bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine Receptors in Myelin Fractions and Subtractions: The Effect of the Agonist (R)‐Phenylisopropyladenosine on Myelin Membrane Microviscosity
Authors: Casadó Vincent , Mallol Josefa , Lluis Carmen , Franco Rafael , Canela Enric I.
Published: October 2006

: In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)‐[3H]phenylisopropyladenosme ([3H]R‐PIA) binding to purified myelin fractions was performed. The distribution of high‐and low‐affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R‐PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.
Internal Ca2+ Mobilization by Muscarinic Stimulation Increases Secretion from Adrenal Chromaffin Cells Only in the Presence of Ca2+ Influx
Authors: Yamagami Kazuhiko , Nishimura Shigeto , Sorimachi Masaru
Published: October 2006

: The cytosolic free Ca2+ concentration ([Ca2+]m) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]m and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]m by activating both Ca2+ influx and intracellular Ca2+mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]m by mobilizing intracellular Ca2+but did not cause CA secretion. The MCh‐induced rise in [Ca2+]m in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]m elevated above basal values was maintained by a continuous Ca2+influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]m in a particular region of the cell.
Histamine: A Neurotransmitter Candidate for Drosophila Photoreceptors
Authors: Sarthy P. Vijay
Published: October 2006

: Recent experimental evidence suggests that histamine might be the synaptic transmitter used by invertebrate photoreceptors. In the present study, we have examined whether histamine is a transmitter candidate for Drosophila photoreceptors. Our findings are as follows; (a) Large amounts of histamine are synthesized by wild‐type heads, whereas heads from the eye‐deficient mutants, eyes absent and sine oculis, show reduced histamine synthesis, (b) Histidine de‐carboxylase activity is ⋍ 10‐fold higher in extracts of normal heads compared with that ia the mutants, (c) Histamine taken up by fly heads is metabolized into N‐acetylhistamine and imidazole‐4‐acetic acid, (d) Immunostaining of normal and sevenless heads with histamine‐specific antisera demonstrates that histamine is present in photoreceptors R1–6 and R8. (e) Histamine synthesized from exogenously supplied [3H]‐histidine can be released by depolarization with 50 mM K+, and the release is Ca2+ dependent. These observations strongly suggest that histamine is a major neurotransmitter used by Drosophila photoreceptors.
Cannabinoid Receptor‐Regulated Cyclic AMP Accumulation in the Rat Striatum
Authors: Bidaut‐Russell Michelle , Hewlett Allyn C.
Published: October 2006

: The present study demonstrates that desacetyllevo‐nantradol, a synthetic cannabinoid analog, reduces cyclic AMP levels in rat striatal slices stimulated with vasoactive intestinal peptide or SKF 38393, a D1‐dopamine agonist. Desacetyllevonantradol and the D2 agonist LY 171555 both inhibited D1‐stimulated cyclic AMP accumulation in the striatum. Spipcrone, a specific D2‐dopamine antagonist, fully reversed the inhibitory effect of LY 171555 but not that of desacetyllevonantradol, indicating that this cannabinoid response is not occurring through a D2‐dopaminergic mechanism. Morphine also inhibited cyclic AMP accumulation in striatal slices stimulated with either SKF 38393 or vasoactive intestinal peptide. Naloxone, an opioid antagonist, fully reversed the effect of morphine but not that of desacetyllev‐onantradol, indicating that cannabinoid drugs are not acting via a mechanism involving opioid receptors. The response to maximally effective concentrations of desacetyllevonan‐tradol was not additive to that of maximally effective concentrations of either morphine or LY 171555, suggesting that dopaminergic, opioid, and cannabinoid receptors may be present on the same populations of cells.
Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium
Authors: Bacic Fatima , Uematsu Sumio , McCarron Richard M. , Spatz Maria
Published: October 2006

: Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (±)‐SKF‐82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose‐dependent manner. The selective D, antagonist, (±)SCH‐23390, inhibited in a dose‐dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP‐ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP‐ribosylation enhanced the DA‐inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of α1‐adrenergic receptors (phenylephrine, 6‐fluoronorepinephrine) or serotonin (5‐HT), which stimulated the production of cyclic AMP, had no additive effect on DA‐stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of α1‐adrenergic agonists or 5‐HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(‐)‐sulpiride, or ketanserin (5‐HT2 > α1 > H1 antagonists), respectively. These findings represent the first demonstration of D1‐(stimulatory) and D2‐(inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either α1adrenergic or 5‐HT receptors in endothelium on the adenylate cyclase level. These results provide strong support for the previously contemplated existence of a central dopaminergic mechanism in cerebral vessels, a notion that is clinically important.
Stimulation of Transglutaminase Activity by GM1‐Ganglioside and α‐Sialylcholesterol in Superior Cervical and Nodose Ganglia Excised from Adult Rat
Authors: Ando Masato , Nakashima Yuhzo , Nagata Yutaka
Published: October 2006

Changes in transglutaminase (TG) activity in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined following application of selected membrane transport‐altering agents, including GM1‐ganglioside (GM1) and α‐sialylcholesterol (α‐SC). Although TG activity of freshly dissected SCG and NG was relatively low, it increased gradually during 30 min of incubation, and it stayed at this elevated level for 2 h. Addition of α‐SC at its maximal effective concentration, 20 μM, stimulated TG activity more than eightfold in SCG and more than twofold in NG by 30 min. Addition of GM1 at its most effective concentration, 5 nM, had similar effects, but of lesser magnitude. Cydoheximide, a potent inhibitor of protein biosynthesis, did not affect the GM1‐or α‐SC‐evoked increases in ganglionic TG activity, suggesting that enzyme activation rather than synthesis of new enzyme was occurring. The stimulation of TG activity in both ganglia caused by either GM1 or α‐SC was associated with a decrease in Km and an increase in Vmax values. Addition of cholera toxin B. which specifically masks the oligosaccharide chain of GM1. reduced the GM1‐induced increase in TG activity by approximately 60% in SCG and 88% in NG. The α‐SC‐induced increase in TG activity was only partially mimicked by free cholesterol. Although application of either dibutyryl cyclic AMP or dibutyryl cyclic GMP produced little change in TG activity of either ganglion, phorbol ester clearly inhibited the enzymic activity. Because TG is a calcium‐dependent enzyme, we measured 45Ca2+ influx into either ganglion, and found that it was reduced by GM1 and a‐SC in SCG and by α‐SC in NG. The GM1‐induced decrease in 45Ca2+ influx was prevented by cholera toxin B in SCG, whereas the α‐SC‐induced decrease was not mimicked by cholesterol in either ganglion. These results suggest that the sialic acid moiety of GM1 and α‐SC is responsible for the activation of ganglionic TG and that activation may occur via a protein kinase C‐regulated pathway linked with calcium ion movement.
The Cysteine Residue in the Carboxyl‐Terminal Domain of the m2 Muscarinic Acetylcholine Receptor Is Not Required for Receptor‐Mediated Inhibition of Adenylate Cyclase
Authors: Van Koppen Chris J. , Nathanson Neil M.
Published: October 2006

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide‐binding protein‐coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl‐terminal region of the protein. Because elimination of this cysteine in the β2‐adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR‐effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild‐type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild‐type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor‐mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl‐terminal domain of the m2 mAChR is not required for ligand binding or mAChR‐mediated inhibition of adenylate cyclase in CHO cells.
Ryanodine Inhibits Caffeine‐Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells
Authors: Teraoka Hiroki , Nakazato Yoshikazu , Ohga Akira
Published: October 2006

The effects of ryanodine, a selective inhibitor of the Ca2+‐induced Ca2+ release mechanism, on caffeine‐evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and cate‐cholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5–40 mM) caused a concentration‐dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg2+‐free medium containing 0.2 mM EGTA. Ryanodine (5 × 10–5M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 × 10–7–5 × 10–5M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2/Mg2+‐free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin‐permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.
Pergolide Presynaptically Inhibits Calcium‐Stimulated Release of γ‐Aminobutyric Acid
Authors: Drew Kelly L. , Ungerstedt Urban
Published: October 2006

The release of γ‐aminobutyric acid (GABA) in rat dorsolateral striatum was studied using in vivo microdialysis. Dialysis was conducted 2 days after probe implantation in awake, freely moving rats using a modified Ringer solution. Calcium induced a reversible increase in GABA release that was abolished by tetrodotoxin but was only slightly attenuated by a maximally effective dose of pergolide, a D2 receptor agonist. It was thus concluded that pergolide inhibits calcium‐stimulated release of GABA presynaptically by a mechanism distinct from that of tetrodotoxin.
Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury
Authors: Rothe Thomas , Müller Hans W.
Published: October 2006

