abstract: The effects of a potent adenosine deaminase inhibitor, deoxycoformycin, on purine and amino acid neuro‐transmitter release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four‐vessel occlusion. Purine and amino acid releases were compared from control ischemic animals and deoxycoformycin‐pretreated ischemic rats. Ischemia enhanced the release of glutamate, aspartate, and γ‐aminobutyric acid into cortical perfusates. The levels of adenosine, inosine, hypoxanthine, and xanthine in the same perfusates were also elevated during and following ischemia. Deoxycoformycin (500 μ/kg) enhanced ischemia‐evoked release of adenosine, indicating a marked rise in the adenosine content of the interstitial fluid of the cerebral cortex. Inosine, hypoxanthine, and xanthine levels were depressed by deoxycoformycin. Deoxycoformycin pretreatment failed to alter the pattern of amino acid neurotransmitter release from the cerebral cortex in comparison with that observed in control ischemic animals. The failure of deoxycoformycin to attenuate amino acid neurotransmitter release, even though it markedly enhanced adenosine levels in the extracellular space, implies that the amino acid release during ischemia occurs via an adenosine‐insensitive mechanism. Inhibition of excitotoxic amino acid release is unlikely to be responsible for the cerebroprotective actions of deoxycoformycin in the ischemic brain.
Evidence for Differing Origins of the Serotonergic Innervation of Major Cerebral Arteries and Small Pial Vessels in the Rat
Authors: Bonvento G. , Lacombe P. , MacKenzie E. T. , Fage D. , Benavides J. , Rouquier L. , Scatton B.
Published: October 2006

Abstract: We have studied the nature and origin of the serotonergic innervation of two distinct anatomical cerebrovascular compartments, namely, small pial vessels and major cerebral arteries, in the rat. To this end, the levels of serotonin [5‐hydroxytryptamine (5‐HT)] and 5‐hydroxyindoleacetic acid (5‐HIAA) were measured by HPLC in both cerebrovas‐cular compartments after either bilateral sympathectomy or destruction of the ascending serotonergic pathways, which originate from the raphe nuclei. We first showed that the small pial vessel samples were not contaminated by underlying cortical tissues through the use of an immunohistochemical approach that revealed the glia limitans, the most superficial cortical layer. Superior cervical ganglionectomy caused a marked decrease in noradrenaline concentrations in major cerebral arteries (−77%), although the reduction was less pronounced (−34%) in small pial vessels. Sympathectomy decreased by 33% 5‐HT concentrations in the major cerebral arteries but was without effect on 5‐HT levels in the small pial vessels. Destruction of the ascending serotonergic pathways (via local administration of 5,7‐dihydroxytryptamine into the ventral tegmental area) produced a dramatic fall in 5‐HT and 5‐HIAA concentrations in both vascular compartments. To establish the authenticity of the serotonergic innervation, the synthesis of 5‐HT [as assessed by measuring the accumulation of 5‐hydroxytryptophan (5‐HTP) after decarboxylase inhibition] was measured in the two vascular beds under control conditions and after destruction of the ascending serotonergic pathways. The rate of accumulation of 5‐HTP was higher in the small pial vessels than in major cerebral arteries, an observation that indicates an important de novo synthesis of 5‐HT in small pial vessels. The neurotoxic lesion of the ascending serotonergic fibers caused a marked reduction of 5‐HTP accumulation in small pial vessels (−67%) and a slight but significant decrease (−24%) in major cerebral arteries. These results collectively point to the existence of a true serotonergic innervation of small pial vessels that originates in the rostral raphe nuclei; in contrast, most of the 5‐HT present in major cerebral arteries seems to originate from sources other than these nuclei.
Cocaethylene: A Unique Cocaine Metabolite Displays High Affinity for the Dopamine Transporter
Authors: Hearn W. Lee , Flynn Donna D. , Hime George W. , Rose Stefan , Cofino Julio C. , Mantero‐Atienza Emilio , Wetli Charles V. , Mash Deborah C.
Published: October 2006

