bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Voltage‐Sensitive Calcium Channel Antagonists on the Release of 5‐Hydroxytryptamine from Rat Hippocampus In Vivo
Authors: Pullar Ian A. , Findlay Jeremy D.
Published: October 2006

Abstract: The effect of calcium channel antagonists on the release of 5‐hydroxytryptamine from the hippocampus of the chloral hydrate‐anaesthetised rat was studied using the technique of intracerebral microdialysis. As the basal concentration of 5‐hydroxytryptamine was close to the limit of detection of the HPLC method (8 fmol), the 5‐hydroxytryptamine reuptake inhibitor, fluoxetine (10 μM], was included in the perfusion fluid. The l‐type voltage‐sensitive calcium channel antagonists, PN200‐110, diltiazem, and verapamil, all passed through the dialysis membrane, giving a recovery of 20‐30%. The N‐type voltage‐sensitive calcium channel antagonist, ω‐conotoxin, penetrated less readily (12% recovery). The dihydropyridine, PN200‐110, adhered to the probe, resulting in an effective concentration at the membrane 30% of that in the perfusion fluid. The concentration of 5‐hydroxytryptamine in the dialysate samples was reduced by 60% in the absence of calcium. The L channel antagonists had little effect on the release of 5‐hydroxytryptamine, which was inhibited, in a dose‐dependent manner, to a maximum of 40% by w‐conotoxin. It is concluded that, under physiological conditions, the release of 5‐hydroxytryptamine from the rat hippocampus is dependent on the entry of calcium through N‐type voltage‐sensitive calcium channels, although another calcium channel may also be involved.
Characterization of Two Cerebellar Binding Sites of[3H]Ro 15‐4513
Authors: Uusi‐Oukari Mikko
Published: October 2006

Abstract: Ro 15‐4513 (ethyl‐8‐azido‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5‐a][1,4]benzodiazepine‐3‐carboxylate), a partial inverse agonist of central benzodiazepine receptors, binds to two distinct sites in the cerebellum. The binding to diazepam‐sensitive (DZ‐S) sites is displaced by different benzodiazepine receptor ligands, whereas the other site is insensitive to benzodiazepine agonists [diazepam‐insensitive (DZ‐IS)]. The binding of [3H]Ro 15‐4513 was studied in pig cerebellar membranes and in receptors solubilized and purified from these. Micromolar concentrations of γ‐aminobutyric acid (GABA) decreased DZ‐S binding at both 0 and 37°C, whereas it had no effect on DZ‐IS binding at 0°C and was stimulatory at 37°C. The pH profiles of [3H]Ro 15‐4513 binding were quite similar in both binding sites in the pH range of 5.5‐10.5 but differed at acidic pH values from those reported for flunitrazepam and Ro 15‐1788 (flumazenil; ethyl‐8‐fluoro‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5‐a][1,4]benzodiazepine‐3‐carboxylate) binding in DZ‐S sites, suggesting that [3H]Ro 15‐4513 does not interact with a histidine residue apparently present in the binding site. Zn2+, Cu2+, Co2+, and Ni2+ enhanced the binding to DZ‐S sites, and the first three mentioned also enhanced the binding to DZ‐IS sites. [3H]Ro 15‐4513 binding activity was solubilized by various detergents. All detergents tested were more efficient in solubilizing DZ‐S binding activity. High ionic strength improved especially the solubility of DZ‐IS binding activity. Cerebellar GABAA receptors were purified by benzodiazepine affinity chromatography using Ro 7‐1986 [1‐(2‐aminoethyl)‐7‐chloro ‐ 5 ‐ (o ‐ fluorophenyl) ‐1,3 ‐ dihydro ‐ 2H ‐1,4 ‐benzodiazepin‐2‐one] or 1012‐S as immobilized ligands. Only DZ‐S binding sites were purified by the procedure used. Because these two binding sites were differentially solubilized and they were not copurified, the results suggest a predominance of the binding sites in distinct isooligomeric receptors. Different coupling of the sites with GABA binding sites suggests that these two receptor subtypes are differently modulated by benzodiazepine receptor ligands.
Amino Acid Release from Cerebral Cortex in Experimental Acute Liver Failure, Studied by In Vivo Cerebral Cortex Microdialysis
Authors: Bosman D. K. , Deutz N. E. P. , Maas M. A. W. , van Eijk H. M. H. , Smit J. J. H. , de Haan J. G. , Chamuleau R. A. F. M.
Published: October 2006

