bluuebunny/crossref_metadata_2025 huggingface dataset:
Lithium Ions Up‐Regulate mRNAs Encoding Dense‐Core Vesicle Proteins in Nerve Growth Factor‐Differentiated PC12 Cells
Authors: Cordeiro Mara L. , Umbach Joy A. , Gundersen Cameron B.
Published: March 2003

Abstract: We recently reported that lithium ions induced anup‐regulation of cysteine string protein (CSP) gene expression in nerve growthfactor (NGF)‐differentiated PC12 cells but not in undifferentiated cells.Concomitantly, expression of two other proteins of regulated secretorypathways, synaptophysin (SY) and SNAP‐25, was unaffected by lithium. To assessfurther the specificity of this effect of lithium, we used cDNA arrays. Ourdata indicate that lithium ions increase the level of mRNA for proteins suchas secretogranin II and vesicular monoamine transporter 1 that arepreferentially associated with large densecore secretory vesicles (LDCVs)without affecting mRNAs for proteins predominantly affiliated with smallsynaptic‐like vesicles, including the vesicular acetylcholine transporter andSY. This action of lithium is detected in NGF‐differentiated PC12 cells butnot in undifferentiated cells. These observations suggest that lithium ionsmodulate the turnover of LDCVs, and this may play a role in mediating thetherapeutic action of lithium in manic‐depressive illness.
Sodium—Calcium Exchange Affects Local Calcium Signal Decay and the Rate of Exocytotic Secretion in Single Chromaffin Cells
Authors: Tang Y. M. , Travis E. R. , Wightman R. M. , Schneider A. S.
Published: March 2003

Abstract: The effects of Na+ deprivation on local calcium signal decay and the rate of exocytotic secretion were measured in single bovine chromaffin cells to determine whether Na‐Ca exchange influences the local cytosolic Ca2+ signal for neurohormone release. Na+ replacement with N‐methylglucamine caused a marked slowing of the decay of the local Ca2+ signal near points of its initiation, as measured by high‐resolution fluorescent Ca2+ imaging in the confocal laser scanning microscope. Na+ replacement also resulted in a doubling of the rate and magnitude of exocytotic secretion measured in single cells by high‐resolution microamperometry. Release rates provide an independent measure of local active zone Ca2+. Five repetitive stimulations of the same cell in Na+‐free, but not in Na+‐containing, medium resulted in a progressively increasing rate of catecholamine release, suggesting an increasing level of active zone Ca2+ and a role of Na‐Ca exchange activity in Ca2+ clearance between stimulations. As secretory activity and its triggering Ca2+ signals are known to be co‐localized in active zones along the plasma membrane, the results suggest that Na‐Ca exchange may influence the decay of the local Ca2+ signal for exocytotic secretion. This would be consistent with a contribution to local Ca2+ clearance by a novel mechanism utilizing the insertion of secretory vesicle Na‐Ca exchangers into the plasma membrane during exocytosis.
Scavenger receptor class B, type I is expressed in porcine brain capillary endothelial cells and contributes to selective uptake of HDL‐associated vitamin E
Authors: Goti Daniel , Hrzenjak Andelko , Levak‐Frank Sanja , Frank Sasa , Van Der Westhuyzen Deneys R. , Malle Ernst , Sattler Wolfgang
Published: March 2003

It is clearly established that an efficient supply to the brain of α‐tocopherol (αTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of αTocH into cells constituting the blood–brain barrier (BBB) is obscure, we previously demonstrated that high‐density lipoprotein (HDL) plays a major role in the supply of αTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL‐associated αTocH in a similar manner to that described for HDL‐associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of αTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13‐fold. The observation that selective uptake of HDL‐associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR‐BI (pSR‐BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT‐PCR and northern and western blot analyses revealed the presence of pSR‐BI in cells constituting the BBB. Adenovirus‐mediated overexpression of the human analogue of SR‐BI (hSR‐BI) in pBCECs resulted in a fourfold increase in selective HDL‐associated αTocH uptake. In accordance with the proposed function of SR‐BI, selective HDL–CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR‐BI (mSR‐BI). Most importantly stable mSR‐BI overexpression mediated a twofold increase in HDL‐associated [14C]αTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR‐BI overexpression resulted in a twofold increase in endogenous HDL3‐associated αTocH uptake. The results of this study indicate that SR‐BI promotes the uptake of HDL‐associated αTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.
Neuroprotective effects of MK‐801 on l‐2‐chloropropionic acid‐induced neurotoxicity
Authors: Williams R. E. , Lock E. A. , Bachelard H. S.
Published: March 2003

