bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of Thiamine Triphosphate in Rat Brain: Correlation with Chloride Permeability
Authors: Bettendorff L. , Peeters M. , Wins P. , Schoffeniels E.
Published: October 2006

Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10‐fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β‐32P]TDP and [γ‐32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein‐bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent‐solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.
A Dystrophin‐Immunoreactive Protein in Mammalian Brain
Authors: Clerk Angela , Muntoni Francesco , Strong Peter N.
Published: October 2006

Abstract: Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of ∼230 kDa that is recognised by anti‐dystrophin antibodies raised to the N‐terminal region of the rod‐like domain. Although the N‐terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230‐kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230‐kDa dystrophin‐immunoreactive protein.
Endothelin‐Mediated Calcium Response and Inositol 1,4,5‐Trisphosphate Release in Neuroblastoma‐Glioma Hybrid Cells (NG108‐15): Cross Talk with ATP and Bradykinin
Authors: Chau L.‐Y. , Lin T. A. , Chang W.‐T. , Chen C.‐H. , Shue M.‐J. , Hsu Y.‐S. , Hu C.‐Y. , Tsai W.‐H. , Sun G. Y.
Published: October 2006

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108‐15) induced increases in cytosolic free Ca2+ ([Ca2+]i) levels of labeled inositol monophosphates and inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. The increases in [Ca2+]i elicited by the three ETs (ET‐1, ET‐2, and ET‐3) were transient and did not show a sustained phase. Chelating extracellular Ca2+ in the medium by adding excess EGTA decreased the ET‐mediated Ca2+ response by 40‐50%. This result indicates that a substantial portion of the increase in [Ca2+]i was due to influx from an extracellular source. However, the increase in [Ca2+]i was not affected by verapamil or nifedipine (10−5M). A rank order potency of ET‐1 ET‐2 ET‐3 is shown for the stimulated increase in [Ca2+]i, as well as labeled inositol phosphates, in these cells. ATP (10−4M) and bradykinin (10−7M) also induced the increases in [Ca2+]i and Ins(1,4,5)P3 in NG108‐15 cells, albeit to a different extent. When compared at 10−7M, bradykinin elicited a five‐ to sixfold higher increase in the level of Ins(1,4,5)P3, but less than a twofold higher increase in [Ca2+]i than those induced by ET‐1. Additive increases in both Ins(1,4,5)P3 and [Ca2+]i were observed when ET‐1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET‐1 exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization to the Ca2+ response elicited by ATP. On the contrary, ET‐1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET‐1. Taken together, these results illustrate that, in NG108‐15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.
Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats
Authors: LoPachin Richard M. , Castiglia Carolyn M. , Saubermann Albert J. , Eichberg Joseph
Published: October 2006

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin‐induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na+,K+‐ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.
Sequence Similarities of Myelin Basic Protein Promoters from Mouse and Shark: Implications for the Control of Gene Expression in Myelinating Cells
Authors: Fors Lance , Hood Leroy , Saavedra Raul A.
Published: October 2006

Abstract: To better understand the cell type‐specific and coordinated regulation of the myelin protein genes, we cloned and sequenced the shark myelin basic protein (MBP) promoter. An alignment of the shark sequence with the corresponding mouse sequence showed striking similarities. These similarities, together with the results from expression experiments, define two major regions (A and B) within the MBP promoter. Region A is located immediately 5’to the transcription initiation sites and includes five sequences thought to be cis‐acting domains. These domains include two boxes of 13 and 12 nucleotides, respectively, separated from each other by 10 nucleotides, an MBP enhancer, and GC, CCAAT, and TATA boxes. Region A also contains a putative exon that codes for 35 amino acids of an unidentified polypeptide. Region B, which is located adjacent to the 5’end of region A, contains two boxes that are 10 and 11 nucleotides long, respectively, and are identical in mouse and shark. We have previously cloned and sequenced the shark glycoprotein zero (P0) promoter. A comparison between the sequences of the rat and shark P0 promoters shows three conserved regions in addition to CCAAT and TATA boxes. The shark P0 promoter is active in the CNS and PNS, and contains a sequence of 13 nucleotides that is located at ‐159 from the initiation of transcription and is similar to that of the MBP enhancer. The mammalian P0 promoter is active exclusively in the adult PNS and contains a sequence similar to that of the MBP enhancer located adjacent to the 3′ side of the transcription initiation site. Sequence similarities and differences between the promoters of the mammalian and shark myelin protein genes will help to identify the basis for the cell type‐specific and coordinated expression of these genes.
Calcium‐Dependent and ‐Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin
Authors: Arribas Mònica , Blasi Joan , Lazarovici Philip , Marsal Jordi
Published: October 2006

