bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium‐Dependent Evoked Release of N[3H]Acetylaspartylglutamate from the Optic Pathway
Authors: Tsai Guochuan , Forloni Gianluigi , Robinson Michael B. , Stauch Barbara L. , Coyle Joseph T.
Published: October 2006

Abstract: N‐Acetylaspartylglutamate (NAAG) is a neuropeptide localized to several putative glutamatergic neuronal systems, including the rodent optic pathway. To determine whether the peptide is released by depolarization, the superior colliculus of the rat was perfused with 2 μCi of [3H]NAAG, then with Krebs‐bicarbonate buffer for 1 h, using a microdialysis system. Subsequently, 10‐min fractions were collected and analyzed by HPLC for [3H]NAAG. Addition of 100 μM veratridine resulted in a several‐fold increase in the evoked release of [3H]NAAG that was virtually abolished by coperfusion with Ca2+‐free Krebs buffer containing 1 mM EGTA. When [3H]glutamate was used as the precursor, veratridine depolarization resulted in only an 80% increase in the release of [3H]NAAG. Prior enucleation of the right eye reduced the spontaneous release of [3H]NAAG by 50%, and the veratridine‐evoked release by greater than 85%, from the left superior colliculus. These results suggest that NAAG is released upon depolarization and may serve as a neurotransmitter/neuromodulator in the optic tract.
Regional Levels of Neurotransmitter Markers in the Pigeon Telencephalon: A Comparison with Possibly Homologous Areas of the Rat Telencephalon
Authors: Bissoli Rosanna , Battistini Sabrina , Guarnieri Tiziana , Contestabile Antonio
Published: October 2006

Abstract: The levels of cholinergic, γ‐aminobutyric acidergic (GABAergic), and excitatory amino acid neurotransmitter markers have been measured in 18 regions of the pigeon telencephalon as well as in supposedly homologous areas of the rat telencephalon. Among the basal telencephalic areas, some similar patterns of regional distribution were observed, with the noticeable exception of the ratio of levels of cholinergic markers between the striatum and globus pallidus, which was much larger in the rat than in the pigeon. In the rat cortical areas, some interesting differences were noticed among the archicortex, the paleocortex, and various parts of the neocortex. In particular, the area identified as prefrontal cortex by previous studies was significantly richer in cholinergic and excitatory amino acid markers and poorer in GABAergic activity than other neocortical regions. In the pigeon, presumedly neocortical equivalent areas—in particular, those constituting the dorsal ventricular ridge—were quite variable in levels of cholinergic markers, and some apparently well‐established areas homologous to mammalian neocortex showed exceptionally low levels of cholinergic markers. The higher variability in levels of neurotransmitter‐related markers shown by cortically equivalent areas of the avian dorsal ventricular ridge, as compared with the more uniform pattern present in basal telencephalic regions, may be the result of a greater plasticity of these structures during evolution, in response to different selective pressures.
Calcium‐Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs
Authors: Doležal Vladimír , Françoise Diebler Marie , Lazereg Sylvie , Israël Maurice , Tuček Stanislav
Published: October 2006

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+‐independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2‐(4‐phenylpiperidino)cyclohexanol (10 μM), ouabain (104 μM), atropine (10μM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+‐independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35‐fold at 203 mM K+). Carbamoylcholine (104 μM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 μM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1–3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin. Under the conditions used. the Ca2+‐dependent K+‐evoked release of ACh was not changed by 104 μM carbamoylcholine. It is apparent that the spontaneous Ca2+‐independent liberation of ACh from Torpedo synaptosomes is not a simple diffusion and is mediated by a carrier. The carrier is inhibited by muscarinic activation; the molecular mechanism of the inhibition is unlikely to involve Ca2+ ions or hyperpolarization. During depolarization, muscarinic inhibition of ACh liberation is ineffective, but carbamoylcholine blocks the Ca2+‐independent depolarization‐stimulated ACh output, probably by a direct action on the carriers.
Inability of [125I]Sar1, Ile8‐Angiotensin II to Move Between the Blood and Cerebrospinal Fluid Compartments
Authors: Harding J. W. , Sullivan M. J. , Hanesworth J. M. , Cushing L. L. , Wright J. W.
Published: October 2006