l‐2‐Chloropropionic acid is selectively toxic to the cerebellum in rats; the granule cell necrosis observed within 48 h can be prevented by prior administration of MK‐801. Short‐term treatment (2 h) with l‐2‐chloropropionic acid has also been shown to activate the mitochondrial pyruvate dehydrogenase complex in fasted adult rats. This study aimed to investigate the effect of prior exposure to MK‐801 on the biochemical and neurotoxicological effects of l‐2‐chloropropionic acid. Extracts were prepared from the forebrain and cerebellum of animals that had been treated with l‐2‐chloropropionic acid, with and without prior treatment with MK‐801, and were analysed using magnetic resonance spectroscopy and amino acid analysis. Glucose metabolism was studied by monitoring the metabolism of [1‐13C]‐glucose using GC/MS. l‐2‐Chloropropionic acid caused increased glucose metabolism in both brain regions 6 h after administration, confirming activation of the pyruvate dehydrogenase complex, which was not prevented by MK‐801. After 48 h an increase in lactate and a decrease in N‐acetylaspartate was observed only in the cerebellum, whereas phosphocreatine and ATP decreased in both tissues. MK‐801 prevented the changes in lactate and N‐acetylaspartate, but not those on the energy state. These studies suggest that l‐2‐chloropropionic acid‐induced neurotoxicity is only partly mediated by the NMDA subtype of glutamate receptor.
Inhibition of protein synthesis in cortical neurons during exposure to hydrogen peroxide
Authors: Alirezaei Mehrdad , Marin Philippe , Nairn Angus C. , Glowinski Jacques , Prémont Joël
Published: March 2003

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H2O2 formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate‐induced increase in cytosolic Ca2+ is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn2+ preferentially inhibits the initiation step. In this study, we provide evidence indicating that H2O2 and thapsigargin mobilized a common intracellular Ca2+ pool. H2O2 treatment stimulated a slow increase in intracellular Ca2+, and precluded the effect of thapsigargin on Ca2+ mobilization. H2O2 stimulated the phosphorylation of both eIF‐2α and eEF‐2, in a time‐ and dose‐dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H2O2‐induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H2O2 treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF‐2. In conclusion, H2O2 inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF‐2α and eEF‐2 and the relative contribution of these two events depends on the duration of H2O2 treatment.
Insulin‐like growth factor‐I and Bcl‐XL inhibit c‐jun N‐terminal kinase activation and rescue Schwann cells from apoptosis
Authors: Cheng Hsin‐Lin , Steinway Matthew L. , Xin Xiping , Feldman Eva L.
Published: March 2003

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin‐like growth factor‐I, via phosphatidylinositol‐3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal‐induced apoptosis. In this study, we examined the role of c‐jun N‐terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal‐induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac‐DEVD‐CHO. Because insulin‐like growth factor‐I and Bcl‐XL are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin‐like growth factor‐I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol‐3 kinase inhibitor, blocked insulin‐like growth factor‐I inhibition on JNK activation, suggesting that phosphatidylinositol‐3 kinase mediates the effects of insulin‐like growth factor‐I. Overexpression of Bcl‐XL also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin‐like growth factor‐I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.
Antisense inhibition of BCL‐2 expression induces retinoic acid‐mediated cell death during differentiation of human NT2N neurons
Authors: Wang Shu , Rosengren Lars E. , Hamberger Anders , Haglid Kenneth G.
Published: March 2003

Changes in expression of the proto‐oncogene Bcl‐2are well known in the developing brain, with a high expression level in young post‐mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl‐2 up‐regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl‐2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 µm retinoic acid for 3–5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up‐regulate the BCL‐2 protein in 6 days. The BCL‐2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL‐2 with vectors expressing the antisense mRNA of Bcl‐2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron‐like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL‐2 to prevent retinoic acid‐induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl‐2 cDNA in sense orientation and exposed to toxic doses (40–80 µm) of retinoic acid. Furthermore, down‐regulation of BCL‐2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid‐induced apoptosis. These results indicate that one function of the up‐regulation of endogenous BCL‐2 during neuronal differentiation is to regulate the sensitivity of young post‐mitotic neurons to retinoic acid‐mediated apoptosis.
Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C‐terminal construct
Authors: Marsh Daniel R. , Holmes Kevin D. , Dekaban Gregory A. , Weaver Lynne C.
Published: March 2003