Abstract: The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 × 10−7M), the release was calcium‐dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 × 10−7M to 10−5M, the pardaxin‐induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10−6M pardaxin‐treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations ( 10−5M), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium‐dependent mechanisms of neurotransmitter release in the nervous system.
Antisense GAP‐43 Inhibits the Evoked Release of Dopamine from PC12 Cells
Authors: Ivins Kathryn J. , Neve Kim A. , Feller Daniel J. , Fidel Seth A. , Neve Rachael L.
Published: October 2006

Abstract: To investigate the role of the neuronal growth‐associated protein GAP‐43 (neuromodulin, B‐50, F1, P‐57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP‐43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP‐43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP‐43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP‐43 RNA in PC12 cells leads to a decrease in GAP‐43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP‐43 in calcium‐dependent neurotransmitter release.
Extracellular Somatostatin Measured by Microdialysis in the Hippocampus of Freely Moving Rats: Evidence for Neuronal Release
Authors: Vezzani A. , Ruiz R. , Monno A. , Rizzi M. , Lindefors N. , Samanin R. , Brodin E.
Published: October 2006

Abstract: Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin‐like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect <1 fmol/ml. The basal concentration of SLI in 20‐min dialysate fractions (5 μl/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 ± 6.4% (p < 0.05) when Ca2+ was omitted and 1 mM EGTA added to the Krebs‐Ringer solution and by 65.5 ± 3.2% (p < 0.05) in the presence of 1 μM tetrodotoxin. Depolarizing concentrations of the Na+ channel opener veratridine (6.25, 25, 100 μM) induced 11 ± 2 (p < 0.05), 17 ± 2 (p < 0.05), and 21 ± 5 (p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 μM veratridine was blocked by coperfusion with 5 μM tetrodotoxin (p < 0.01) and reduced by 79% (p < 0.01) in the virtual absence of Ca2+. Neuronal depolarization by 20 min of perfusion with Krebs‐Ringer solution containing 25 and 50 mM KCl and proportionally lowered Na+ increased the dialysate SLI 4.4 ± 1 (p < 0.05) and 17 ± 3 (p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na+,K+‐ATPase, increased the dialysate SLI 15‐fold baseline, on average (p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.
Characterization and Genetic Analysis of Functional Corticotropin‐Releasing Hormone Receptors in Primary Cerebellar Cultures
Authors: Barthel F. , Loeffler J. P.
Published: October 2006