The angiotensin II competitive antagonist [125I]‐Sar1,Ile8‐angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1,Ile8‐angiotensin II lacked the capacity to move in the opposite direction over a 20‐min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood‐borne and cerebroventricular angiotensins access different receptor populations.
Chromosomal Location of the Mouse Gene that Encodes the Myelin‐Associated Glycoproteins
Authors: D'Eustachio Peter , Colman David R. , Salzer James L.
Published: October 2006

The two myelin‐associated glycoprotein (MAG) species, designated large MAG (L‐MAG) and small MAG (S‐MAG), are believed to be generated by differential splicing from a single RNA transcript. We have now defined the genetic locus encoding the two MAG proteins in the mouse. Analysis of a panel of interspecies somatic cell hybrids indicated that all MAG coding sequences reside on chromosome 7. Following the inheritance of a restriction fragment length polymorphism associated with MAG coding sequences allowed the locus to be positioned 0.5 centimorgans from the locus Abpa (androgen binding protein α) on proximal chromosome 7. These data strongly support the hypothesis that a single gene encodes the two MAG proteins, and we propose the name Mag for this locus. This localization places Mag in close proximity to the neurological mutant locus qv (quivering) and raises the possibility of a functional relationship or identity between Mag and qv. However, an analysis of the MAG gene, its RNA transcripts, and its protein products revealed no abnormalities in homozygous qv mutant mice, suggesting that this chromosomal linkage is not etiologically significant.
Muscarinic Acetylcholine Receptors from Avian Retina and Heart Undergo Different Patterns of Molecular Maturation
Authors: Cho Nam J. , Klein William L.
Published: October 2006

Abstract: Muscarinic acetylcholine receptors (mAChRs) from the avian CNS exist in two molecular weight forms whose concentrations change during development. Here, we have compared the development of mAChRs from embryonic hearts with those of the CNS. Analysis of [3H]‐propylbenzilylcholine mustard (PrBCM)‐labeled retina and heart mAChRs by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed two atropine‐sensitive peaks for each tissue. Apparent molecular masses of retina mAChRs, 86 ± 0.7 kilodaltons (kDa) and 72 ± 0.7 kDa, were different from those of heart mAChRs, 77 ± 1.0 kDa and 52 ± 0.9 kDa. During retina development, the major receptor type changed from 86 kDa to 72 kDa. No such change occurred during heart development. Furthermore, the 52‐kDa species appeared to be generated by endogenous proteolysis, as prolonged incubation of heart membranes at 37°C increased the amount of 52‐kDa peptide with a decrease of 77‐kDa peptide. Protease inhibitors blocked this conversion. Incubation of retina membranes at 37°C did not result in a conversion of the 86‐kDa peptide into the 72‐kDa peptide, but it did cause the appearance of a minor amount of 52‐kDa peptide. The proteolysis of retina mAChRs was not enhanced by cohomogenizing them with heart tissue, arguing against the presence of releasable proteases in heart. Membrane‐bound retina and heart mAChRs displayed similar sensitivity to exogenous (Staphylococcus aureus V8) protease, indicating that heart receptors were not unusually susceptible to proteolytic attack; analysis of the labeled polypeptides with the V8 protease showed different patterns of digestion for the retina and heart receptors. Chronic treatment of embryos with a muscarinic agonist reduced the concentration of receptors, but the Mr profile remained unaffected. The fact that muscle mAChRs, unlike neural mAChRs, do not change molecular forms during development may reflect different mechanisms of synaptogenesis in CNS and PNS.
Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons
Authors: Hofmann Hans‐Dieter
Published: October 2006

It has been demonstrated that cultured choliner‐gic retinal neurons from 8‐day‐old chicken embryos respond to a polypeptide factor present in retinal cell‐conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl‐CoAxholine O‐acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 ± 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sul‐fate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.
Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP‐Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina
Authors: Lombardini John B.
Published: October 2006

