bluuebunny/crossref_metadata_2025 huggingface dataset:
Effluxes of 3,4‐Dihydroxyphenylalanine, 3,4‐Dihydroxyphenylglycol, and Norepinephrine from Four Blood Vessels During Basal Conditions and During Nerve Stimulation
Authors: Tyce Gertrude M. , Hunter Larry W. , Ward Lawrence E. , Rorie Duane K.
Published: July 2010

Abstract: Effluxes of 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenyglycol, and norepinephrine from four superfused canine blood vessels (saphenous and portal veins and mesenteric and pulmonary arteries) were studied under basal conditions and during nerve stimulation. From quantification of the compounds a series of indices of activities at neuroeffector junctions are proposed. These are (a) basal overflow of 3,4‐dihydroxyphenylglycol as an index of vesicular‐cytoplasmic translocation of norepinephrine, (b) the increase in 3,4‐dihydroxyphenyglycol overflow attributable to nerve stimulation as an index of neuronal reuptake of norepinephrine released by stimulation, (c) the sum of the increases in overflows of norepinephrine and 3,4‐dihydroxyphenylglycol attributable to nerve stimulation as an index of evoked release of norepinephrine, and (d) the efflux of 3,4‐dihydroxyphenylalanine as an index of the activity of tyrosine hydroxylase, the rate‐limiting enzyme in the synthesis of norepinephrine. There were clear differences between these indices in the vessels. Correlation coefficients of the indices among vessels indicated that a high tissue norepinephrine level was associated with high biosynthetic capacity and high vesicular‐cytoplasmic exchange but not with high release. There was no evidence suggesting feedback inhibition of synthesis by neuroplasmic norepinephrine—whether arising from vesicular‐cytoplasmic translocation or from reuptake from the junctional cleft. The major value of these indices will probably be in determining the intergrated effects of pharmacologic agents at neuroeffector junctions in different blood vessels.
Interleukin‐6 Production by Schwann Cells and Induction in Sciatic Nerve Injury
Authors: Bolin Laurel M. , Verity A. Neil , Silver Jon E. , Shooter Eric M. , Abrams John S.
Published: July 2010

Abstract: Interleukin‐6 (IL‐6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell‐derived IL‐6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL‐6. Cocultured iSC transcribe IL‐6 message as confirmed by northern analysis. Stimuli that induce IL‐6 production in the hematopoietic lineage induced transcription and production of IL‐6 by iSC cells. Lipopolysaccharide, tumor necrosis factor‐α, IL‐1α, IL‐6, and serum withdrawal induced iSC cell IL‐6 mRNA. The kinetics of IL‐6 production was confirmed in the mouse IL‐6‐dependent B9 bioassay and that activity could be neutralized with antisera to IL‐6. Expression of both the IL‐6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL‐6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL‐6 message is induced. IL‐6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL‐6 in response to PC12 cell coculture and to stimuli that induce IL‐6 production in the hematopoietic lineage. The induction of IL‐6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.
Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases
Authors: Hilmi Souad , Fournier Michel , Valeins Henri , Gandar Jean Claude , Bonnet Jacques
Published: July 2010

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO43− from [γ‐32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P‐phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C‐terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.
Enzymatic Oxidation of Dopamine: The Role of Prostaglandin H Synthase
Authors: Hastings Teresa G.
Published: July 2010