Abstract: We demonstrate that granular cerebellar neurons express functional corticotropin‐releasing hormone (CRH) receptors. Activation of these receptors with CRH receptor agonists leads to a dose‐dependent increase in cyclic AMP (cAMP) levels with an apparent EC50 close to 10−9M. Using the c‐fos protooncogene as a system to evaluate genomic effects of CRH, we show that activation of CRH receptors regulates gene expression at the transcriptional level. CRH rapidly induced c‐fos mRNA accumulation. Genetic studies, using chimera genes containing human c‐fos promoter sequences coupled to a chloramphenicol acetyltransferase (CAT) reporter gene, confirmed and extended this observation. When protein kinase A (PKA) was specifically inactivated by gene transfer of a mutated regulatory subunit of PKA lacking cAMP binding sites, CRH‐stimulated c‐fos transcription was suppressed but the increase in cAMP level was not affected, indicating a key role of PKA in mediating CRH‐stimulated transcription. As CRH clearly modulates gene expression via the cAMP pathway, we analyzed the genomic effect of this neurohormone on a deleted c‐fos‐CAT construct containing only the cAMP‐responsive element (CRE) and on a heterologous promoter construct bearing the minimal palindromic consensus CRE (core sequence TGACGTCA). These minimal cAMP‐responsive genes are induced by CRH. These inductions are dependent on functional PKA. Taken together, our results demonstrate the presence of functional CRH receptors in primary cerebellar cultures. Activation of these receptors stimulates gene expression via the cAMP/PKA pathway and the transacting factor CREB (cAMP‐responsive element binding protein).
Phosphatidylethanol Affects Inositol 1,4,5‐Trisphosphate Levels in NG108‐15 Neuroblastoma × Glioma Hybrid Cells
Authors: Lundqvist C. , Rodriguez F. D. , Simonsson P. , Alling C. , Gustavsson L.
Published: October 2006

Abstract: Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108–15 neuroblastoma × glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5‐trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol‐naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5‐h incubation with 30 μM phosphatidylethanol. Phosphatidylethanol was found to cause a time‐and dose‐dependent increase in the basal levels of inositol 1,4,5‐trisphosphate. The effects on inositol 1,4,5‐trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin‐stimulated inositol 1,4,5‐trisphosphate production could be detected. However, the increase in basal inositol 1,4,5‐trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5‐trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol‐induced effects on cellular processes.
Differential Interaction of 1‐Methyl‐4‐Phenylpyridinium Ion with the Putatively Vesicular Binding Site of [3H]Tyramine in Dopaminergic and Nondopaminergic Brain Regions
Authors: Vaccari A. , Saba P. L. , Gessa G. L. , Del Zompo M.
Published: October 2006

Abstract: The neurotoxin 1‐methyl‐4‐phenylpyridinium ion (MPP+) in the brain striatum has recently been shown to bind at a putatively vesicular site labeled by [3H]tyramine ([3H]TY). Whereas in the rat and mouse striatum MPP+ antagonized TY binding competitively, in the cerebellum there was a mixed‐type antagonism, which suggests the simultaneous occupancy of two different sites. Ki values from displacement curves revealed a fourfold difference in the affinity of MPP+ for TY sites in the two brain regions. The degeneration of central noradrenergic terminals induced by an intraperitoneal injection of the toxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine in rats decreased by 80% the maximal number of cerebellar TY binding sites, while not affecting striatal binding. Furthermore, guanethidine, a marker for noradrenaline (NA) vesicles, potently inhibited TY binding in NA‐innervated regions, such as the cerebellum and the parietal cortex, and poorly in the striatum. It is concluded (a) that both MPP+ and TY may also label NA vesicles and (b) that the vesicular carriers for dopamine and NA have different characteristics, which may underlie a regional specificity in the rate of endovesicular sequestration of MPP+, with either neurodegenerative or neuroprotective consequences, depending on the brain area involved.
Enhancement of the Synthesis and Secretion of Nerve Growth Factor in Primary Cultures of Glial Cells by Proteases: A Possible Involvement of Thrombin
Authors: Neveu Isabelle , Jehan Frédéric , Jandrot‐Perrus Martine , Wion Didier , Brachet Philippe
Published: October 2006