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C‐terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C‐terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle‐mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1‐1 (rNR1‐1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1‐1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1‐1 protein expression in sensory neurons. Expression of a construct containing only the C‐terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co‐expressed with rNR1‐1 in sensory neurons, membranous localization of rNR1‐1 was disrupted. In contrast, co‐expression of a C‐terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1‐1 did not alter the membranous distribution of rNR1‐1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1‐1 content by expressing a construct containing the C1 exon cassette.
Antisense suppression of GABAA receptor β subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression
Authors: Kumar Mallika , Luo Xiang , Quirk Phillip L. , Siegel Ruth E.
Published: March 2003

GABAA receptors in the CNS are pentameric molecules composed of α, β, γ, δ, ε and θ subunits. Studies on transfected cells have shown that GABAA receptor β subunit isoforms can direct α1 subunit localization within the cell. To examine the role of selected subunits in governing GABAA receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the α1, β2 or γ2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT‐PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the β2 antisense ODN reduced the level of the α1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the β2 subunit antisense ODN did not alter γ2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the α1 subunit requires a β subunit for assembly into GABAA receptors in cerebellar granule neurons.
Bone morphogenetic proteins (BMP6 and BMP7) enhance the protective effect of neurotrophins on cultured septal cholinergic neurons during hypoglycemia
Authors: Nonner Doris , Barrett Ellen F. , Kaplan Paul , Barrett John N.
Published: March 2003

The effects of two bone morphogenetic proteins (BMP6, BMP7), alone and in combination with neurotrophins, were tested on cultures of embryonic day 15 rat septum. A week‐long exposure to BMP6 or BMP7 in the optimal concentration range of 2–5 n m increased the activity of choline acetyltransferase (ChAT) by 1.6–2‐fold, in both septal and combined septal−hippocampal cultures. The increase in ChAT activity reached significance after 4 days and continued to increase over an 11‐day exposure. Under control culture conditions neither BMP significantly altered the number of cholinergic neurons, and BMP effects on ChAT activity were less than linearly additive with those of nerve growth factor. The effects of BMPs and BMP + neurotrophin combinations were also assayed under two stress conditions: low‐density culture and hypoglycemia. In low‐density cultures BMPs and BMP + neurotrophin combinations preserved ChAT activity more effectively than neurotrophins alone. During 24 h hypoglycemic stress, BMPs alone did not preserve ChAT activity, but BMP + neurotrophin combinations preserved ChAT activity much more effectively than neurotrophins alone. These results demonstrate that BMP6 and BMP7 enhance ChAT activity under control and low‐density stress conditions, and that during a hypoglycemic stress their trophic effect requires and complements that exerted by neurotrophins.
γ‐Aminobutyric acidA receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development
Authors: Brooks‐Kayal Amy R. , Shumate Melissa D. , Jin Hong , Rikhter Tatiana Y. , Kelly Mary Ellen , Coulter Douglas A.
Published: March 2003

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABAA receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABAA receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABAA receptor subunit mRNA expression, including greater than two‐fold lower expression of α1‐, α4‐ and γ2‐subunit mRNAs and 10‐fold higher expression of α5‐mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABAA receptor function in immature dentate granule cells, including two‐fold higher blockade by zinc and three‐fold lower augmentation by type‐I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABAA receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABAA receptor function during postnatal development.
[3H](2S,4R)‐4‐Methylglutamate: a novel ligand for the characterization of glutamate transporters
Authors: Apricó Karina , Beart Philip M. , Lawrence Andrew J. , Crawford Duncan , O'Shea Ross D.
Published: March 2003