ATP‐dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 μM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP‐dependent and taurine‐stimulated ATP‐dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP‐dependent and taurine‐stimulated ATP‐dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP‐dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP‐dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine‐stimulated ATP‐dependent Ca2+ uptake in synaptosomal preparations.
Sequence of Two mRNAs Encoding Active Rat Tryptophan Hydroxylase
Authors: Darmon Michèle C. , Guibert Bernard , Leviel Vincent , Ehret Mireille , Maitre Michel , Mallet Jacques
Published: October 2006

Two full‐length cDNA clones that encode functional rat tryptophan hydroxylase (EC 1.14.16.4), the key enzyme in serotonin synthesis, have been isolated from a rat pineal gland library. These tv/o clones correspond to the 1.8 and 4‐kilobase mRNA species, respectively. They contain the same coding sequence corresponding to a 51,010 dalton protein and differ in the length of their 3’ untranslated regions.
Oxidative Metabolism of Nonsynaptic Mitochondria Isolated from Rat Brain Hippocampus: A Comparative Regional Study
Authors: Dagani F. , Marzatico F. , Curti D.
Published: October 2006

Abstract: Nonsynaptic mitochondria isolated from rat brain hippocampus were compared with those obtained by means of the same preparative procedure from cerebral cortex and striatum. Protein recovery, marker enzyme activities (lactate dehydrogenase, citrate synthase, and acid phosphatase), state 4 respiration, and response to hypoosmotic shock showed no difference among the three cerebral regions, suggesting homogeneous behavior during the subfractionation procedure. Cholinergic markers—choline acetyltransferase, acetylcholinesterase activities, and high‐affinity choline uptake—evaluated on synaptosomes showed the classic regional pattern with an enrichment in the striatum (striatum cortex > hippocampus). The coupling state of the mitochondrial fractions was maintained (respiratory control ratios ranging from 3.62 to 5.08 with glutamate + malate as oxidizable substrates), showing a metabolic competence sufficient to perform metabolic studies. Regional differences were found in state 3, uncoupled state of respiration, and cytochrome oxidase activity. Hippocampus showed the lower values (hippocampus < striatum < cortex). A possible role of this lower capacity of mitochondrial energy metabolism in determining the sensitivity of hippocampal neurons to ischemia or epileptic seizures is suggested.
Relationship Between Opioid‐Receptor Occupancy and Stimulation of Low‐Km GTPase in Brain Membranes
Authors: Clark Mary J. , Nordby Gordon L. , Medzihradsky Fedor
Published: October 2006

Abstract Treatment of rat brain membranes with the irreversible opioid ligand cis‐3‐methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the δ‐selective agonist [3H][d‐Ser2, Leu5lenkephalin‐Thr6 ([3H]DSLET). Subsequently, the correlation between ligand binding and low‐Km GTPase was investigated. Alkylation with 10 μM and 25 μM Superfit inactivated 66% and 71% of high‐affinity (KD, 1 nM) binding sites without decreasing the affinity of the remaining sites and the stimulation of low‐.Km GTPase by DSLET. Following exposure of the membranes to 50 μM and 75 μM Superfit, ligand binding was confined to the low‐affinity (KD, 20 nM) sites. In these membranes, the δ‐agonists DSLET and [d‐Pen2,D‐Pen5]enkephalin still stimulated low‐Km GTPase, and these effects were blocked by ICI 174864 (N,N‐ diallyl‐Tyr‐AIB‐AIB‐Phe‐Leu‐OH; AIB, α‐aminoisobutyric acid), a δ‐selective antagonist. A similar relationship between low‐affinity ligand binding and GTPase stimulation was observed following alkylation of the δ‐opioid receptor with the nonselective irreversible antagonist β‐chlomaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the δ‐opioid receptor to low‐Km GTPase in brain and identify low‐affinity ligand binding as a functional component in the process.
Metabolism of Angiotensin Peptides by Neuronal and Glial Cultures from Rat Brain
Authors: Hermann Klaus , Phillips M. Ian , Raizada Mohan K.
Published: October 2006