Abstract: Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as α‐thrombin or collagenase induce a continuous, dose‐dependent enhancement of the levels of cell‐secreted NGF. Incubation of astrocytes for a 24‐h period with 300 ng/ml of α‐thrombin (∼9 nM, or 1 U/ml) results in an increase of the levels of cell‐secreted NGF by a factor of three‐ to fourfold, and at doses 10 times higher, stimulation by a factor of up to four‐to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP‐1 family, such as c‐fos, c‐jun or junB. Trypsin, plasmin, α‐chymotrypsin, or elastase also enhanced, to different extents, the levels of cell‐secreted NGF. However, unlike α‐thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose‐response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell‐substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of α‐thrombin‐indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of α‐thrombin with D‐phenyl‐alanyl‐L‐propyl‐L‐arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of α‐thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of α‐thrombin require binding to the cell surface. We found that γ‐thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as α‐thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity. Part of this effect may be due to the cleavage of a transmembrane receptor for a heptapeptide that acts as an agonist of this receptor and displays a limited but significant action on the levels of cell‐secreted NGF. These data suggest that proteolytic enzymes might induce astrocytes to produce NGF in vivo.
Morphine Activates ω‐Conotoxin‐Sensitive Ca2+ Channels to Release Adenosine from Spinal Cord Synaptosomes
Authors: Cahill C. M. , White T. D. , Sawynok J.
Published: October 2006

Abstract: Morphine‐induced release of adenosine from the spinal cord is believed to contribute to spinal antinociception. Although this release is Ca2+ dependent, little is known of the nature of this dependence. In this study, the effects of the dihydropyridine L‐type Ca2+ channel agonist Bay K 8644 and the antagonist nifedipine, the N‐type Ca2+ channel antagonist ω‐conotoxin, and ruthenium red, a blocker of Ca2+ influx induced by capsaicin, on release of adenosine evoked by morphine were determined. The effect of partial depolarization with a minimally effective concentration of K+ on morphine‐evoked release of adenosine also was examined. Morphine 10−5‐10−4M produced a dose‐dependent enhancement of adenosine release from dorsal spinal cord synaptosomes. Following the addition of 6 mM K+ (total K+ concentration of 10.7 mM), 10−6M morphine also enhanced release, and an additional component of action at 10−8M was revealed. Release was Ca2+‐dependent as it was not observed in the absence of Ca2+ and presence of EGTA. Bay K 8644 (10 nM) and nifedipine (100 nM) had no effect on the release of adenosine evoked by morphine, but ω‐conotoxin (100 nM) markedly reduced such release in both the absence and the presence of the additional 6 mM K+. Morphine‐evoked adenosine release was not altered in the presence of a partially effective dose of capsaicin, nor by ruthenium red. These results indicate that morphine can stimulate two distinct phases of adenosine release from the spinal cord (nanomolar and micromolar), and that both phases of release are due to Ca2+ entry via ω‐conotoxin‐sensitive N‐type Ca2+ channels.
Additive Induction of Egr‐1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108‐15 Cells via Cholinergic Muscarinic, α2‐Adrenergic, and Bradykinin Receptors
Authors: Katayama Noriko , Iwata Emi , Sakurai Hiroaki , Tsuchiya Tomofusa , Tsuda Masaaki
Published: October 2006

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr‐1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr‐1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α2‐adrenergic receptors, respectively. At their saturation levels for Egr‐1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr‐1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr‐1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr‐1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr‐1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr‐1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr‐1 induction, depending on the times of stimulation and the combinations of receptors stimulated.
Parallel Up‐Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells
Authors: Kim Kyong‐Tai , Park Dong H. , Joh Tong H.
Published: October 2006

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time‐dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L‐amino acid decarboxylase (AADC), and dopamine γ‐hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μM) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0‐, 2.4‐, and 7.0‐fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6‐, 1.8‐, and 8.0‐fold increases, respectively. Low, but detectable, phenylethanolamine N‐methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6‐fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up‐regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.
Antibodies to the Human γ2 Subunit of the γ‐Aminobutyric AcidA/Benzodiazepine Receptor
Authors: Khan Zafar U. , Fernando Lawrence P. , Escribá Pablo , Busquets Xavier , Mallet Jacques , Miralles Celia P. , Filla Michael , De Blas Angel L.
Published: October 2006