[3H](2S,4R)‐4‐Methylglutamate ([3H]4MG), used previously as a ligand for low‐affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na+ ions at 4°C, specific binding of [3H]4MG was rapid and saturable (t1/2 ≈ 15 min), representing > 90% of total binding. Dissociation of [3H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (nH = 0.85) and a Kd of 6.2 ± 0.8 µm with a Bmax of 111.8 ± 23.8 pmol/mg protein. Specific binding of [3H]4MG was Na+‐dependent and inhibited by K+ and HCO3−. Pharmacological inhibition with compounds acting at GluTs revealed that Glu, d‐ and l‐aspartate, l‐serine‐O‐sulfate and ltrans‐pyrrolidine‐2,4‐dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and lthreo‐3‐methylglutamate, all inhibited ≈ 40% of specific binding. The inhibition pattern of l‐serine‐O‐sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [3H]4MG also labelling GLAST. 6‐Cyano‐7‐nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [3H]4MG binding. The pharmacological profile of binding of [3H]4MG resembled that found for [3H]d‐aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [3H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GluTs.
The role of putative intragenic control elements in c‐fos regulation by calcium and growth factor signalling pathways
Authors: Lange Claudia M. , Bading Hilmar
Published: March 2003

Sequences in the transcribed region of the c‐fos gene have been suggested to control c‐fos induction following exposure of cells to mitogens or stimuli that increase intracellular calcium concentrations. Using a mutational analysis we show that putative regulatory elements present in the first intron of the human c‐fos gene and the fos‐intragenic‐regulatory‐element (FIRE) are not required for c‐fos regulation by growth factor and calcium signalling pathways in AtT20 and PC12 cells. Removal of the c‐fos first intron and the FIRE did not increase the basal level of c‐fos mRNA and only moderately reduced the magnitude of calcium‐induced transcription mediated by either the entire c‐fos promoter or the cAMP response element (CRE). Intragenic mutations did not affect serum response element (SRE)‐dependent gene expression induced by calcium signals but caused a superinduction of c‐fos expression in nerve growth factor‐stimulated PC12 cells. These results indicate that c‐fos promoter elements, rather than intragenic sequences, are the principal targets of transcription‐regulating signalling pathways. This suggests that CRE‐ and SRE‐bound activators of transcription initiation may also enhance, in a signal‐dependent manner, c‐fos transcript elongation beyond promoter–proximal pause sites.
Cerebrin prohormone processing, distribution and action in Aplysia californica
Authors: Li L. , Floyd P. D. , Rubakhin S. S. , Romanova E. V. , Jing J. , Alexeeva V. Y. , Dembrow N. C. , Weiss K. R. , Vilim F. S. , Sweedler J. V.
Published: March 2003

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17‐residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F‐ and C‐clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral‐buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command‐like neuron CBI‐2, dramatically shortening the duration of the radula protraction in a concentration‐dependent manner, mimicking the motor‐pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food‐induced arousal in the animal. Cerebrin‐like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin‐like peptides may be widespread throughout gastropoda.
Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue
Authors: Kruchkova Yelena , Ben‐Dror Iris , Herschkovitz Avia , David Magda , Yayon Avner , Vardimon Lily
Published: March 2003

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c‐Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c‐Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down‐regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down‐regulation of glutamine synthetase.
Morphine‐induced in vivo release of spinal cholecystokinin is mediated by δ‐opioid receptors – effect of peripheral axotomy
Authors: Gustafsson Henrik , Afrah Abdullahi Warsame , Stiller Carl‐Olav
Published: March 2003

Morphine and other opioid agonists induce spinal in vivo release of cholecystokinin (CCK), a neuropeptide with anti‐opioid properties. However, so far the opioid receptor subtype responsible for this effect has not been determined. In the present in vivo microdialysis study, the morphine‐induced release of cholecystokinin‐like immunoreactivity (CCK‐LI) in the dorsal horn was completely blocked by the δ‐opioid antagonist naltrindole (10 µm in the perfusion fluid). Neither the µ‐opioid receptor antagonist d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr amide (CTOP; 10 µm in the perfusion fluid), nor the κ‐opioid receptor antagonist nor‐binaltorphimine (nor‐BNI); 10 µm in the perfusion fluid) had any significant effect in this respect. In addition, systemic administration of the δ‐opioid receptor agonist BW373U86 (1 mg/kg, s.c.) and spinal administration of the δ2‐opioid receptor agonist, Tyr‐d‐Ala‐Phe‐Glu‐Val‐Val‐Gly amide ([d‐Ala2] deltorphin II) (1 µm in the perfusion fluid) induced a significant increase of the CCK‐LI level. The effect of BW373U86 on spinal CCK‐LI release was completely blocked by spinal administration of naltrindole. The µ‐opioid receptor agonist [D‐ala2‐N‐Me‐Phe4‐Gly5‐ol]‐enkephalin (DAMGO) (1 µm in the perfusion fluid or 1 mg/kg, s.c.) failed to alter the CCK‐LI level. Peripheral nerve lesions have previously been shown to down‐regulate µ‐ and δ‐opioid receptors in the dorsal horn, to increase the gene‐expression of CCK and CCK‐receptor mRNA in dorsal root ganglion neurons and to alter the potassium‐induced spinal CCK‐LI release. After complete sciatic nerve transection, administration of the two selective δ‐opioid receptor agonists induced a significant release of CCK‐LI, which was comparable to controls. In contrast, neither systemic nor spinal administration of morphine and DAMGO altered the spinal CCK‐LI release in axotomized animals. The present data indicate that the δ‐opioid receptor mediates morphine‐induced CCK‐LI release in the spinal cord.
Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation
Authors: Bobrovskaya Larisa , Odell Adam , Leal Rodrigo B. , Dunkley Peter R.
Published: March 2003