Abstract: An enzyme responsible for the oxidation of dopamine and formation of neuromelanin in brain has not been identified. Prostaglandin H synthase is prominent in brain and possesses peroxidase activity that may cooxidize dopamine to reactive dopamine quinones. This study examined the ability of purified prostaglandin H synthase to catalyze the oxidation of dopamine in vitro. Dopamine oxidation was determined by monitoring the formation of aminochrome and by examining catechol‐modified residues on protein present in the reaction mixture. Aminochrome was formed from dopamine in the presence of prostaglandin H synthase, and the reaction rate was dependent on the concentration of substrate and enzyme in the reaction mixture. Both arachidonic acid and hydrogen peroxide could serve as substrates for the prostaglandin H synthase‐catalyzed oxidation of dopamine. Indomethacin blocked the reaction when arachidonic acid was used as a substrate, but not when hydrogen peroxide was used. Enzymatically oxidized dopamine covalently bound to protein, as indicated by the presence of cysteinyl‐dopamine residues. Binding was significantly reduced in the absence of enzyme or in the presence of antioxidants. These results suggest that the peroxidase activity of prostaglandin H synthase is responsible for catalyzing the oxidation of dopamine to reactive dopamine quinones. It is possible that prostaglandin H synthase is responsible for the oxidation of dopamine and formation of neuromelanin in vivo, which may have implications for the development of Parkinson's disease. Furthermore, drugs such as aspirin that modulate the activity of this enzyme may provide a potential therapeutic approach for the prevention of Parkinson's disease.
Inhibition of Neuronal Nitric Oxide Synthase by 7‐Nitroindazole Protects Against MPTP‐Induced Neurotoxicity in Mice
Authors: Schulz Jörg B. , Matthews Russell T. , Muqit Miratul M. K. , Browne Susan E. , Beal M. Flint
Published: July 2010

Abstract: Several studies suggest that nitric oxide (NO•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7‐nitroindazole (7‐NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7‐NI dose‐dependently protected against MPTP‐induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7‐NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP‐induced depletions of both homovanillic acid and 3,4‐dihydroxyphenylacetic acid. 7‐NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO−), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3‐nitrotyrosine, which was attenuated by treatment with 7‐NI. Our results suggest that NO• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.
Cloning and Expression of a Betaine/GABA Transporter from Human Brain
Authors: Borden Laurence A. , Smith Kelli E. , Gustafson Eric L. , Branchek Theresa A. , Weinshank Richard L.
Published: July 2010

Abstract: A cDNA clone encoding a human γ‐aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT‐1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT‐1. The transporter displays high affinity for GABA (IC50 of 30 µM) and is also sensitive to phloretin, l‐2,4‐diaminobutyric acid, and hypotaurine (IC50 values of ∼150–400 µM). The osmolyte betaine is ∼25‐fold weaker than GABA, displaying an IC50 of ∼1 mM. The relative potencies of these inhibitors at human BGT‐1 differ from those of mouse and dog BGT‐1. Northern blot analysis reveals that BGT‐1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT‐1 will further our understanding of the roles of GABA and betaine in neural function.
Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis
Authors: Svensson B. , Ekström P. A. R. , Edström A.
Published: July 2010

Abstract: Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation‐dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury‐induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 nM) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating‐dephosphorylating reactions are critical for regeneration of peripheral nerves.
τ and Microtubule‐Associated Protein 2c Transfection and Neurite Outgrowth in ND 7/23 Cells
Authors: Langkopf Anja , Guilleminot Jocelyne , Nunez Jacques
Published: July 2010

Abstract: Neuronal hybrid ND 7/23 cells, which display sensorylike properties, develop neurites when cultured in the presence of either dibutyryl cyclic AMP plus nerve growth factor (DBcAMP + NGF) or retinoic acid or a phorbol ester derivative, although they express only trace amounts of the microtubule‐associated τ proteins and low levels of microtubule‐associated protein 2c (MAP2c). Nondifferentiated ND cells transfected with τ cDNAs did not develop neurites, whereas very short cell processes were formed in MAP2c‐transfected cells. τ and MAP2 antibodies labeled microtubule bundles displayed in a ring array underneath the surface of the transfected cells and short microtubules starting from the cell center. After differentiation in the presence of DBcAMP + NGF, the same bundle organization was observed in the transfected cells. In addition, τ and MAP2 antibodies stained a short section of the formed neurites. These data demonstrate that the expression of τ protein is not sufficient to induce neurite extension and that other proteins induced by morphogens are more important to initiate morphological differentiation of this cell line.
Inhibition of Drug‐Induced Apoptosis by Survival Factors in PC12 Cells
Authors: Lindenboim Liora , Haviv Ronit , Stein Reuven
Published: July 2010