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E‐containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine‐labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4°C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross‐react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor‐mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.
Inhibitory Modulation by Sodium Ions of the N‐Methyl‐D‐Aspartate Recognition Site in Brain Synaptic Membranes
Authors: Yoneda Yukio , Ogita Kiyokazu
Published: October 2006

Specific binding of radiolabeled L‐glutamic acid (Glu) was examined using rat brain synaptic membranes treated with a low concentration of Triton X‐100. The binding drastically increased in proportion to increasing concentrations of the detergent used up to 0.1%. Addition of 100 mM sodium acetate significantly potentiated the binding in membranes not treated with Triton X‐100, whereas it markedly inhibited the binding in Triton‐treated membranes. The binding in Triton‐treated membranes was inversely dependent on incubation temperature and reached a plateau within 10 min after the initiation of incubation at 2°C, whereas the time required to attain equilibrium at 30°C was <1 min. Sodium acetate invariably inhibited the binding detected at both temperatures independently of the incubation time via decreasing the affinity for the ligand. The binding was significantly displaced by agonists and antagonists for an N‐methyl‐d‐aspartate (NMDA)‐sensitive subclass of brain excitatory amino acid receptors, but not by those for the other subclasses. Inclusion of sodium acetate reduced the potencies of NMDA agonists to displace the binding without virtually affecting those of NMDA antagonists. Moreover, sodium ions inhibited the ability of Glu to potentiate the binding of N‐[3H][1‐(2‐thienyl)cyclohexyl]piperidine to open NMDA channels in Triton‐treated membranes. These results suggest that sodium ions may play an additional modulatory role in the termination process of neurotransmission mediated by excitatory amino acids via facilitating a transformation of the NMDA recognition site from a state with high affinity for agonists to a state with low affinity.
Modulation of γ‐Aminobutyric AcidA Receptor‐Operated Chloride Channels by Benzodiazepine Inverse Agonists Is Related to Genetic Differences in Ethanol Withdrawal Seizure Severity
Authors: Buck Kari J. , McQuilkin Susan J. , Harris R. Adron
Published: October 2006

To determine whether genetic differences in development of ethanol dependence are related to changes in γ‐aminobutyric acidA (GABAA) receptor function, we measured 36Cl uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Musci‐mol‐stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15–4513 (ethyl‐ 8‐azido‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5a]‐ [1,4]benzodiazepine‐3‐carboxylate) and methyl‐6,7‐dime‐ thoxy‐4‐ethyl‐β‐carboIine‐3‐carboxylate (DMCM) was not different for ethanol‐naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15–4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15–4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.
The Cholinergic Regulation of Intracellular Calcium in the Human Neuroblastoma, SH‐SY5Y
Authors: Murphy Nuala P. , Vaughan Peter F. T. , Ball Stephen G. , McCormack James G.
Published: October 2006

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH‐SY5Y, loaded with fura‐2. The resting free Ca2+ concentration in this cell line was 199 ± 14 nM (mean ± SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 μM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 ± 52 (mean ± SEM, n = 12) and 422 ± 48 nM (mean ± SEM, n = 7), respectively, in <4 s. This initial calcium transient declined to a plateau of 268 ± 36 and 240 ± 27 nM for carbachol and acetylcholine, respectively, in ∼40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol‐evoked initial calcium transient with Ki values of 0.85 ± 0.05, 8.3 ± 1.6, 411 ± 36, and 240 ± 46 nM (mean ± SEM, n = 3), respectively, and the acetylcholine‐induced initial calcium transient with Ki values of 0.48 ± 0.18, 13.5 ± 8.5, 192 ± 32, and 414 ± 25 nM (mean ± SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects. Furthermore, atropine, HHSD, and pirenzepine inhibited the plateau phase of the carbachol‐ evoked change in intracellular Ca2+ concentration with Ki values of 0.25, 1, and 861 nM, respectively, which are consistent with an M3 receptor being coupled to this effect. The plateau was abolished in the presence of EGTA, thereby converting the biphasic signal into a monophasic response. Under these conditions, the initial calcium transient or its pharmacology was not altered. However, if the cells were washed in calcium‐free buffer for 3 min, the initial peak was decreased by∼30%. The subsequent readdition of calcium caused a further increase in fura‐2 fluorescence. The influx of calcium and hence the plateau were also blocked by nickel but were insensitive to verapamil. We may conclude, therefore, on pharmacological grounds, that an M3 muscarinic receptor is coupled both to the peak response, presumably due to inositol trisphosphate mobilization of intracellular calcium, and to the plateau due to an influx of extracellular calcium.
Exogenous GM3 Ganglioside Stimulates Process Formation and Glycoprotein Release by Cultured Bovine Oligodendrocytes
Authors: Yim Sung Hye , Yavin Ephraim , Hammer Jeffrey A. , Quarles Richard H.
Published: October 2006