Abstract: Both increased γ‐aminobutyric acid (GABA)‐ergic and decreased glutamatergic neurotransmission have been suggested relative to the pathophysiology of hepatic encephalopathy. This proposed disturbance in neurotransmitter balance, however, is based mainly on brain tissue analysis. Because the approach of whole tissue analysis is of limited value with regard to in vivo neurotransmission, we have studied the extracellular concentrations in the cerebral cortex of several neuroactive amino acids by application of the in vivo microdialysis technique. During acute hepatic encephalopathy induced in rats by complete liver ischemia, increased extracellular concentrations of the neuroactive amino acids glutamate, taurine, and glycine were observed, whereas extracellular concentrations of aspartate and GABA were unaltered and glutamine decreased. It is therefore suggested that hepatic encephalopathy is associated with glycine potentiated glutamate neurotoxicity rather than with a shortage of the neurotransmitter glutamate. In addition, increased extracellular concentration of taurine might contribute to the disturbed neurotransmitter balance. The observation of decreasing glutamine concentrations, after an initial increase, points to a possible astrocytic dysfunction involved in the pathophysiology of hepatic encephalopathy.
Lack of In Vitro Ceramide Formation in the PNS of Trembler Mice
Authors: Boiron‐Sargueil Françoise , Heape Anthony , Cassagne Claude
Published: October 2006

Abstract: The amidification of sphingosine by acyl donors has been investigated in a microsomal fraction prepared from sciatic nerves of normal and Trembler mice. In the control, a ceramide synthesis is observed in the presence of acyl‐CoAs and not with free fatty acids. The synthesis increases as a function of the protein amount and the time and is dependent on acyl‐CoA concentration. The level of synthesis is highly similar to that observed in vivo after palmitate injection into the sciatic nerves of normal mice. In the mutant, there is a major abnormality because a weak synthesis (20% of the control) is observed only with high acyl‐CoA concentration (>200 μM), whereas in the range of the physiological acyl‐CoA concentrations (<20 μM), there is no ceramide formation from stearoyl‐CoA or lignoceroyl‐CoA.
Maitotoxin‐Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine‐Sensitive and ω‐Conotoxin‐Sensitive Calcium Channels and Phosphoinositide Breakdown
Authors: Meucci Olimpia , Grimaldi Maurizio , Scorziello Antonella , Govoni Stefano , Bergamaschi Stefania , Yasumoto Takeshi , Schettini Gennaro
Published: October 2006

Abstract: The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage‐sensitive calcium channel blockers (nicardipine and ω‐conotoxin) on maitotoxin‐induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC 12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium‐free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose‐dependent manner. The maitotoxin‐induced calcium rise was also reduced by pretreating cells with w‐conotoxin. Pretreatment of cells with maitotoxin did not modify 125,I‐ω‐conotoxin and [3H]PN 200‐110 binding to PC 12 membranes. Nicardipine and ω‐conotoxin inhibition of maitotoxin‐evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC 12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or ω‐conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose‐dependent manner. This effect was reduced by pretreating the cells with 1 μM nicardipine and was completely abolished in a calcium‐free EGTA‐containing medium. The findings on maitotoxin‐induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage‐sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and ω‐conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that l‐and N‐type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin. and not that a depolarization of unknown nature causes the opening of calcium channels.
Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay
Authors: Paul Surojit , Das Sumantra , Sarkar Pranab Kumar
Published: October 2006

Abstract: An improved DNase I inhibition assay for the filamentous actin (F‐actin) and monomeric actin (G‐actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F‐actin/G‐actin ratio for at least 4‐5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F‐actin in the Triton‐soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton‐insoluble cytoskeletal F‐actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton‐insoluble cytoskeletal F‐actin in cultures from hypothyroid brain relative to normal controls. In the Triton‐soluble fraction, containing the G‐actin and the noncytoskeletal F‐actin, cultures from hypothyroid brain showed a 15% increase in G‐actin, whereas the F‐actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G‐actin. The mean F‐actin/G‐actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F‐actin relative to G‐actin.
K‐252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin‐3
Authors: Knüsel Beat , Kaplan David R. , Winslow John W. , Rosenthal Arnon , Burton Louis E. , Beck Klaus D. , Rabin Stuart , Nikolics Karoly , Hefti Franz
Published: October 2006