Abstract: A γ‐aminobutyric acidA (GABAA) receptor (GABAAR) γ2 subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261‐bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX‐3X. Both γ‐galactosidase (LacZ) and glutathione‐S‐transferase (GST) fusion proteins containing the γ2IL were purified, and a rabbit antibody to the LacZ–γ2IL was made. The antibody reacted with the γ2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABAAR/ benzodiazepine receptor (GABAAR/BZDR) from bovine and rat brain. The antibody reacted in affinity‐purified GABAAR/BZDR immunoblots with a wide peptide band of 44,000–49,000 Mr. Immunoprecipitation studies with the anti‐γ2IL antibody suggest that in the cerebral cortex, 87% of the GABAARs with high affinity for benzodiazepines and 70% of the GABAARs with high affinity for muscimol contain at least a γ subunit, probably a γ2. These results indicate that there are [3H]muscimol binding GABAARs that do not bind [3H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti‐GABAAR/BZDR antibodies indicate that the most abundant combination of GABAAR subunits in the cerebral cortex involves α1, γ2 (or other γ), and β2 and/or β3 subunits. These subunits coexist in >60% of the GABAAR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABAAR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α6, also contain γ2 (or other γ) subunit(s). The α1 and β2 or β3 subunits are also frequently associated with γ2 (or other γ) and α6 in these cerebellar receptors.
Neurite Outgrowth and Protein Phosphorylation in Chick Embryonic Sensory Ganglia Induced by a Brief Exposure to 12‐O‐Tetradecanoylphorbol 13‐Acetate
Authors: Mehta Sujata , Hsu Linda , Jeng Arco Y. , Chen Kuang Yu
Published: October 2006

Abstract: An exposure to 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) at 20 nM for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagencoated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse‐labeling with [35S]‐methionine indicated no significant difference in protein synthesis between control and TPA‐treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66‐kDa protein were blocked. Biochemical analysis of the phosphorylated 66‐kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross‐reacted with an antiserum raised against a 66‐kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66‐kDa protein may be involved in neuritogenesis.
Effect of Reducing Brain Glutamine Synthesis on Metabolic Symptoms of Hepatic Encephalopathy
Authors: Hawkins Richard A. , Jessy J. , Mans Anke M. , De Joseph M. Regina
Published: October 2006

Abstract: Liver failure, or shunting of intestinal blood around the liver, results in hyperammonemia and cerebral dysfunction. Recently it was shown that ammonia caused some of the metabolic signs of hepatic encephalopathy only after it was metabolized by glutamine synthetase in the brain. In the present study, small doses of methionine sulfoximine, an inhibitor of cerebral glutamine synthetase, were given to rats either at the time of portacaval shunting or 3–4 weeks later. The effects on several characteristic cerebral metabolic abnormalities produced by portacaval shunting were measured 1–3 days after injection of the inhibitor. All untreated portacaval‐shunted rats had elevated plasma and brain ammonia concentrations, increased brain glutamine and tryptophan content, decreased brain glucose consumption, and increased permeability of the blood–brain barrier to tryptophan. All treated rats had high ammonia concentrations, but the brain glutamine content was normal, indicating inhibition of glutamine synthesis. One day after shunting and methionine sulfoximine administration, glucose consumption, tryptophan transport, and tryptophan brain content remained near control values. In the 3–4‐week‐shunted rats, which were studied 1–3 days after methionine sulfoximine administration, the effect was less pronounced. Brain glucose consumption and tryptophan content were partially normalized, but tryptophan transport was unaffected. The results agree with our earlier conclusion that glutamine synthesis is an essential step in the development of cerebral metabolic abnormalities in hyperammonemic states.
Glutamate Binding to Brain Membranes Is Increased in Pentylenetetrazole‐Kindled Rats
Authors: Schröder Helmut , Becker Axel , Lössner Bernd
Published: October 2006