Angiotensin II (AII, 100 nm) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)‐mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress‐activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen‐activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1–50 µm) and UO126 (0.1–10 µm), dose‐dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 µm, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 µm SB203580 did not affect AII‐stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 µm SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 µg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non‐specific effects of 20 µm SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.
Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury
Authors: Beer Ronny , Franz Gerhard , Krajewski Stanislaw , Pike Brian R. , Hayes Ronald L. , Reed John C. , Wang Kevin K. , Klimmer Christian , Schmutzhard Erich , Poewe Werner , Kampfl Andreas
Published: March 2003

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact‐induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT‐PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8‐ and caspase 3‐immunopositive cells with apoptotic‐like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2–5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.
Multiple ecto‐nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression
Authors: Vollmayer Petra , Koch Marco , Braun Norbert , Heine Petra , Servos Jörg , Israr Eltahmash , Kegel Birgit , Zimmermann Herbert
Published: March 2003

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface‐located enzymes (ecto‐nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto‐nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface‐located ATPase and ADPase activity with apparent Km‐values of 283 µm and 243 µm, respectively. Using PCR we identified the mRNA of all members of the ecto‐nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto‐nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue‐non‐specific alkaline phosphatase and ecto‐5′‐nucleotidase. The surface‐located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto‐nucleoside triphosphate diphosphohydrolase family. Activity of ecto‐5′‐nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto‐nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto‐nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto‐nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.
Prevalence between different α subunits performing the benzodiazepine binding sites in native heterologous GABAA receptors containing the α2 subunit
Authors: Carlos del Río Juan , Araujo Francisco , Ramos Blanca , Ruano Diego , Vitorica Javier
Published: March 2003

The presence of two heterologous α subunits and a single benzodiazepine binding site in the GABAA receptor implicates the existence of pharmacologically active and inactive α subunits. This fact raises the question of whether a particular α subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of α subunits with different benzodiazepine binding properties (α1, α2 and α5). Thus, we first demonstrated the existence of α2–α1 (36.3 ± 5.2% of the α2 population) and α2–α5 (20.2 ± 2.1%) heterologous receptors. A similar α2–α1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABAA receptors containing a common (α2) and a different (α1 or α5) α subunit. The α2 subunit pharmacologically prevailed over the α1 subunit in both cortex and hippocampus (there was an absence of high‐affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in α2–α1 double immunopurified receptors (Ki = 295 ± 56 nm and 200 ± 8 nm in hippocampus and cortex, respectively). On the contrary, the α5 subunit pharmacologically prevailed over the α2 subunit (low‐ and high‐affinity binding sites for zolpidem and [3H]L‐655,708, respectively). This prevalence was probed in α2–α5 double immunopurified receptors. Zolpidem displayed a single low‐affinity binding site (Ki = 1.73 ± 0.54 µm). These results demonstrated the existence of a differential dominance between the different α subunits performing the benzodiazepine binding sites in the native GABAA receptors.
Intermediate filaments regulate astrocyte motility
Authors: Lepekhin Eugene A. , Eliasson Camilla , Berthold Claes‐Henric , Berezin Vladimir , Bock Elisabeth , Pekny Milos
Published: March 2003

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell‐type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.
Inhibition of Rho‐kinase‐induced myristoylated alanine‐rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H‐1152, a novel and specific Rho‐kinase inhibitor
Authors: Ikenoya Mami , Hidaka Hiroyoshi , Hosoya Takamitsu , Suzuki Masaaki , Yamamoto Naoki , Sasaki Yasuharu
Published: March 2003