Isolated adult bovine Oligodendrocytes maintained in vitro for 10 days were treated for 1 day with 50 μg/ml of GM3 ganglioside (NeuNAcα2‐3Galβ1–4Glcβ1‐l'ceramide) in serum‐free culture medium. The treated Oligodendrocytes had significantly longer processes with more branching than control cells in the same medium without GM3. The treatment also stimulated the release of a series of 22‐ 100‐kDa, [3H]glucosamine‐labeled glycoproteins into the culture medium. Treatment of Oligodendrocytes maintained in vitro for 50 days with GM3 for 1 day resulted in a thickening of the processes and the appearance of many fine branches on existing processes as well as a similar stimulation of glycoprotein release into the medium.
Proteolytic Cleavage of the Alzheimer's Disease Amyloid A4 Precursor Protein
Authors: Ishiura Shoichi
Published: October 2006

Abstract: Amyloid A4 protein (ß‐protein) is deposited in the brain of a patient with Alzheimer's disease (AD) as one of the main components of extracellular cerebrovascular amyloid, as well as neurofibrillary tangles. It is derived from a precursor protein, and its formation has been considered to be a rate‐limiting step for brain degeneration in AD. In this article, proteolytic cleavage events that can degrade amyloid precursor protein are reviewed with respect to how the topographical distribution of the proteinase and its substrates disturbs normal processing steps in AD brain.
The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin‐Deficient (mld) Mutant Mice
Authors: Kuchler Sabine , Zanetta Jean‐Pierre , Zaepfel Marlyse , Badache Ali , Sarliève Louis L. , Vincendon Guy , Matthieu Jean‐Marie
Published: October 2006

Abstract: The myelin‐deficient (mld) mutation is an autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mid. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin‐associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low‐molecular‐mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.
Isolation of Hippocampal Synaptosomes on Percoll Gradients: Cholinergic Markers and Ligand Binding Sites
Authors: Thorne B. , Wonnacott S. , Dunkley P. R.
Published: October 2006

Abstract: The S1 Percoll procedure, devised empirically for cortical tissue, provides highly purified, functionally viable synaptosomes on a four‐step Percoll gradient. Here, for the first time, the procedure has been applied to rat hippocampus, and the gradient fractions have been analysed with respect to cholinergic markers and the synaptosomal index, lactate dehydrogenase. The presynaptic cholinergic markers choline acetyltransferase and [3H]choline uptake were most enriched in fraction 4. In contrast, acetylcholinesterase activity was broadly distributed across the gradient, consistent with the separation of synaptic plasma membranes (in fractions 1 and 2) from synaptosomes (in fractions 3 and 4). This is supported by the recovery of muscarinic binding sites labelled with [3H]quinuclidinylbenzilate in fractions 1 and 2. (−)‐[3H]‐Nicotine binding sites, however, were most enriched in fraction 4, consistent with their predominantly presynaptic localisation in the CNS. These results demonstrate the applicability of the S1 Percoll method to discrete brain regions for the recovery of homogeneous and viable synaptosome fractions. The separation of presynaptic terminals from post‐synaptic membranes is a further advantage of this technique.
[3H]Harman Binding Experiments. I: A Reversible and Selective Radioligand for Monoamine Oxidase Subtype A in the CNS of the Rat
Authors: May Torsten , Rommelspacher Hans , Pawlik Monika
Published: October 2006