Abstract:Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.
Two Forms of the γ‐Aminobutyric Acid Synthetic Enzyme Glutamate Decarboxylase Have Distinct Intraneuronal Distributions and Cofactor Interactions
Authors: Kaufman Daniel L. , Houser Carolyn R. , Tobin Allan J.
Published: October 2006

Abstract: Glutamate decarboxylase (GAD) catalyzes the production of γ‐aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Ms of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo‐GAD65 and holo‐GAD65 in synaptic terminals may couple GABA production to neuronal activity.
Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes
Authors: Wang James K. T.
Published: October 2006

The wide‐ranging neuronal actions of glutamate arc thought to be mediated by postsynaptic N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non‐NMDA agonists in the presence of Mg2+, results in Ca2+‐depeodent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non‐NMDA agonists is blocked by a non‐NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non‐NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important rote in transmitter interactions in die striatum.
Tumor Necrosis Factor‐Induced Proliferation of Astrocytes from Mature Brain Is Associated with Down‐Regulation of Glial Fibrillary Acidic Protein mRNA
Authors: Selmaj Krzysztof , Shafit‐Zagardo Bridget , Aquino Dennis A. , Farooq Muhammad , Raine Cedric S. , Norton William T. , Brosnan Celia F.
Published: October 2006

Previous results from this laboratory have shown that tumor necrosis factor (TNF) is mitogenic for bovine astrocytes in chemically defined (CO) medium. The maximum mitogenic response was detected with 200 U/ml at 48 h. We have now extended these studies to assess the effect of TNF on message levels for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The results have shown that, whereas TNF had only a slight effect on vimentin mRNA, TNF induced a marked decrease to 4.3 ± 2.0% of controls in GFAP mRNA which was both time and dose dependent. The lowest effective dose was 50 U/ml and the maximal effective dose was 200 U/ml. Kinetic analysis of this response demonstrated that a marked decrease in GFAP mRNA was present at 12 h and continued to decrease through 72 h. To determine the reversibility of the TNF effect, astrocyte cultures were exposed to 200 U/ml TNF for varying periods of tee and then cultured in fresh CD medium. A 1‐h pulse with TNF was sufficient to reduce GFAP mRNA levels when measured 24 h later. However, cultures incubated with 200 U/ml TNF for 48 h followed by incubation in CD medium without TNF for 7 days showed that GFAP mRNA levels had returned to 60% of the control values. Nuclear runoff assays showed that the effect of TNF on GFAP mRNA was at the posttranscriptional level. Polyacrylamide gel electrophoretic analysis of astrocyte cytoskeletal proteins demonstrated that GFAP levels were reduced after a 5‐day incubation with 200 U/ml TNF whereas protein levels of vimentin and actin were not significantly changed. Western blots confirmed that GFAP levels were reduced to 36% of the control values. Thus the effect of TNF on GFAP mRNA expression was not due to a generalized effect on intermediate filament metabolism.
Developmental Expression of HNK‐1‐Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS
Authors: Chou Denise K. H. , Prasadarao Nemani , Koul Omanand , Jungalwala Firoze B.
Published: October 2006

Monoclonal antibody HNK‐1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high‐performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolac‐totetraosylceramide (SGGL‐1), from CNS was compared with those of SGGL‐1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL‐1 species were identical, SGGL‐1 from CNS contained mainly short‐chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL‐1 from PNS contained large proportions (59%) of long‐chain fatty acids (> 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK‐1‐reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of > 160 kDa were HNK‐1 reactive in the rat cerebral cortex at different ages. The major HNK‐1 reactivity to the 160‐kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK‐1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N‐CAMs) during development of the rat cortex with a polyclonal anti‐N‐CAM antibody showed that the major HNK‐1‐reactive protein bands were not N‐CAMs. Between PD 1 and 10, 190–200‐kDa N‐CAM was the major N‐CAM, and between PD 15 to adulthood, 180‐kDa N‐CAM was the only N‐CAM present in the rat cortex.
Activation of Excitatory Amino Acid Receptors Cannot Alone Account for Anoxia‐Induced Impairment of Protein Synthesis in Rat Hippocampal Slices
Authors: Carter Adrian J. , Müller R. Enzio
Published: October 2006