The functions of small G protein Rho‐associated kinase (Rho‐kinase) have been determined in muscle and non‐muscle cells, but, particularly in neuronal cells, its effector(s) has not been well known. Recently, we preliminarily reported that Rho‐kinase phosphorylates the Ser159 residue in myristoylated alanine‐rich C kinase substrate (MARCKS)in vitro, but it remains obscurein vivo. To further clarify this point, we developed an isoquinolinesulfonamide derivative, H‐1152, that is a more specific, stronger and membrane‐permeable inhibitor of Rho‐kinase with aKivalue of 1.6 nm, but poor inhibitor of other serine/threonine kinases. H‐1152 dose‐ dependently inhibited the phosphorylation of MARCKS in human neuroteratoma (NT‐2) cells stimulated by Rho‐activator lysophosphatidic acid (LPA), which was determined by phosphorylation site‐specific antibody against phospho‐Ser159 in MARCKS, whereas it hardly inhibited the phosphorylation stimulated by phorbol‐12,13‐dibutyrate (PDBu). In contrast, two other Rho‐kinase inhibitors, HA‐1077 at 30 µmand Y‐27632 at 10–30 µm, inhibited the phosphorylation of MARCKS in the cells stimulated by LPA and PDBu. A PKC inhibitor Ro‐31‐8220 selectively inhibited PDBu‐induced phosphorylation of MARCKS. Taken together with our previous results, the present findings strongly suggest that Rho/Rho‐kinase phosphorylates MARCKS at Ser159 residue in neuronal cells in response to LPA stimulation and that H‐1152 is a useful tool to confirm Rho‐kinase function(s) in cells and tissues.
NS 1231, a novel compound with neurotrophic‐like effects in vitro and in vivo
Authors: Dagø Lone , Bonde Christian , Peters Dan , Møller Arne , Bomholt Signe Farsø , Hartz Barbara P. , Meyer Morten , Drejer Jørgen , Grønborg Mette
Published: March 2003

NS 1231 [5‐(4‐chlorophenyl)‐6,7,8,9‐tetrahydro‐1H‐pyrrolo‐[3.2‐h]naphthalene‐2,3‐dione‐3‐oxime] belongs to a chemical series of compounds, which exhibit neurotrophic‐like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)‐ differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF‐induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF‐induced signalling events, such as TrkA phosphorylation at the Shc‐binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA‐induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.
Increased brain histamine levels in Parkinson's disease but not in multiple system atrophy
Authors: Rinne J. O. , Anichtchik O. V. , Eriksson K. S. , Kaslin J. , Tuomisto L. , Kalimo H. , Röyttä M. , Panula P.
Published: March 2003

We investigated histamine concentration in post‐mortem brain samples of patients with Parkinson's disease (PD, n = 24), multiple system atrophy (MSA, n = 8) and age‐matched controls (n = 27). Histamine concentrations were significantly increased in the putamen (to 159% of the control mean), substantia nigra pars compacta (to 201%), internal globus pallidus (to 234%) and external globus pallidus (to 200%), i.e. in areas which play a crucial role in the motor behaviour and which show typical functional alterations in PD. In MSA no significant differences were seen. Tele‐methylhistamine (histamine metabolite) concentrations were unchanged in PD. These results indicate that histamine concentration, but not its metabolism is increased in PD, but not in MSA. This finding may have implications in developing new drug therapies for PD and in differential diagnosis between PD and MSA.
Nordihydroguaiaretic acid potently breaks down pre‐formed Alzheimer's β‐amyloid fibrils in vitro
Authors: Ono Kenjiro , Hasegawa Kazuhiro , Yoshiike Yuji , Takashima Akihiko , Yamada Masahito , Naiki Hironobu
Published: March 2003