Abstract: Harman (1‐methyl‐β‐carboline) is an endogenous compound with neurotropic properties in rats and humans. In a novel in vitro binding assay, the binding site of [3H]harman has been characterized in the rat crude mitochondrial (P2) fraction. The binding was saturable and reversible. Only a single high‐affinity binding site was detected by kinetic, saturation, and displacement analyses in the cerebral cortex of the rat. The linear Scatchard plots revealed equilibrium dissociation constant (KD) values of ∼2.5 nM at 0°C, ∼9 nM at 23°C, and ∼30 nM at 37°C. Among six CNS regions (hypothalamus, hippocampus, cerebral cortex, striatum, cerebellum, and spinal cord), the highest density of binding sites (Bmax) was determined in the hypothalamus (∼5.5 pmol/mg of protein) and the lowest in the spinal cord (∼2.0 pmol/mg of protein). Several drugs known to affect serotonergic, adrenergic, dopaminergic, cholinergic, or GA‐BAergic neurotransmission inhibited specific binding at best in the micromolar range. In contrast, potent and selective inhibitors of monoamine oxidase subtype A were active in the lower and middle nanomolar range. The displacing potency (apparent Ki) of substrates and inhibitors of monoamine oxidase correlated positively and highly significantly with the corresponding values of the inhibition of monoamine oxidase activity of subtype A (r= 0.92, p < 0.001, n = 17) but not of subtype B (r=−0.47, p > 0.05, n = 15). In conclusion, [3H]harman was identified as a specific ligand of the active site of the A subtype of monoamine oxidase in rat brain.
Enhanced Rate of Expression and Biosynthesis of Neuropeptide Y After Kainic Acid‐Induced Seizures
Authors: Bellmann Romuald , Widmann Rudolf , Olenik Claudia , Meyer Dieter K. , Maas Dagmar , Marksteiner Josef , Sperk Günther
Published: October 2006

Abstract: Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse‐labeling of the peptide and by determining prepro‐NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed‐phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by ∼380% in the cortex. In addition, concentrations of prepro‐NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P‐labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro‐NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.
L‐[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes
Authors: Gu J. G. , Delaney S. , Sawka A. N. , Geiger J. D.
Published: October 2006

Abstract: The stereoenantiomers D‐[3H]adenosine and L‐[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L‐Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 μM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L‐[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor‐stop centrifugation or with rapid filtration methods were 82 ± 14 and 75 ± 10, respectively. Using the filtration method, the rates of L‐[3H]adenosine accumulation were not significantly different from the value of 105 ± 15 fmol/mg of protein/s measured for D‐[3H]adenosine transport. Unlabeled D‐adenosine and nitrobenzylthioinosine, both at a concentration of 100 μM, reduced the levels and rates of L‐[3H]adenosine accumulation by >44%. These findings suggest that L‐adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D‐adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L‐adenosine may represent an important new probe with which adenosine uptake may be studied.
Ca2+‐Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes
Authors: Shao Z. , Suszkiw J. B.
Published: October 2006

Abstract: The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin‐permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 μM Pb2+ to intact synaptosomes in Ca2+‐deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2+‐induced ACh release can be augmented by activation of the voltage‐gated Ca2+ channels in nerve terminals. The lead‐induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1–50 nM (EC50± 4 nM) free Pb2+ to digitonin‐permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2–5 μM (EC50± 0.5 μM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.
3‐[(3‐Cholamidopropyl)dimethylammonio]‐1‐Propane Sulfonate‐Solubilized Binding Sites for 2‐[125I]Iodomelatonin in Chick Brain Retain Sensitivity to Guanine Nucleotides
Authors: Ying Shui‐Wang , Niles Lennard P.
Published: October 2006

Abstract: Binding of 2‐[125I]iodomelatonin to 3‐[(3‐chol‐amidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS)‐solubilized sites from chick forebrain was rapid, reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2‐(125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 ± 22 (n = 4) and 302 ± 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 ± 2.0 and 49.5 ± 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2‐iodomelatonin > melatonin > 6‐chloromelatonin ± prazosin > N‐acetylserotonin ± serotonin > metergoline > ketanserin > propranolol > phentolamine > cyproheptadine. Guanyl nucleotides inhibited binding of 2‐[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high‐affinity to a lowaffinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane‐bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.
Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures
Authors: Atkinson Brian N. , Minneman Kenneth P.
Published: October 2006