We have investigated the contribution of excitatory amino acid receptor activation to the inhibition of protein synthesis observed after anoxia in rat hippocampal slices. Protein synthesis was assessed in normoxic medium by measuring the incorporation of [14C]lysine into perchloric acid‐insoluble tissue extracts. Protein synthesis was impaired after anoxia; the extent of inhibition was dependent on the duration of anoxia and on the time allowed for postanoxic recovery. There was a similar impairment under normoxic conditions when the N‐methyl‐D‐aspartate (NMDA) receptor channel was activated by removing Mg2+ and adding NMDA. This was prevented by noncompetitive antagonists of the NMDA receptor channel (MK‐801, phencyclidine, and N‐allylnor‐metazocine). In contrast, incubation with the NMDA antagonists failed to prevent the protein synthesis inhibition caused by anoxia, although it moderately facilitated the postanoxic recovery. Protein synthesis was also impaired under normoxic conditions after incubation with quisqualate and kainate, agonists of non‐NMDA glutamate receptors. This impairment was prevented by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist of these receptors. Although 6‐cyano‐7‐nitroquin‐oxaline‐2,3‐dione alone failed to prevent anoxic damage, when used in combination with an NMDA antagonist it did partially enhance the later recovery of protein synthesis. These results indicate that the activation of excitatory amino acid receptors cannot alone account for anoxia‐induced impairment of protein synthesis in rat hippocampal slices.
Different Mechanisms of β‐Adrenoceptor Down‐Regulation by Chronic Imipramine and Electroconvulsive Treatment: Possible Role for Protein Kinase C
Authors: Nalepa Irena , Vetulani Jerzy
Published: October 2006

The aim of this study was to find out how protein kinase C (PKC) is involved in down‐regulation of the β‐adrenoceptor in cortical slices of rats subjected to antidepressant treatments. The responses of the cyclic AMP generating system to forskolin, isoproterenol, and noradrenaline were tested in the absence and presence of a PKC activator, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). The anti‐depressive treatments applied were chronic administration of imipramine and electroconvulsive shock. The potentiating effect of the phorbol ester on cyclic AMP response to isoproterenol was retained in imipramine‐treated animals and even accentuated in rats subjected to electroconvulsive treatment; the TPA effect on noradrenaline‐induced cyclic AMP response was blunted in rats receiving imipramine. but augmented in those receiving electroconvulsive treatment. In imipramine‐treated rats the β‐down‐regulation was still evident in the presence of TPA; after electroconvulsive treatment the phorbol ester‐induced potentiation was so high that no significant β‐downregulation could be observed. No procedure affected the response to forskolin. The β‐down‐regulation that develops during chronic imipramine treatment differs from that caused by chronic electroconvulsive treatment; in both cases it is not related to the direct effect on adenylate cyclase.
Kininogen and Kinin in Experimental Spinal Cord Injury
Authors: Xu Jian , Hsu Chung Y. , Junker Herman , Chao Steven , Hogan Edward L. , Chao Julie
Published: October 2006

Activation of the kallikrein‐kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time‐dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low‐molecular‐weight kininogen or T‐ kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T‐kinin, which were separated by HPLC and quantified with a kinin radioimmu‐ noassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40‐fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.
Prolonged Alterations in Canine Striatal Dopamine Metabolism Following Subtoxic Doses of l‐Methyl‐4‐Phenyl‐ 1,2,3,6‐Tetrahydropyridine (MPTP) and 4′‐Amino‐MPTP Are Linked to the Persistence of Pyridinium Metabolites
Authors: Johannessen An N. , Sobotka Thomas J. , Weise Virginia K. , Markey Sanford P.
Published: October 2006