Abstract: K‐252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K‐252b was found to prevent trophic actions of two other neurotrophins, brain‐derived neurotrophic factor, and neurotrophin‐3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 μM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1‐100 nM, K‐252b selectively enhanced the trophic action of neurotrophin‐3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC 12 cells, K‐252b potentiated the neurotrophin‐3‐induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K‐252a, K‐252b, and staurosporine, only the first two also mediated neurotrophin‐3 potentiation. These findings indicate that K‐252b generally and selectively potentiates the neurotrophic action of neurotrophin‐3 and suggest that this action involves trk‐type neurotrophin receptors.
Diadenosine Tetraphosphate Is Co‐Released with ATP and Catecholamines from Bovine Adrenal Medulla
Authors: Castillo Carlos J. F. , Moro María A. , Valle Mercedes Del , Sillero Antonio , Garcia Antonio G. , Sillero Maria A. G.
Published: October 2006

Abstract: Secretion of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co‐release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1‐2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.
Analysis of Muscarinic Receptor Subtypes in the Mouse Cochlea by Means of the Polymerase Chain Reaction
Authors: Drescher Dennis G. , Upadhyay Sanjiv , Wilcox Edward , Fex Jörgen
Published: October 2006

Abstract: Total RNA was extracted with guanidine thiocyanate from the cochleas of 16‐day‐old CBAJ mice. The mRNA was purified from the total RNA using oligo‐dT cellulose, and the mRNA was treated with DNase to degrade genomic DNA. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR), using primers specific for the nucleotide sequences m1‐m5, representing subtypes of muscarinic acetylcholine receptors. PCR products corresponding to subtypes m1, m3, and m5, but not to m2 and m4, were amplified. These results suggest that muscarinic acetylcholine receptors of these odd‐numbered subtypes are expressed in the mammalian cochlea.
Brain Cytochrome Oxidase in Alzheimer's Disease
Authors: Kish Stephen J. , Bergeron Catherine , Rajput Ali , Dozic Slobodan , Mastrogiacomo Frank , Chang Li‐Jan , Wilson Julie M. , DiStefano Linda M. , Nobrega Jose N.
Published: October 2006

Abstract: A recent demonstration of markedly reduced (‐50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (‐26%; p < 0.01), temporal (‐17%; p < 0.05), and parietal (‐16%; not significant, p= 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region‐specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16‐26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy‐metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.
Cytokine Regulation of Neuronal Survival
Authors: Brenneman Douglas E. , Schultzberg Marianne , Bartfai Tamas , Gozes Illana
Published: October 2006

Abstract: Interleukin‐1 is a cytokine involved in the immune response to infection and inflammation as well as a growth promotor for several cell types. Interleukin‐1‐like immunoreactive material has been found in the nervous system. We now show that antisera, which blocked the T‐cell proliferative effects of interleukin‐1α, decreased neuronal cell counts (to 40% of control) in dissociated spinal cord cultures derived from fetal mice. This neuronal loss was prevented by addition of interleukin‐1α, and to a lesser extent by interleukin‐1β. Exogenous interleukin‐1α increased the survival of neurons when added to cultures in which the electrical activity was blocked with tetrodotoxin, whereas no such cytokine‐related increase in neuronal survival was observed in electrically active cultures. The antiserum‐induced death could also be prevented by cotreatment of the cultures with 0.1 nM vasoactive intestinal peptide, a substance that induces the secretion of neuronal trophic factors from nonneuronal spinal cord cells and thereby increases neuronal survival in electrically inactive cultures. These studies indicate that the cytokine interleukin‐1, or an immunologically cross‐reactive protein, can increase neuronal survival.
Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation
Authors: Johnson C. L. , Johnson C. G.
Published: October 2006