Abstract: The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the β‐adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the α‐adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by α‐adrenergic receptor antagonists (phentolamine > yohimbine > prazosin). α2‐Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 > NE > 6‐fluoro‐NE > epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by α‐adrenergic receptor antagonists with a different order of potency (prazosin > phentolamine > yohimbine). α1‐Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE > phenyiephrine > 6‐fluoro‐NE > methoxamine). In all three tissue preparations, large responses to both α1‐receptor activation (increases in inositol phosphate accumulation) and α2‐receptor activation (decreases in forskolin‐stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (β, α1, α2) are present in each tissue preparation. However, the cyclic AMP response to NE is mediated through β‐adrenergic receptors in neuronal cultures, through combinations of β‐ and α2‐adrenergic receptors in glial cultures, and through combinations of β‐ and α1‐adrenergic receptors in cortical slices.
Characterization and Regulation of β1‐Adrenergic Receptors in a Human Neuroepithelioma Cell Line
Authors: Fishman Peter H. , Nussbaum Elise , Duman Ronald S.
Published: October 2006

Abstract: Intact human neuroepithelioma SK‐N‐MC cells bound the β‐adrenergic antagonist (–)‐[3H]‐CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to β‐adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol norepinephrine > epinephrine, which is indicative of a β1‐subtype receptor. Membranes prepared from the cells bound (–)‐3‐[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist‐stimulated cyclic AMP production and competition binding experiments indicated that the β1‐selective antagonists CGP 20712A and ICI 89,406 were much more potent than the β2‐selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only β1‐adrenergic receptors. Northern blot analysis of SK‐N‐MC mRNA using cDNA probes for the β1‐ and β2‐adrenergic receptors revealed the presence of a very strong β1‐adrenergic receptor mRNA signal, while under the same conditions no β2‐adrenergic receptor mRNA was observed. Thus, SK‐N‐MC cells appear to express a pure population of β1‐adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down‐regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.
Rat Brain Free Glucose and Lactate Measurement by a Novel Method Using Bisecting Decapitation‐Extrusion and Enzyme Denaturation at Five Seconds
Authors: Oldendorf William H. , Stoller Barret E.
Published: October 2006

Abstract: This study investigates a novel method as a means for animal decapitation with rapid brain removal and enzyme denaturation. Briefly, the rat head is simultaneously decapitated and bisected. Either half of the in situ brain is aspirated under —250 mm Hg pressure into a modified small plastic syringe and then extruded through a needle as a fine strand into a relatively large volume of 2 M urea at 95°C. After cooling, sonication, and centrifugation of the brain homogenate, the supernatant is measured enzymatically for brain free glucose and lactate concentration. Enzyme denaturation is effected within 4–6 s. The results are in good agreement with published values for glucose and lactate using other rapid enzyme inactivation techniques.
N‐Acetylhistidine, Glutamate, and β‐Alanine Are Concentrated in a Receptor Cell Layer of the Trout Inner Ear
Authors: Drescher Marian J. , Drescher Dennis G.
Published: October 2006

Abstract: An epithelial sheet isolated from the trout saccular macula, highly enriched in acousticolateralis receptor cells (hair cells), has been analyzed for primary amine‐containing compounds. The hair cell preparation, compared to the saccular nerve, was found to contain elevated levels of the presumptive receptoneural transmitter, glutamate, as well as β‐alanine, and components eluting in the positions of the standards phosphoserine and phosphoethanolamine on cation‐exchange HPLC. Saccular nerve contained a different spectrum of primary amines and was elevated specifically in carnosine/homocarnosine. Acid hydrolysis of perchlorate extracts of both hair cell and nerve fractions yielded large amounts of histidine. For the saccular nerve fraction, production of histidine by acid hydrolysis was matched by production of β‐alanine and γ‐aminobutyric acid (GABA) and disappearance of carnosine/homocarnosine. The dipeptides carnosine and homocarnosine have been chromatographically resolved by expanded HPLC and found to be present in saccular nerve in a ratio of approximately 10:1, respectively. Production of histidine in the hair cell extract was not coupled with production of β‐alanine and GABA. The hair cell histidine‐containing unknown, present in millimolar concentration, has been identified as N‐acetylhistidine by the hydrolysis and rechromatography of fractions from cation‐exchange HPLC. The large and specific presence of N‐acetylhistidine in the hair cell preparation, together with electrophysiological evidence for its facilitatory action on afferent fibers in the frog semicircular canal, is suggestive of a role for this molecule as well as glutamate in acousticolateralis receptoneural transmission.
Regional Distribution of Extracellular 5‐Hydroxytryptamine and 5‐Hydroxyindoleacetic Acid in the Brain of Freely Moving Rats
Authors: Adell Albert , Carceller Alicia , Artigas Francesc
Published: October 2006