Inhibition of the accumulation of amyloid β‐peptide (Aβ) and the formation of β‐amyloid fibrils (fAβ) from Aβ, as well as the degradation of pre‐formed fAβ in the CNS would be attractive therapeutic objectives for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) inhibited fAβ formation from Aβ(1–40) and Aβ(1–42) dose‐dependently in the range of 10–30 µmin vitro. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that NDGA dose‐dependently breaks down fAβ(1–40) and fAβ(1–42) within a few hours at pH 7.5 at 37°C. At 4 h, the fluorescence of fAβ(1–40) and fAβ(1–42) incubated with 50 µm NDGA was 5% and 10% of the initial fluorescence, respectively. The activity of NDGA to break down these fAβs was observed even at a low concentration of 0.1 µm. At 1 h, many short, sheared fibrils were observed in the mixture incubated with 50 µm NDGA, and at 4 h, the number of fibrils reduced markedly, and small amorphous aggregates were observed. We next compared the activity of NDGA to break down fAβ(1–40) and fAβ(1–42), with other molecules reported to inhibit fAβ formation from Aβ and/or to degrade pre‐formed fAβ both in vivo and in vitro. At a concentration of 50 µm, the overall activity of the molecules examined in this study was in the order of: NDGA >> rifampicin = tetracycline > poly(vinylsulfonic acid, sodium salt) = 1,3‐propanedisulfonic acid, disodium salt > β‐sheet breaker peptide (iAβ5). In cell culture experiments, fAβ disrupted by NDGA were less toxic than intact fAβ, as demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Although the mechanisms by which NDGA inhibits fAβ formation from Aβ, as well as breaking down pre‐formed fAβin vitro, are still unclear, NDGA could be a key molecule for the development of therapeutics for AD.
Mitochondrial voltage‐dependent anion channel is involved in dopamine‐induced apoptosis
Authors: Premkumar A. , Simantov R.
Published: March 2003

Neuronal NMB cells were used to determine changes in gene expression upon treatment with dopamine. Twelve differentially expressed cDNAs were identified and cloned, one of them having 99.4% sequence homology with isoform 2 of a voltage‐dependent anion channel (VDAC‐2). The known role of VDAC, a mitochondrial outer‐membrane protein, in transport of anions, pore formation, and release of cytochrome C prompted us to investigate the possible role of VDAC gene family in dopamine‐induced apoptosis. Semi‐quantitative PCR analysis indicated that expression of the three VDAC isoforms was reduced by dopamine. Immunoblotting with anti‐VDAC antibodies detected two VDAC protein bands of 33 and 34 kDa. Dopamine decreased differentially the immunoreactivity of the 34 kDa protein. Whether the decrease in VDAC expression influence the mitochondrial membrane potential (ΔΨm) was determined with the dye Rhodamine‐123. Dopamine indeed decreased the mitochondrial ΔΨm, but the maximum effect was observed within 3 h, prior to the decrease in VDAC mRNA or protein levels. Cyclosporin A, a blocker of the mitochondrial pore complex, prevented the decrease in ΔΨm, but did not rescue the cells from dopamine toxicity. To elucidate possible involvement of protease caspases in dopamine‐induced apoptosis, the effect of the caspase inhibitor z‐Val‐Ala‐Asp(Ome)‐FMK (zVAD) was determined. zVAD decreased dopamine toxicity, yet it did not rescue the mitochondrial ΔΨm drop. Dopamine also decreased ATP levels. Finally, transfection of NMB cells with pcDNA–VDAC decreased the cytotoxic effect of dopamine. These findings are in agreement with the notion that the mitochondria, and VDAC, are important participants in dopamine‐induced apoptosis.
Rafts in adult peripheral nerve myelin contain major structural myelin proteins and myelin and lymphocyte protein (MAL) and CD59 as specific markers
Authors: Erne B. , Sansano S. , Frank M. , Schaeren‐Wiemers N.
Published: March 2003

The myelin and lymphocyte protein (MAL) proteolipid is localized in central and peripheral compact myelin membranes, as well as in apical membranes of particular polarized cells. In this study, we addressed the question whether MAL and other peripheral myelin proteins are sorted and targeted to myelin membranes using mechanisms similar to those observed in polarized epithelial cells. To investigate the presence of raft‐mediated sorting pathways in Schwann cells, we have isolated and analysed their composition in myelin membranes. Here, we show that rafts are present in adult human and rat peripheral compact myelin membranes and contain MAL, the GPI‐anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59, and MAL have an almost identical expression pattern within compact myelin. Moreover, immuno‐electron microscopy revealed that MAL, besides its localization in compact myelin, is also localized to Schmidt–Lanterman incisures. Taken together, our results demonstrate the presence of detergent‐insoluble glycolipid‐enriched complexes (DIGs) in different compartments of myelin membranes and indicate an important role for DIG‐mediated transport mechanisms in the maintenance of the adult myelin sheath.