Abstract: UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin‐1 receptor ligand [3H][Sar9,Met(O2)11]‐substance P. Kinetic binding experiments conducted at 4°C yielded an association rate constant k1 of 1.86 X 107M–1 min–1, a dissociation rate constant k–1 of 0.00478 min–1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD= 447 ± 103 pM, Bmax= 862 ± 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]‐substance P yielded average values of KD= 491 ± 48 pM and Bmax= 912 ± 67 fmol/mg of protein. In [3H]inositol‐labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20‐fold within ∼15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60–120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin‐1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.
Heterogeneity in the Allosteric Interaction Between the γ‐Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System
Authors: Ruano Diego , Vizuete Marisa , Cano Josefina , Machado Alberto , Vitorica Javier
Published: October 2006

Abstract: In the present communication we have investigated the allosteric coupling between the γ‐aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high‐ and low‐affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very‐low‐affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high‐ and low‐affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very‐low‐affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very‐low‐affinity binding site for zolpidem.
Regulation of Aromatic l‐Amino Acid Decarboxylase by Dopamine Receptors in the Rat Brain
Authors: Zhu M. Y. , Juorio A. V. , Paterson I. A. , Boulton A. A.
Published: October 2006

Abstract: Decarboxylation of phenylalanine by aromatic l‐amino acid decarboxylase (AADC) is the rate‐limiting step in the synthesis of 2‐phenylethylamine (PE), a putative modulator of dopamine transmission. Because neuroleptics increase the rate of accumulation of striatal PE, these studies were performed to determine whether this effect may be mediated by a change in AADC activity. Administration of the D1 antagonist SCH 23390 at doses of 0.01–1 mg/kg significantly increased rat striatal AADC activity in an in vitro assay (by 16–33%). Pimozide, a D2‐receptor antagonist, when given at doses of 0.01–3 mg/kg, also increased AADC activity in the rat striatum (by 25–41%). In addition, pimozide at doses of 0.3 and 1 mg/kg increased AADC activity in the nucleus accumbens (by 33% and 45%) and at doses of 0.1, 0.3, and 1 mg/kg increased AADC activity in the olfactory tubercles (by 23%, 30%, and 28%, respectively). Analysis of the enzyme kinetics indicated that the Vmax increased with little change in the Km with l‐3,4‐dihydroxyphenylalanine as substrate. The AADC activity in the striatum showed a time‐dependent response after the administration of SCH 23390 and pimozide: the activity was increased within 30 min and the increases lasted 2–4 h. Inhibition of protein synthesis by cycloheximide (10 mg/kg, 0.5 h) had no effect on the striatal AADC activity or on the increases in striatal AADC activity produced by pimozide or SCH 23390. The results indicate that the increases in AADC activity induced by dopamine‐receptor blockers are not due to de novo synthesis of the enzyme. These results show that AADC activity in the striatum is regulated by D1 and D2 receptors and that the activities in the nucleus accumbens and olfactory tubercles are regulated by D2 receptors. The observation that dopamine‐receptor antagonists stimulate the synthesis of PE may be explained by the increase in AADC activity.
6‐Methylmercaptopurine Riboside Is a Potent and Selective Inhibitor of Nerve Growth Factor‐Activated Protein Kinase N
Authors: Volonté Cinzia , Greene Lloyd A.
Published: October 2006

Abstract: Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6‐thioguanine and 2‐aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a finding suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6‐methylmercaptopurine riboside (6‐MMPR) inhibits NGF‐stimulated PKN activity in vitro with an apparent Ki of ∼5 nM. This is ∼1,000‐fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6‐MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6‐MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF‐stimulated PKN, a PKN‐like activity stimulated in PC12 cells in response to activation of cyclic AMP‐dependent protein kinase was nearly insensitive to 6‐MMPR. Application of 6‐MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6‐MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.
Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ‐ and δ‐Opioid Peptides: A Microdialysis Study
Authors: Dourmap N. , Michael‐Titus A. , Costentin J.
Published: October 2006

Abstract: The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by μ‐ and δ‐opioid peptides. The effects of [d‐Pen2, d‐Pen5]‐enkephalin (DPDPE) and [d‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin (DAGO), two highly selective δ‐ and μ‐opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the δ agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that δ‐opioid receptors modulating the striatal DA release, in contrast to μ receptors, are not located on neurons that may be lesioned by kainic acid.
Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations
Authors: Siuciak Judith A. , Gamache Paul H. , Dubocovich Margarita L.
Published: October 2006