Abstract: The specific binding of L‐[3H]glutamate to its receptors was investigated on crude membrane preparations from different brain regions of pentylenetetrazole‐kindled rats using a binding assay technique. Pentylenetetrazole kindling induced by 10 intraperitoneal applications of 45 mg/kg over a period of 20 days resulted in a significant increase of both the convulsive susceptibility of animals to the convulsant and the specific L‐[3H]glutamate binding in hippocampus and in motor, frontal, and inferiotemporal (acoustic) cortex tested with a L‐[3H]glutamate concentration of 50 nM. No differences were observed in the other brain structures studied. Kinetic studies indicated that the enhanced L‐[3H]glutamate binding to hippocampal membranes from kindled rats reflects changes in the density of the glutamate binding sites rather than an increase in receptor affinity. To study the effect of acute generalized convulsions on L‐[3H]glutamate binding to synaptosomal membranes of hippocampus and visual cortex, rats were treated 24 h before the experiment with 60 mg/kg of pentylenetetrazole, i.p. Under these conditions, no differences between treated and control rats were observed. From these findings, it is concluded that the increase in glutamate receptor density demonstrated in hippocampus and several neocortical brain structures of pentylenetetrazole‐kindled rats may be the expression of a specific enhancement of susceptibility of glutamatergic systems to this excitatory amino acid developing in the course of formation of pentylenetetrazole‐induced kindling.
Dopamine D1 Agonist SKF 38393 Increases the State of Phosphorylation of ARPP‐21 in Substantia Nigra
Authors: Tsou Kang , Girault Jean‐Antoine , Greengard Paul
Published: October 2006

Abstract: ARPP‐21 is a cyclic AMP‐regulated phosphoprotein (Mr= 21,000) that has a distribution in brain similar to that of DARPP‐32 (dopamine‐ and cyclic AMP‐regulated phosphoprotein, Mr= 32,000). It is enriched in the medium‐sized spiny neurons in the striatum and in the striatonigral nerve terminals in the pars reticulata of the substantia nigra. The present study shows that dopamine D1 agonist SKF 38393 increases the state of phosphorylation of ARPP‐21 by 26% in nigral slices and that pretreatment of the slices with D1 antagonist SCH 23390 blocks this effect. These results demonstrate that ARPP‐21 is a dopamine‐regulated phosphoprotein. Because D1 receptors are localized on nerve terminals of striatonigral pathway, the phosphorylation of ARPP‐21 is likely to mediate some of the intracellular effects of dopamine on these terminals.
Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C‐Mediated Decoupling
Authors: Banie Anne P. , Nicholls David G.
Published: October 2006

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4‐aminopyridine‐evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A1 receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4‐aminopyridine (where K+‐channel activation would inhibit release) and by elevated KC1 (where K+‐channel activation would have no effect), arguing for a central role of Ca2+‐channel modulation. In support of this, the plateau phase of depolarization‐evoked free Ca2+ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1‐ or 4‐aminopyridine‐stimulated synaptosomes. The interaction of protein kinase C with the A1 receptormediated inhibition is examined. Activation of protein kinase C by 4β‐phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K+‐channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.
Age‐Related Changes in Tyrosine Hydroxylase and Choline Acetyltransferase in Sympathetic Ganglia of a Rat Strain with Reduced Sympathetic Neuron Numbers
Authors: Hendry Ian A. , Bell Christopher
Published: October 2006