Abstract: Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl‐2. We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum‐deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl‐2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl‐2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.
Accumulation of 3H‐Phosphoinositides Mediated by Muscarinic Receptors in the Developing Chick Retina: Inhibition of Carbachol‐Induced Response by Glutamate Receptors
Authors: Calvet Guilherme Amaral , Marques Ventura Ana Lucia
Published: July 2010

Abstract: In the present work we show the development of carbachol‐induced accumulation of 3H‐inositol phosphates (3H‐InsPs) in the chick embryonic retina and its regulation by glutamate receptors. Although basal levels of 3H‐InsPs increased during development, the retinal response to carbachol was high in the early developing stages and decreased after synaptogenesis in the retina. Eserine also stimulated the turnover of phosphoinositides in the embryonic but not in the mature retina. The effect of eserine could be blocked by atropine, suggesting that acetylcholine could be released from developing retina cells and further stimulate the turnover of InsPs in the embryonic tissue. Our data also show that muscarinic stimulation of turnover of 3H‐InsPs could be blocked by stimulation of glutamatergic ionotropic receptors. Moreover, the effect of glutamate agonists did not seem to be mediated by the release of other neurotransmitters such as GABA, glycine, adenosine, or dopamine from the tissue because these neurotransmitters did not interfere with the retinal response to carbachol. These results suggest that muscarinic activation of phosphoinositide turnover occurs mainly in the embryonic retina and that activation of glutamate receptors can inhibit directly the muscarinic stimulation of hydrolysis of 3H‐InsPs in this tissue.
Signal Flows from Two Phospholipase C‐Linked Receptors Are Independent in PC12 Cells
Authors: Suh Byung‐Chang , Lee Chin‐Ok , Kim Kyong‐Tai
Published: July 2010

Abstract: Bradykinin (BK) receptor and P2‐purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC‐linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca2+ spikes caused by BK and ATP in a single fura‐2‐loaded cell. BK‐ and ATP‐induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK‐induced homologous desensitization of CA secretion did not block the ATP‐induced secretion, and vice versa. Each agonist‐induced an increase in inositol 1,4,5‐trisphosphate (IP3) production and intracellular free Ca2+ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP3 production, internal Ca2+ mobilization, and Ca2+ influx were all additive. We also found that both IP3‐induced Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from extracellular space were able to release [3H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca2+. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.
Natriuretic Peptides Inhibit Nicotine‐Induced Whole‐Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors
Authors: Babinski K. , Haddad P. , Vallerand D. , McNicoll N. , De Léan A. , Ong H.
Published: July 2010

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C‐type natriuretic peptide (CNP) and found that these cells specifically express the ANF‐R1C but not the ANF‐R1A receptor subtype. Evidence for the existence of ANF‐R2 receptors was obtained from patch‐clamp experiments where C‐ANF, an ANF‐R2‐specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF‐R2 receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide‐bridged structure of C‐ANF. This linearized form of C‐ANF also displayed a lower binding affinity for ANF‐R2 receptors. Like the patch‐clamp studies, secretion experiments demonstrated that both CNP and C‐ANF are equally effective in reducing nicotine‐evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF‐R2 and not the ANF‐R1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine‐ nor KCI‐induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF‐R1C and ANF‐R2 receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole‐cell currents as well as nicotine‐induced catecholamine secretion, (3) the possible mediation of these effects by the ANF‐R2 class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF‐R2 receptor subtype.
Morphine Tolerance and Physical Dependence: Reversal of Opioid Inhibition to Enhancement of Cyclic AMP Formation
Authors: Wang Lin , Gintzler Alan R.
Published: July 2010