Abstract: The degradation pattern and rate of [Ile5]‐Angio‐tensin (Ang) I, II, and III were studied in neuron‐enriched and glia‐enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des‐Asp1‐[Ile5]‐Ang I, des‐Asp1‐[Ile5]‐Ang II, [Ile5]‐Ang II (3–8) hexapeptide, [Ile5]‐Ang II (4–8) pentapeptide, and [Ile5]‐Ang II (5–8) tetrapeptide. Glia‐enriched cells degraded [Ile5]‐Ang I and [Ile5]‐Ang III significantly faster than neuron‐enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]‐Ang II. Although the half‐lives of [Ile5]‐Ang I and [Ile5]‐Ang III in neuron‐enriched cells from normotensive Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron‐enriched cultures from WKY rats metabolized [Ile5]‐Ang II about 2.6 times faster than neuron‐enriched cells derived from SHR.
High‐Pressure Extraction of Membrane‐Associated Protein Kinase C from Rat Brain
Authors: Lester David S.
Published: October 2006

Abstract: Extraction of rat brain membrane‐associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain‐associated activity was extracted from membranes. The pressure‐extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure‐extracted activity. The enzyme is extracted at > 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane‐associated protein kinase C‐lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.
Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex
Authors: Becker Cord‐Michael , Hoch Werner , Betz Heinrich
Published: October 2006

Abstract: The inhibitory glycine receptor of mammalian spinal cord is a ligand‐gated chloride channel that, on affinity purification, contains two subunits of 48‐kilodalton (kD) and 58‐kD molecular mass in addition to an associated 93‐kD protein. Ligand‐binding 48‐kD subunit and 93‐kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit ≤ 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93‐kD protein was found to co‐distribute at a fixed stoichiometry with the 48‐kD subunit throughout the CNS of the rat. Moreover, the 93‐kD protein cofractionated with the ligand‐binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93‐kD polypeptide is selectively associated with the membrane core of the strychnine‐sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.
Ganglioside Modulation of Cyclic AMP‐Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro
Authors: Yates Allan J. , Walters John D. , Wood Catherine L. , Johnson David
Published: October 2006

Abstract: Purified cyclic AMP‐dependent protein kinase (cAK) catalytic subunit phosphorylated 180‐, 49‐, 31‐, 19‐, and 14‐kilodalton (kDa) proteins of rabbit sciatic nerve membranes. The ability of cAK to phosphorylate these membrane substrate proteins was inhibited by gangliosides GM1, GDIa, and GTlb with half‐maximal inhibitory concentration (I50) = 7–25 μM. Neutral glycolipids and lyso‐phosphatidylcholine were much less effective. Cyclic AMP (cAMP) kinase phosphorylation of histone IIA was inhibited by GM1, GDla, and GTlb (I50 = 115 μM, 75 μM, and 75 μM, respectively). Inhibition by GM1 was competitive with respect to histone (K‐, = 108 μM). Autophosphorylation of cAMP kinase was inhibited by GM1 (I50 = 15 μM). GTlb, GDla, and GM1 half‐maximally stimulated calmodulin‐de‐pendent cyclic nucleotide phosphodiesterase at 0.1 μM, 0.2 μM, and 0.3 μM, respectively. Although GTlb stimulated phosphodiesterase by increasing Vmax and decreasing Km (similar to calmodulin), GDla and GM1 produced only an increase in Vmax. These results suggest that ganglioside can modulate the activity of cAMP kinase by both direct inhibition of the enzyme and indirect reduction of cAMP levels through activation of phosphodiesterase. Through these mechanisms, gangliosides may alter cAMP‐dependent protein phosphorylation and cell function within the nervous system.
Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N‐Methyl‐D‐Aspartate Receptor Complex
Authors: Monihan Joseph B. , Corpus Valerie M. , Hood William F. , Thomas John W. , Compton Robert P.
Published: October 2006