Abstract: In Wistar rats, a subpopulation of sympathetic ganglionic neurons dies during ageing, but in the GH strain, these same neurons die during the period of perinatal maturation. We have compared tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in superior cervical ganglia of GH and control rats at different ages. Ganglionic TH rose to near adult levels between postnatal weeks 1 and 2. No significant differences in TH values were seen between GH and control ganglia at any age, indicating that reduced neuron numbers are compensated for by increased cellular activity, Ganglionic ChAT rose initially in parallel with TH and then more slowly over postnatal weeks 3–4, reaching adult levels that were about 20° lower in GH than in normal ganglia. During ageing, TH remained constant but ChAT continued to rise slowly in GH ganglia, whereas ChAT in normal ganglia fell by about 10°. Both the strain difference in ChAT during development and the fall in ChAT during ageing in normal animals parallel the differences in ganglion cell numbers seen under these circumstances.
Diminished Functional Activity of Newly Synthesized Muscarinic Acetylcholine Receptors in Stably Transfected Y1 Adrenal Cells
Authors: Ikegaya Takayoshi , Nathanson Neil M.
Published: October 2006

Abstract: We have examined the functional responsiveness of newly synthesized m2 muscarinic acetylcholine receptors in stably transfected Y1 adrenal cells. After inactivation of preexisting receptors with the covalent alkylating antagonist propylbenzilylcholine mustard, the number of cell surface receptors returned to control values over a 3‐h period. After a 3‐h recovery, the cells exhibited diminished sensitivity for muscarinic receptor‐mediated inhibition of adenylyl cyclase activity, with much higher concentrations of agonist being required to elicit a response. The functional sensitivity returned to control values over a 12–18‐h period. The decreased functional activity was not due to a decreased affinity of the newly synthesized receptors for agonist or to a decrease in the levels of inhibitory G proteins in the cells. The results suggest that muscarinic receptors may be synthesized in a form with diminished functional activity. The ability to study the maturation of receptor function in a transfected cell system should allow a combination of biochemical and molecular genetic approaches to analyze the synthesis and functional responsiveness of muscarinic receptors.
Long‐Term Opiate Exposure Leads to Increase in Synapsin I in Rat Spinal Cord–Dorsal Root Ganglion Cocultures
Authors: Nah Seung‐Yeol , Saya Danielle , Vogel Zvi
Published: October 2006

Abstract: Cocultures of spinal cord and dorsal root ganglion cells contain relatively high concentrations of k‐opiate receptors. We have previously shown that acute k‐opiate agonist treatment reduces phosphorylation of synapsin I stimulated by depolarizing agents (such as 60 mM KCl). Here we show that prolonged opiate treatment increases the levels of synapsin I immunoreactivity in the cells. Several opiate agonists, such as U50488, ethylketocyclazocine, dynorphin, and [D‐Ala2,D‐Leu5]enkephalin, caused a 3.0–3.4‐fold increase in the immunoreactive level of synapsin I. The effect of the k‐agonist U50488 on the up‐regulation of synapsin I was dose dependent and was blocked by the k‐opiate antagonist norbinaltorphimine. The results suggest that continued activation of opiate receptors by chronic agonist treatment up‐regulates the levels of synapsin I. This increase in synapsin I could contribute to the development of tolerance to opiates.
An Amyloid Peptide, βA4 25–35, Mimics the Function of Substance P on Modulation of Nicotine‐Evoked Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells
Authors: Cheung Nam Sang , Small David H. , Livett Bruce G.
Published: October 2006

Abstract: The amyloid protein (βA4) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA4βA4 25–35; Gly‐Ser‐Asn‐Lys‐Gly‐Ala‐Ile‐Ile‐Gly‐Leu‐Met‐NH2), which contains the conserved C‐terminal sequence of substance P (X‐Gly‐Leu‐Met‐NH2), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine‐evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10−5M nicotine was inhibited by SP and βA4 25–35. The IC50 of SP and βA4 25–35 was 3 × 10−6 and 3 × 10−5M, respectively. SP and βA4 25–35 both protected against nicotinic receptor desensitization. However, βA4 25–35 was ∼ 10‐fold less effective than SP in its protective effect. The present work shows that βA4 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.
Stable Expression and Regulation of a Rat Brain K+ Channel
Authors: Critz Stuart D. , Wible Barbara A. , Lopez Hector S. , Brown Arthur M.
Published: October 2006