Abstract: Levels of norepinephrine, epinephrine, dopamine, and serotonin (5‐HT) and their precursors [tyrosine, l‐3,4‐dihydroxyphenylalanine, tryptophan, and 5‐hydroxytryptophan (5‐HTP)] and metabolites [3,4‐dihydroxyphenylacetic acid (DOPAC), 3‐methoxytyramine (3‐MT), homovanillic acid, 3‐methoxy‐4‐hydroxyphenylglycol, and 5‐hydroxyin‐doleacetic acid (5‐HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5‐HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5‐HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5‐HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5‐HTP, 5‐HT, and 5‐HIAA levels. Retinal levels of 3‐MT and DOPAC were significantly decreased at night.
Phorbol Esters Cause Preferential Secretion of Norepinephrine from Bovine Chromafin Cells
Authors: Cahill Anne L. , Perlman Robert L.
Published: October 2006

Abstract: Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13‐dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine‐containing cells or epinephrine‐containing cells. In response to 55 mM K+, catecholamine release from norepinephrine‐containing cells was two times greater than that from epinephrine‐containing cells. In response to phorbol 12,13‐dibutyrate, secretion from norepinephrine‐containing cells was 13 times greater than that from epinephrine‐containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine‐containing cells.
Ca2+ ‐Dependent Release from Rat Brain of Cannabinoid Receptor Binding Activity
Authors: Evans David M. , Johnson M. Ross , Howlett Allyn C.
Published: October 2006

Abstract: As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid‐like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 μM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to >60 nM to evoke release. The released cannabinoid‐like binding activity displaced [3H]CP‐55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration‐dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca2+‐dependent manner and that binds to the cannabinoid receptor.
Irreversible Inhibition of Mitochondrial Complex I by 1‐Methyl‐4‐Phenylpyridinium: Evidence for Free Radical Involvement
Authors: Cleeter M. W. J. , Cooper J. M. , Schapira A. H. V.
Published: October 2006

Abstract: Incubation of 10 mM I‐methyl‐4‐phenylpyridinium (MPP+) with sonicated beef heart mitochondria caused an irreversible time‐dependent decrease in NADH‐ubiquinone‐l (CoQ1) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH‐CoQ1 reductase activity. These results suggest that the interaction of MPP+ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin‐induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder.
L‐Fucose Is a Potent Inhibitor of myo‐Inositol Transport and Metabolism in Cultured Neuroblastoma Cells
Authors: Yorek Mark A. , Dunlap Joyce A. , Stefani Mark R. , Davidson Eric P.
Published: October 2006

Abstract: It has been proposed that abnormal myo‐inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo‐inositol metabolism and content. Recently, we have shown that L‐fucose, a 6‐deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo‐inositol transport. To examine the effect of L‐fucose on myo‐inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L‐fucose. L‐Fucose is a competitive inhibitor of Na+‐dependent, high‐affinity myo‐inositol transport. The Ki for inhibition of myo‐inositol transport by L‐fucose is about 3 mM. L‐Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L‐fucose is inhibited by Na+ depletion, D‐glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo‐inositol nor L‐glucose inhibits L‐fucose uptake. Chronic exposure of neuroblastoma cells to 1–30 mM L‐fucose causes a decrease in myo‐inositol accumulation and incorporation into inositol phospholipids, intracellular free myo‐inositol content, and phosphatidylinositol levels. Na+,K+‐ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L‐fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo‐inositol metabolism and Na+/K+‐pump activity are maintained when 250 μM myo‐inositol is added to the L‐fucose‐supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L‐fucose. The effect of L‐fucose on cultured neuroblastoma cell properties occurs at concentrations of L‐fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L‐fucose may have a role in myo‐inositol‐related defects in mammalian cells.
Transport and Metabolism of D‐[3H]Adenosine and L‐[3H]Adenosine in Rat Cerebral Cortical Synaptoneurosomes
Authors: Gu J. G. , Geiger J. D.
Published: October 2006