Abstract: A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N‐methyl‐D‐aspartate receptor complex. Inculpation of SPM with [3H]glycine for 10 min at 2°C results in saturable, reversible binding with a KD of 0.234 μMand a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D‐serine (Ki= 0.27 μM), D‐alanine (Ki= 1.02 μM), and D‐cycloserine (Ki= 2.33 /μM) are patent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki= 25.4 μM, 15.9 μM, and > 100 μM, respectively). Inactive at concentrations of up to 100 μM were strychnine, L‐valine, N,N=‐dimethylglycine, ami‐nomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H] l‐[l‐(2‐thienyl)cyclohexyl]piperidine ([3H]TCP) binding to Triton X‐100‐washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate (3H]TCP binding.
Restoration of High Affinity Choline Uptake in the Hippocampal Formation Following Septal Cell Suspension Transplants in Rats with Fimbria‐Fornix Lesions
Authors: Kaseda Yumiko , Simon Jay R. , Low Walter C.
Published: October 2006

Abstract: High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria‐fornix lesions. Nine‐14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria‐fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus. HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and pic‐rotoxin. These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria‐fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system. The activation of HACU by picrotoxin, a γ‐aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus. Lesions of the fimbria‐fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus. This increase in binding was not significantly ameliorated by intrahippo‐campal grafts of cholinergic neurons.
Chronically Administered Lithium Alters Neither myo‐Inositol Monophosphatase Activity nor Phosphoinositide Levels in Rat Brain
Authors: Honchar Michael P. , Ackermann Karen E. , Sherman William R.
Published: October 2006

Abstract: Rats were exposed to either 29 consecutive days of LiCl injections or 27 and 39 days of dietary Li2C03, followed by injected LiCl at the end of the diet to insure a constant level of exposure to the drug. At the end of the period of chronic exposure to lithium, the rats were sacrificed and brain myo‐inositol‐1‐phosphate phosphohydrolase (myo‐inositol monophosphatase) activity was measured. In none of the experiments was there any difference in the lithium‐sensitive activity toward myo‐inositol monophosphatase when comparing the control and chronic groups. These brains and those from another group of rats that had been given Li2C03 in their diet for 41 days, followed by 7 additional days of LiCl injections, were also examined for changes in the levels of the phosphoinositides. No reproducible differences in the absolute tissue levels of those lipids were found when control and chronic lithium groups were compared. These results are contrary to published reports which suggest that myo‐inositol monophosphatase activity increases and that the phospha‐tidylinositol level decreases in rat brain as a result of chronic administration of lithium.
Effects of Phorbol Esters and Forskolin on Basal and Histamine‐induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells
Authors: Wan David C. C. , Bunn Stephen J. , Livett Bruce G.
Published: October 2006

Abstract: The effect of phorbol esters and forskolin pretreat‐ment on basal and histamine‐induced accumulation of inositol phosphates and catecholamine release was Examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose‐dependent, Ca2+‐dependent accumulation of total inositol phosphates with an EC50 at ∼ 1 μMandjan eight‐ to 10‐fold increase at 100 μM within 30 min of jincubation. Histamine (10 μM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20‐min period. Both the inositol phosphite and catecholamine responses were completely blocked by the Hrantagonist mepyramine and were insensitive tojthe H2‐antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphatesj stimulated by histamine revealed an early and sustained rise| in inositol 1,4‐bisphosphate content but not inositol 1,4,5‐trikphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor‐promoting phorbol ester phorbol 12‐myristate 13‐acetate (PMA) resulted in a dose‐dependent, time‐dependent inhibition of histamine‐induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC5o of ∼0.5 nM), an effect not shared by the inactive phorbol ester 4‐a‐phorbol 12,13‐didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine‐induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.
Slow Axonal Transport Impairment of Cytoskeletal Proteins in Streptozociti‐Induced Diabetic Neuropathy
Authors: Macioce P. , Filliatreau G. , Figliomeni B. , Hassig R. , Thiéry Jean , Giamberardin L. Di
Published: October 2006