Single toxic doses of l‐methyl‐4‐phenyl‐l,2,3,6‐ tetrahydropyridine (MPTP)‐HCI (2.5 mg/kg i.v.) and 4′‐ amino‐MPTP · 2HCI (22.5 mg/kg) induce loss of striatal dopamine (DA) and tyrosine hydroxylase (TH) activity and of nigral DA neurons in the dog. To examine the subacute neurochemical changes induced by low doses of MPTP and 4′‐ amino‐MPTP, dose‐response studies of these compounds were carried out in the dog, using 6‐ and 3‐week survival times for these two compounds, respectively. Low single doses of MPTP (1.0, 0.5, and 0.1 mg/kg i.v.) and 4′‐amino‐MPTP (15, 7.5, and 3.75 mg/kg i.v.) did not cause depletion of canine striatal DA or TH or a loss of nigral neurons. However, levels of the DA metabolites 3,4‐dihydroxyphenylacetic acid (DO‐ PAC) and homovanillic acid (HVA) were decreased in a doserelated fashion, with significant loss of DOPAC being evident 6 weeks after the lowest administered dose of MPTP and 3 weeks after 4′‐amino‐MPTP. This selective loss of DA metabolites following nontoxic doses of MPTP and 4′‐amino‐ MPTP led to a shift in the ratio of DA to DOPAC or HVA, which was characteristic for each compound. The measurement of striatal l‐methyl‐4‐phenylpyridinium (MPP+) and 4′‐amino‐MPP+ levels revealed that high concentrations (up to 150 μM) persist in the striatum for weeks following administration of a single nontoxic dose of MPTP or 4′‐amino‐ MPTP. A causal relationship between the striatal concentration of MPP+ or 4‐amino‐MPP+ and the change in DA metabolism as reflected in the DA/DOPAC ratio is suggested by a significant correlation between these measures. It is suggested that presynaptic sequestration and retention of MPP+ and 4′‐amino‐MPP+ by striatal DA terminals result in the inhibition of the monoamine oxidase contained within these terminals.
Nucleotide Sequence and Regulatory Studies of VGF, a Nervous System‐Specific mRNA That Is Rapidly and Relatively Selectively Induced by Nerve Growth Factor
Authors: Salton Stephen R. J.
Published: October 2006

A nervous system‐specific mRNA that is rapidly induced in PC 12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12‐myristate 13‐acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.
Recombinant Human and Rat Ciliary Neurotrophic Factors
Authors: Masiakowski Piotr , Liu Haoxing , Radziejewski Czeslaw , Lottspeich Friedrich , Oberthuer Walter , Wong Vivien , Lindsay Ronald M. , Furth Mark E. , Panayotatos Nikos
Published: October 2006

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.
Biological and Immunological Properties of Recombinant Human, Rat, and Chicken Nerve Growth Factors: A Comparative Study
Authors: Ibáñez Carlos F. , Hallböök Finn , Söderström Stine , Ebendal Ted , Perssdon Håkan
Published: October 2006

Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors (NGFs) were studied and compared. Recombinant NGF proteins were produced in a transient expression system using COS cells and levels of secreted NGF protein were assessed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of conditioned media from in vivo [35S]cysteine‐labeled cell cultures. Antigenic differences among the three NGFs were studied by immunoblotting and immunoprecipitation of secreted cell products using a rabbit polyclonal antiserum against purified mouse NGF, and by a two‐site enzyme im‐munoassay (EIA) with a monoclonal antibody against mouse NGF. Although all three NGFs were recognized equally well in the immunoblotting, only one‐third of the chicken NGF protein could be detected by immunoprecipitation or by the EIA as compared to the rat and human NGFs. Thus, changes in the three‐dimensional structure of the NGF molecule are most likely responsible for the antigenic differences between avian and mammalian NGFs. The three NGF proteins were also compared in their ability to displace 125I‐mouse NGF from low‐affinity NGF receptors on rat pheochromocytoma PC12 cells. Similar displacement curves and values were obtained for each NGF protein, indicating that structural differences among these molecules do not affect low‐affinity binding to NGF receptors. Biological activities were studied by the ability of the conditioned media to promote neurite outgrowth from explants of 9 chick sympathetic ganglia and from PC12 ceils. Although the rat system showed a slight preference for the homologous molecule, the morphological changes, dose‐response curves, and maximal stimulation values obtained with the different NGFs were practically indistinguishable in the chicken bioassay. The observed differences and similarities among the three NGF proteins are discussed in the context of their evolutionary relationship and their potential therapeutic applications.