Abstract: The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D‐[3H]adenosine and L‐[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D‐[3H]adenosine, high‐ and low‐affinity accumulation processes were present. For the high‐affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D‐[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 μM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 μM erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine, an adenosine deaminase inhibitor, and 5′‐iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D‐[3H]adenosine to 6% with 5‐s and 9% with 15‐s incubations. This resulted in significantly higher KT values: 36 μM at 5 s and 44 μM at 5 s. At 10‐min incubations in the presence of these inhibitors, metabolism of accumulated D‐[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L‐[3H]adenosine, apparent KT and Vmax values for 20‐s incubations were 38.7 μM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L‐[3H]adenosine was observed only at incubations of >30 s. Taken together, these results demonstrate that adenosine transport is significantly faster than subsequent metabolism; that accumulated D‐adenosine is rapidly incorporated into and trapped intracellularly as adenine nucleotides, thereby affecting measured kinetic parameters for adenosine transport and giving an “appearance” of concentrative accumulations; and that the apparent KTT value of 39 μM for D‐adenosine transport conducted in the presence of the enzyme inhibitors was the same as the apparent KT value for L‐adenosine transport.
Drug Transfer Across the Blood‐Brain Barrier: Correlation Between In Vitro and In Vivo Models
Authors: Dehouck Marie‐Pierre , Jolliet‐Riant Pascale , Brée Françoise , Fruchart Jean‐Charles , Cecchelli Roméo , Tillement Jean‐Paul
Published: October 2006

Abstract: To assess the drug transport across the blood‐brain barrier (BBB), we compared the maximal brain extraction values at time 0 [E(0) values] obtained using either in vitro or in vivo methods. The in vitro BBB model consisted of a coculture of brain capillary endothelial cells growing on one side of a filter and astrocytes on the other. The in vivo model used intracarotid injection in anesthetized rats. Eleven compounds were tested. They were selected because they exhibit quantitatively different brain extraction rates: very low for inulin and sucrose, low for oxicam‐related nonsteroidal antiinflammatory drugs and diclofenac, and high for propranolol and diazepam. As these compounds are apparently transferred by a passive diffusion mechanism, two others, glucose and leucine, were added that cross the BBB by a known carrier‐mediated process. The in vivo and in vitro E(0) values showed a strong correlation as indicated by the Spearman's correlation coefficient (r= 0.88, p < 0.01). The relative ease with which such cocultures can be produced in large quantities could facilitate the screening of new centrally acting drugs.
Increase in Cytoplasmic Casein Kinase II‐Type Activity Accompanies Neurite Outgrowth After DNA Synthesis Inhibition in NIA‐103 Neuroblastoma Cells
Authors: Díaz‐Nido J. , Armas‐Portela R. , Avila J.
Published: October 2006

Abstract: Whereas cells from most clonal lines derived from the murine neuroblastoma C1300 tumor can be induced to differentiate by serum withdrawal from culture medium, the NIA‐103 clonal cell line has been considered unable to extend axon‐like processes (neurites). Neurite growth depends on microtubule protein assembly, and although NIA‐103 cells have essentially the same amounts of microtubule‐associated protein (MAP)‐1B and the neuronal‐specific class β3‐tubulin isoform as other neuroblastoma cell lines, these proteins are not phosphorylated in NIA‐103 cells on serum withdrawal. The lack of microtubule protein phosphorylation may be due to the different sorting between the nucleus and the cytoplasm of the casein kinase II‐related enzyme that is possibly involved in the modification of microtubule proteins. It is interesting that addition of DNA synthesis inhibitors to serum‐starved NIA‐103 cell cultures induces an increase in the level of cytosolic casein kinase II, an augmented in situ phosphorylation of MAP‐1B, and the extension of neurites. Thus, the level of cytoplasmic casein kinase II appears to be controlled by the growth status of neuroblastoma cells. The shift to an increased cytoplasmic concentration of casein kinase II in nonproliferating, differentiating neuroblastoma cells is consistent with its putative role in the regulation of the cytoskeletal rearrangements underlying neuronal morphogenesis and plasticity.
Adrenal Phenylethanolamine N‐Methyltransferase Induction in Relation to Glucocorticoid Receptor Dynamics: Evidence that Acute Exposure to High Cortisol Levels Is Sufficient to Induce the Enzyme
Authors: Betito Katia , Diorio Josie , Meaney Michael J. , Boksa Patricia
Published: October 2006