Abstract: The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of strep‐tozocin‐diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.
Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long‐Term Culture
Authors: Méresse Stéphane , Dehouck Marie‐Pierre , Delorme Pierre , Bensaïd Mohamed , Tauber Jean‐Pierre , Delbart Christiane , Fruchart Jean‐Charles , Cecchelli Roméo
Published: October 2006

Abstract: The passage of substances across the blood‐brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20‐fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial‐specific characteristics (factor V11I‐related antigen, angiotensin‐converting enzyme, and nonthrombogenic surface) is shown for brain capillary‐derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood‐brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity
(+)‐PN200‐l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells
Authors: Castillo Carlos J. F. , Fonteríz Rosalba I. , López Manuela G. , Rosenheck Kurt , García Antonio G.
Published: October 2006

Abstract: Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10‐ 12‐fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)‐[3H]PN200‐1 10 binding to these membranes amounted to 90% of total binding and was saturabie and of high affinity (KD= 41 pM; Bmax= 119 fmol/mg of protein) with a Hill coefficient close to I, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 × 10′ M‐′ rnin‐l and 0.023 min‐I. Unlabeled (+)‐PN200‐110 displaced (+)‐[3H]PN200‐ 1 10 binding with a potency 100‐fold higher than (‐)‐PN200‐1 10 (ICsO, 0.5 and 45 nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)‐[3H]PN200‐1 10 binding, they exhibited no stereoselectivity (ICsO, 69 and 83 nM, respectively). Whereas (i‐)‐nitrendipine very potently displaced (+)‐[3H]PN200‐l 10 binding (ICsO = 1.3 nM), verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 FM, and diltiazem increased it by 20% at 10 pM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80‐fold higher than the Bmax for (+)‐PN200‐ 1 10. t3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/106 cells. This figure, together with the plasma membrane ouabain/(+)‐PNZOO‐ I I0 ratio of 80, allows the calculation of 1,884 (+)‐PN200‐110 binding sites/cell. Assuming a 1: 1 stoichiometry, this means that intact chromaffin cells contain ‐ 1.5 L‐type, dihydropyridine‐associated, voltage‐dependent Ca2+ channels/pm2 of surface area.
Postmortem Human Brain pH and Lactate in Sudden Infant Death Syndrome
Authors: Butterworth John , Tennant Mara C.
Published: October 2006

Abstract: Lactate and pH were measured in frontal and temporal cortex, cingulate gyrus, and caudate nucleus in brains from sudden infant death syndrome (SIDS) cases, control infants, and control adults. Both the lactate levels and the pH values were significantly correlated (p < 0.001) between the four brain areas, whereas lactate and pH values were significantly correlated within each brain area (p < 0.001) with a value of pH 7.2 for zero lactate. The lactate concentration in heart blood was significantly correlated with brain lactate (p < 0.001). Adult sudden death cases (heart attacks) had low lactate and high pH values, whereas agonal state cases had high lactate and low pH values. Control infants who had died because of accidents also had low lactate and high pH values, but infants who might have been exposed to hypoxia before death had high lactate and low pH values. SIDS cases fell into two groups: the first, consisting of all victims over 30 weeks of age and about one‐half to two‐thirds of those aged less than 30 weeks, had low lactate and high pH values; the second group, consisting of about one‐third to one‐half of those less than 30 weeks old, had high lactate and low pH values. The changes in lactate levels and pH values indicate that the majority of SIDS cases had died suddenly, but that a sizeable minority had been exposed to hypoxia prior to death
Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance
Authors: Brooks Keith J. , Porteous Roderick , Bachelard Herman S.
Published: October 2006

Abstract: Changes in high‐energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2‐deoxyglucose‐6‐phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (Po2= 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.
1‐Methyl‐4‐Phenylpyridinium Uptake by Human and Rat Striatal Synaptosomes
Authors: Willoughby J. , Cowburn R. F. , Hardy J. A. , Glover Vivette , Sandler M.
Published: October 2006