Abstract: This laboratory has previously demonstrated that the μ‐selective opiate receptor agonist sufentanil can produce a naloxone‐reversible increase or decrease in the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus, depending on the concentration of opioid used. On the basis of these results, it was suggested that μ‐opiate receptors are positively as well as negatively coupled to adenylyl cyclase. In the present study, the effect of chronic morphine exposure, in vivo, on the magnitude of electrically stimulated formation of cAMP and its modulation by sufentanil was investigated. In chronic morphine‐treated preparations, the magnitude of electrically stimulated cAMP formation, while in the presence of an inhibitory (10−6M) concentration of sufentanil, is indistinguishable from the formation that occurs in opiate‐naive preparations (in the absence of exogenous opioid). This indicates that the negative modulation of stimulated enteric cAMP formation by sufentanil manifests tolerance. Paradoxically, however, in “addicted tissue” the magnitude of the increase in cAMP formation produced by electrical stimulation in the presence of a previously inhibitory concentration of sufentanil is significantly larger than in its absence. Thus, the equivalence between the magnitude of stimulation‐induced increase in cAMP formation observed in naive versus tolerant/dependent tissue, while in the presence of sufentanil, is due to the ability of an originally inhibitory concentration of opioid to enhance or facilitate stimulated formation of cAMP. It is suggested that tolerance/dependence to the opioid inhibition of stimulated cAMP formation results not only from the loss of inhibitory potency but also from its reversal to enhancement.
Pertussis Toxin‐Sensitive G Protein α‐Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA‐N‐5 Cells
Authors: Li Xiaohua , Mumby Susanne M. , Greenwood Angela , Jope Richard S.
Published: July 2010

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin‐sensitive G protein α‐subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α‐subunits during differentiation of PC12 cells and human neuroblastoma LA‐N‐5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor‐induced differentiation. In contrast, αi2 but not αi1 increased when LA‐N‐5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 increased during differentiation of both PC12 and LA‐N‐5 cells. The level of 43‐kDa growth‐associated protein, a protein known to associate with αo, increased similarly to that of αo1. ADP‐ribosylation of αo, αi1, and αi2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of αq, which is not ADP‐ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.
N‐Terminal‐Specific Anti‐B‐50 (GAP‐43) Antibodies Inhibit Ca2+‐Induced Noradrenaline Release, B‐50 Phosphorylation and Dephosphorylation, and Calmodulin Binding
Authors: Hens Jacques J. H. , De Wit Marina , Boomsma Frans , Mercken Marc , Oestreicher A. Beate , Gispen Willem Hendrik , De Graan Pierre N. E.
Published: July 2010

Abstract: B‐50 (GAP‐43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin‐binding domain of B‐50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B‐50 into streptolysin O‐permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N‐terminal residues 39–43 of rat B‐50 dose‐dependently inhibited Ca2+‐induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C‐terminal‐directed (residues 132–213) anti‐B‐50 antibodies were without effect in the same dose range. NM2 inhibited PKC‐mediated B‐50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P‐B‐50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B‐50 in the absence of Ca2+. Similar concentrations of NM6 did not affect B‐50 phosphorylation or dephosphorylation or B‐50/calmodulin binding. We conclude that the N‐terminal residues 39–43 of the rat B‐50 protein play an important role in the process of Ca2+‐induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca2+‐ and phosphorylation‐dependent fashion.
Mechanism Underlying the ATP‐Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons
Authors: Sorimachi Masaru , Abe Yumiko , Furukawa Katsutoshi , Akaike Norio
Published: July 2010

Abstract: We examined the mechanism underlying the ATP‐induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons, using fura‐2 microfluorometry. The ATP‐induced increase in [Ca]in was dependent on external Ca2+, was blocked in a dose‐dependent manner by reactive blue 2, and was substantially inhibited by both L‐ and N‐type Ca2+ channel blockers. ATP was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine (100 µM), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na+ concentration as determined by sodium‐binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na+ influx through P2 purinoceptor‐associated channels resulting in membrane depolarization, which in turn increases Ca2+ influx through voltage‐dependent Ca2+ channels. However, ATP still caused a small increase in [Ca]in under Na+‐free conditions, and this [Ca]in increase was little affected by Ca2+ channel blockers. ATP also increased Mn2+ influx under Na+‐free conditions, as indicated by quenching of fura‐2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca2+ but also to Mn2+, in addition to monovalent cations.
Mobilization of Inositol 1,4,5‐Trisphosphate‐Sensitive Ca2+ Stores Supports Bradykinin‐ and Muscarinic‐Evoked Release of [3H]Noradrenaline from SH‐SY5Y Cells
Authors: Purkiss John R. , Nahorski Stefan R. , Willars Gary B.
Published: July 2010