Abstract: The extracellular concentrations of 5‐hydroxytryptamine (5‐HT) and its metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) have been determined in six brain areas of awake rats (frontal cortex, striaturn, hypothalamus, hippocampus, inferior colliculus, and raphe nuclei) using intracerebral microdialysis. The extracellular levels of 5‐HT showed no significant differences among the brain regions studied. The tissue levels of 5‐HT and 5‐HIAA as well as the extracellular concentration of 5‐HIAA were significantly higher in raphe nuclei. The regional distribution of tissue and extracellular 5‐HIAA were very similar, suggesting that extracellular 5‐HIAA depends mainly on the output from the intracellular compartment. On the other hand, extracellular 5‐HT and tissue 5‐HT showed a different distribution pattern. The tissue/extracellular ratio for 5‐HT ranged from 739 in frontal cortex to 2,882 in raphe, whereas it only amounted to 1.8–3.6 for 5‐HIAA. The relationship between the present results and the density of 5‐HT uptake sites in these areas is discussed.
Characterization and Pharmacological Responsiveness of Dopamine Release Recorded by Microdialysis in the Substantia Nigra of Conscious Rats
Authors: Santiago M. , Westerink B. H. C.
Published: October 2006

The extracellular concentration of dopamine (DA) and 3,4‐dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5 μmol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1 μmol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of γ‐butyrolactonc (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1 μmol/L) of the D2 agonist (‐)‐N‐0437 [2‐(n‐propyl‐N‐2‐thienylethylamino)‐5‐hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (‐)‐N‐0437 was suppressed in the presence of nomifensine. Infusion (1 μmol/L) as well as systemic administration (40 mg/kg) of sulphide caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (‐)‐N‐0437 or (‐)‐sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level. It was possible to estimate the relative contribution of nigral and striatal au‐toreceptors to the typical changes in striatal DA release seen after peripheral administration of D2 agonists or antagonists. These results suggest that the dendritic release of DA fulfills classical release criteria.
Characterization and Partial Purification of AIM: A Plasma Protein That Induces Rat Cerebral Type 2 Astroglia from Bipotential Glial Progenitors
Authors: Levison Steven W. , McCarthy Ken D.
Published: October 2006

Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte‐type 2 astrocyte (O‐2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia‐inducing molecule (AIM). Using cultures enriched in O‐2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240‐fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin‐agarose, followed by gel nitration chromatog‐ raphy. In crude preparations, AIM activity migrated at 50 kDa by gel nitration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of >9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O‐2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects, AIM may be a novel differentiation factor.
The Differential Role of Protein Kinase C Isozyme in the Rapid Induction of Neurofilament Phosphorylation by Nerve Growth Factor and Phorbol Esters in PC12 Cells
Authors: Clark Edwin A. , Lee Virginia M.‐Y.
Published: October 2006

We examined the short‐term regulation of the phosphorylation of the mid‐sized neurofilament subunit (NF‐M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12‐O‐tet‐radecanoylphorbol 13‐acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF‐M and (b) the rate of conversion of NF‐M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF‐M, because NGF alone did not increase NF‐M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF‐M. Further, an increase in calcium/phospholipid‐dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF‐M. This PKC activity was determined to be derived from the PKCα and PKCβ isozyme. Finally, when PC12 cells were rendered PKC‐deficient by treatment with 1 μM TPA for 24 h, NGF maintained the ability to induce an increase in NF‐M phosphorylation, though not to the level attained in cells which were not PKC‐deficient. These data suggest (hat NGF with or without TPA stimulates NF‐M phosphorylation as a result of a complex series of events which include PKC‐in‐dependent and PKC‐dependent pathways.