Abstract: 1‐Methyl‐4‐phenylpyridinium (MPP+) was taken up into human and rat striatal synaptosomes by a saturable system, similar to that for dopamine, with Km values of 0.24 and 0.17 μM, respectively, and similar Vmax values. Uptake of MPP+ and dopamine into both rat and human synaptosomes was inhibited by cocaine and amfonelic acid, with the latter being five to 10 times more potent than the former. MPP+ uptake was potently inhibited by dopamine in preparations from both species. In general, the characteristics of human and rat synaptosomal MPP+ uptake were very similar. It seems unlikely that species differences in toxicity to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine or reaction to dopamine uptake blockers stem from this system.
Sex‐Dependent and Sex‐Independent Distribution of the β‐Subunit of Nerve Growth Factor in the Central Nervous and Peripheral Tissues of Mice
Authors: Katoh‐Semba Ritsuko , Semba Reiji , Kashiwamata Shigeo , Kato Kanefusa
Published: October 2006

Abstract: Levels of the β‐subunit of nerve growth factor (β‐NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich‐type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. β‐NGF‐specific antibody isolated on a column of Sepharose CL‐4B coupled with purified β‐NGF reacted only with β‐NGF. The assay for β‐NGF was performed by incubation of F(ab′)2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab’ fragments labeled with β‐D‐galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of β‐NGF in almost all organs of mice. The amount of β‐NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3–5 mg/g of tissue). In serum, some of the 2‐month‐old males, but none of the females, exhibited a fairly high level of β‐NGF (> 100 pg/ml). The level of β‐NGF at 4 months of age was relatively high (2–5 ng/g) in the pancreas, spleen, and ovary among the organs examined, excluding the submandibular glands. An unexpected finding was that levels of β‐NGF not only in submandibular glands, but also in the brain, adrenal glands, and spinal cord, of males were three to seven times as high as levels in the analogous organs of females. Possible mechanisms regulating the level of β‐NGF in neural and paraneural tissues are discussed.
Neuroactive Sulphur Amino Acids Evoke a Calcium‐Dependent Transmitter Release from Cultured Neurones That Is Sensitive to Excitatory Amino Acid Receptor Antagonists
Authors: Dunlop John , Grieve Angus , Schousboe Arne , Griffiths Roger
Published: October 2006

Abstract: A dose‐dependent, saturable, and calcium‐dependent release of γ‐[3H]aminobutyrate ([3H]GABA) from cortical neurones and D‐[3H]aspartate from cerebellar granule cells following stimulation by a range of l‐enantiomers of neuroactive acidic sulphur amino acids has been demonstrated. Moreover, the sulphur amino acid‐evoked release of the transmitter amino acids was found to be sensitive to the presence of both selective N‐methyl‐D‐aspartate and quisqualate/kainate receptor antagonists. Following the recent demonstration of an endogenous location for several of the acidic sulphur amino acids and their excitotoxic involvement in several neuropathological states and coupled with the knowledge that many important CNS connections are still undefined as far as their excitatory transmitter or transmitters are concerned, the present findings are of immediate importance in the continued search for endogenous excitatory amino acid agonists in addition to glutamate and aspartate.
Effect of Guanosine 5′‐O‐(3‐Thiotriphosphate) and Calcium on γ‐Aminobutyric AcidB Binding as a Function of Postnatal Development
Authors: Al‐Dahan Muna I. , Thalmann Robert H.
Published: October 2006

Abstract: We have examined the development of γ‐aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of ∼50% with the GABAB ligand (–)‐[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium‐ and guanosine triphosphate‐binding protein (G protein)‐regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTPγS was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptor affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTPγS may affect the GABAB receptor by different mechanisms.
Translational Regulation of Myelin Protein Synthesis by Steroids
Authors: Verdi Joseph M. , Kampf Kathy , Campagnoni Anthony T.
Published: October 2006