Abstract: The Shaw‐type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K+ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to Kv 3.1 channels expressed transiently in Xenopus oocytes. Kv 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13‐dibutyrate decreased Kv 3.1 currents. Based on these results, we find that the stable expression of voltage‐gated K+ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.
Modulation of the Serine Base Exchange Enzyme Activity of Rat Brain Membranes by Amphiphilic Cations and Amphiphilic Anions
Authors: Kanfer Julian N. , McCartney Douglas G.
Published: October 2006

Abstract: The biosynthesis of phosphatidylserine in mammalian tissues is catalyzed by the serine base exchange enzyme. The activity of this membrane‐bound enzyme can be manipulated by amphiphiles. Amphiphilic cations, such as oleylamine, W‐7, chlorpromazine, and didodecyldimethylamine, stimulate the serine base exchange activity. Amphiphilic anions, such as bis(2‐ethylhexyl) hydrogen phosphate and cholesterol sulfate, inhibit the serine base exchange activity. These effects are more pronounced at pH 7.0 than at the pH optimum of 8.5 for this enzyme. Both the stimulators and the inhibitors alter the Vmax values without changing the Km value for serine, suggesting that their mechanism of action is related to interactions of the membrane‐bound cosubstrate, phosphatidylethanolamine, with the membrane‐bound enzyme. The optimal concentration of stimulator varies with the amount of membrane protein present; however, supraoptimal concentrations cause inhibitions. It is proposed that the amphiphilic cations enhance the interaction of the phosphorylethanolamine moiety of the membrane‐bound cosubstrate with the enzyme and the amphiphilic anions interfere with such an interaction. Some of the pharmacological properties of these amphiphilic cations, employed clinically as antidepressants, may be mediated by modulation of the serine base exchange enzyme activity.
Physiological Stimulation Increases Nonoxidative Glucose Metabolism in the Brain of the Freely Moving Rat
Authors: Fellows L. K. , Boutelle M. G. , Fillenz M.
Published: October 2006

Abstract: The effects of mild stress on nonoxidative glucose metabolism were studied in the brain of the freely moving rat. Extracellular lactate levels in the hippocampus and striatum were monitored at 2.5‐min intervals with microdialysis coupled with an enzyme‐based flow injection analysis system. Ten minutes of restraint stress led to a 235% increase in extracellular lactate levels in the striatum. A 5‐min tail pinch caused an increase of 193% in the striatum and 170% in the hippocampus. Local application of tetrodotoxin in the striatum blocked the rise in lactate following tail pinch and inhibited the subsequent clearance of lactate from the extracellular fluid. Local application of the noncompetitive N‐methyl‐d‐aspartate receptor antagonist MK‐801 had no effect on the tail pinch‐stimulated increase in lactate in the striatum. These results show that mild physiological stimulation can lead to a rapid increase in nonoxidative glucose metabolism in the brain.
1H Nuclear Magnetic Resonance Spectroscopy Study of Cerebral Glutamate in an Ex Vivo Brain Preparation of Guinea Pig
Authors: Pirttilä Tiina‐R. M. , Hakumäki Juhana M. , Kauppinen Risto A.
Published: October 2006

Abstract: Cerebral glutamate was monitored in a superfused cerebral cortical preparation by 1H NMR spectroscopy using a semiselective spin‐echo sequence N‐acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral 1H NMR‐detected glutamate‐to‐NAA ratio was ∼ 20–30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the 1H NMR glutamate‐to‐NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 mM glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide‐m‐fluorophenylhydrazone, increased 1H NMR‐detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin‐spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 ± 89 and 729 ± 94 ms, respectively, and thus, the portion of glutamate that could not be detected by 1H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate‐to‐NAA ratio from 0.55 ± 0.02 to 0.67 ± 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.