Abstract: Glucocorticoids (GCs) are thought to regulate, in a permissive fashion, the basal activity of adrenal medullary phenylethanolamine N‐methyltransferase (PNMT). However, it is unclear whether a large short‐term increase in GC release, such as occurs during an acute stress response, may also play a role in PNMT regulation. The present study investigated how the GC influence over PNMT activity varies in relation to dynamic changes in the hormone‐receptor signal. Using [3H]dexamethasone (DEX) and [3H]RU 28362 as radioligands, we have confirmed the presence of GC receptors in bovine adrenal medullary cells. A concentration‐dependent decline in soluble GC receptor sites and an increase in nuclear uptake of [3H]DEX were found in response to GC levels as low as 5 × 10−8M. The loss of soluble sites plateaued between 5 × 10−8 and 10−6M cortisol, with further losses occurring at 10−5 and at 10−4M. The functional consequence of GC receptor binding was confirmed by measuring PNMT activity following 3‐day exposure to cortisol. The pattern of PNMT induction was similar to that seen with GC receptor occupancy; at cortisol concentrations between 10−8 and 10−5M, PNMT induction was at a plateau, with a further increase in activity at 10−4M. The increase in PNMT activity following 3‐day exposure to low (10−7M) and high (5 × 10−5, 10−5M) cortisol was blocked by the GC receptor antagonist RU 38486, suggesting a GC receptor‐mediated event. Finally, a short (2 h) pulse of GC, which mimics the time course of physiological elevation of GC following acute stress, elevated adrenal medullary PNMT activity measured 3 days later. Therefore, our results provide novel evidence that short‐term exposure of adrenal medullary cells to high cortisol levels can elevate PNMT activity.
Role of Calmodulin and Protein Kinase C in Astrocytic Cell Volume Regulation
Authors: Bender Alex S. , Neary Joseph T. , Blicharska Janina , Norenberg Luz‐Oliva B. , Norenberg Michael D.
Published: October 2006

Abstract: We investigated the role of Ca2+‐dependent protein kinases in the regulation of astrocytic cell volume. Calmodulin (CaM) antagonists were used to inhibit CaM and thus Ca2+/CaM‐dependent protein kinase. The effect of these inhibitors as well as activators and inhibitors of protein kinase C (PKC) on astrocytic volume was measured in response to hypoosmotic stress and under isoosmotic conditions. In conditions of hypoosmolarity, CaM antagonists had no effect on swelling, but inhibited the regulatory volume decrease. PKC activation facilitated the swelling induced by hypoosmotic stress. PKC inhibitors induced cell shrinkage and inhibited the initial phase of regulatory volume decrease, whereas PKC down‐regulation caused pronounced swelling and partial inhibition of regulatory volume decrease. In isoosmotic conditions, CaM antagonists and PKC activation did not affect astrocytic volume, but PKC inhibitors caused shrinking and PKC down‐regulation led to swelling of these cells. These studies indicate the importance of Ca2+‐dependent protein kinases in the regulation of astrocytic cell volume.
Glutamate Metabolism in Rat Cortical Astrocyte Cultures
Authors: Farinelli Stephen E. , Nicklas William J.
Published: October 2006

Abstract: Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1‐14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a >70% decrease in 14CO2 production from [1‐14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.
Effect of Chronic Ethanol Treatment on the γ‐Aminobutyric Acid‐Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus
Authors: Negro Montserrat , Chinchetru Miguel A. , Fernández‐López Arsenio , Calvo Pedro
Published: October 2006

Abstract: The mechanism by which ethanol affects the γ‐aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the CI− influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of >10−6M for the various cortical and hippocampal areas studied from both control and ethanol‐treated animals; this enhancement peaked at 10−4M GABA but decreased at 10−3M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanoltreated animals.
Receptor‐Linked Hydrolysis of Phosphoinositides and Production of Prostacyclin in Cerebral Endothelial Cells
Authors: Xu Jian , Qu Zhi‐Xiang , Moore Steven A. , Hsu Chung Y. , Hogan Edward L.
Published: October 2006

Abstract: The receptor agonist‐mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H‐inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 μM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol‐induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE‐induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.