Abstract: Various steroids, including glucocorticoids, were observed to exert a direct effect on the rates of translation of several myelinprotein specific transcripts in a cell‐free, reticulocyte lysate system. Hydrocortisone caused a twofold stimulation in the translation of mRNAs of myelin basic protein and proteolipid protein. It inhibited the translation of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase mRNA by 50%, and had no effect on the translation of a number of other mRNAs. The data suggest that steroid‐mediated translational regulation may serve as a novel mechanism to modulate the expression of myelin protein genes at the translational level.
β‐N‐Oxalylamino‐L‐Alanine Action on Glutamate Receptors
Authors: Ross Stephen M. , Roy Dwijendra N. , Spencer Peter S.
Published: October 2006

β‐N‐OxalyJamino‐L‐aJanine (L‐BOAA) is a nonprotein excitatory amino acid present in the seed of Lathyrus sativus L. This excitotoxin has been characterized as the causative agent of human neurolathyrism; an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue‐culture, and in vitro receptor binding investigations revealed that L‐BOAA might mediate acute neurotoxicity through quisqualate (QA)‐preferring glutamate receptors. The present study demonstrates the stereospecific action of L‐BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L‐BOAA was most active in displacing thiocyanate (KSCN)‐sensitive specific tritiated (RS)‐α‐3‐hy‐droxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) binding (i.e., QA receptor) (Ki= 0.76 μM) with a rank‐order potency of QA > kainate > N‐methyl‐D‐aspartate (NMDA). By contrast, the nonneurotoxic D‐BOAA isomer (100 μM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L‐BOAA. Scatchard analysis of L‐BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L‐BOAA, 265 nM) without a significant change in QA‐receptor density, and Hill plots yielded coefficients approaching unity. Differential L‐BOAA concentration‐dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. Comparative experiments for inhibition of specific [3H]‐AMPA binding in cortex revealed that L‐BOAA was approximately five‐ and 10‐fold less potent than QA and AMPA, respectively, but twofold greater than the endogenous neurotransmitter, glutamate. Parallel studies with KSCN‐treated spinal cord synaptic membranes (compared to cortex controls) indicated that specific [3H]AMPA binding in untreated tissue was (a) approximately 5% of cortex and pharmacologically distinct and (b) differentially sensitive to L‐BOAA and stimulation of binding by KSCN. These data suggest that low levels of L‐BOAA in vivo might initially exert excitotoxicity through sensitive cortex QA‐preferring neurons while eliciting little interference with QA sites in the spinal cord. Although the precise role(s) of the excitatory amino acid synapse in neurodegenerative diseases remains unclear, the cortex QA receptor could be preferentially sensitive to L‐BOAA attack and might represent the initial molecular recognition site responsible for the pathogenesis of this upper motor neuron disease.
Stathmin Is a Major Phosphoprotein and Cyclic AMP‐Dependent Protein Kinase Substrate in Mouse Brain Neurons but Not in Astrocytes in Culture: Regulation During Ontogenesis
Authors: Chneiweiss Hervé , Beretta Laura , Cordier Jocelyne , Boutterin Marie‐Claude , Glowinski Jacques , Sobel André
Published: October 2006

Stathmin is a ubiquitous soluble protein (Mr˜ 19,000; pI ˜ 6.2–5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. It is present in various tissues and cell types and has several nonphosphorylated and increasingly phosphorylated forms, and it is particularly abundant in brain. Very high concentrations of stathmin were also detected in mouse embryo striatal neurons grown in primary culture, whereas stathmin was barely detectable in astrocytes from the same source. Stathmin appeared in neurons as a major substjrate for protein phosphorylation and, in particular, for the cyclic AMP (cAMP)‐dependent protein kinase, because its phosphorylation was stimulated by cAMP in cell‐free preparations and in intact cells by forskolin, a potent activator of adenylate cyclase. During brain ontogenesis, stathmin was first detected at embryonic day 12; its concentration increased until birth and then decreased from postnatal day 10 to adulthood. In parallel, its molecular forms shifted from the least phosphorylated to the more phosphorylated ones. This result may reflect the evolution of the activity of stathmin during development and the subsequent maturation of the brain. In conclusion, our results substantiate the likely role of stathmin as an intracellular relay of extracellular regulations, as they point out its specific importance related to neuronal functions and brain differentiation.