Abstract: The human neuroblastoma cell line SH‐SY5Y, maintained at confluence for 14 days, released [3H]‐noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine‐evoked release was slower. When the extracellular [Ca2+] ([Ca2+]e) was buffered to ∼50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ∼50% with EC50 values of −5.46 ± 0.05 M and −7.46 ± 0.06 M (log10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were −6.00 ± 0.14 M for methacholine and −7.95 ± 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca2+‐ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins(1,4,5)P3‐sensitive stores is a major trigger of methacholine‐ and bradykinin‐evoked [3H]NA release in SH‐SY5Y cells.
Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A
Authors: Lynch David R. , Lawrence John J. , Lenz Shelley , Anegawa Norifusa J. , Dichter Marc , Pritchett Dolan B.
Published: July 2010

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125I‐MK‐801 binding assays and electrophysiological measures. Binding of 125I‐MK‐801 to cells transfected with NR 1a/2B is saturable with a KD of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK‐801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125I‐MK‐801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA‐induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.
Interferons Suppress Nerve Growth Factor Synthesis as a Result of Interference with Cell Growth in Astrocytes Cultured from Neonatal Mouse Brain
Authors: Awatsuji Hirofumi , Furukawa Yoshiko , Hirota Masao , Furukawa Shoei , Hayashi Kyozo
Published: July 2010

Abstract: Interferon (IFN)‐β and IFN‐γ inhibited the DNA synthesis and nerve growth factor (NGF) synthesis in growing astrocytes cultured from neonatal mouse brain, but they did not affect the NGF synthesis in quiescent astrocytes. IFN‐β and IFN‐γ also inhibited the enhanced DNA synthesis and NGF synthesis in growing astrocytes after the administration of basic fibroblast growth factor. These results indicated that NGF synthesis in astrocytes is regulated by IFNs associated with cell growth. The mechanism of IFN action on NGF synthesis/secretion is unknown, but the results that their effects last long after IFN removal from the cultures present the possibility that IFNs destabilize NGF mRNA.
Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons
Authors: Tanner Sandra L. , Storm Elaine E. , Bittner George D.
Published: July 2010

Abstract: Using video‐enhanced microscopy and a pulse‐radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish (Procambarus clarkii) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.
Cytokines Regulate Gelatinase A and B (Matrix Metalloproteinase 2 and 9) Activity in Cultured Rat Astrocytes
Authors: Gottschall Paul E. , Yu Xin
Published: July 2010

Abstract: Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium‐dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1‐day‐old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24‐h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin‐1α or ‐β, or tumor necrosis factor‐α for 24 h, a dose‐dependent increase in the activity of the 58‐ and 66‐kDa gelatinases and the induction of a 94‐kDa gelatinase occurred. All three astrocyte‐derived proteases showed maximal activity in the presence of millimolar levels of Ca2+, their activity was inhibited in the presence of 1,10‐phenanthroline, and their proenzymes were cleaved and activated after incubation with p‐aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58‐kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94‐kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94‐kDa gelatinase by lipopolysaccharide was not influenced when interleukin‐1 receptor antagonist was included during the 24‐h incubation period; however, the antagonist completely blocked interleukin‐1β‐induced 94‐kDa activity and diminished the activity of the 58‐ and 66‐kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin‐1β stimulation of the 94‐kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte‐derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.
β‐Adrenergic Modulation of Glial Inwardly Rectifying Potassium Channels
Authors: Roy M. L. , Sontheimer H.
Published: July 2010

Abstract: Cultured spinal cord astrocytes (2–13 days in vitro) express several different potassium current types, including delayed rectifier, transient A‐type, and inward rectifier (Kir) K+ currents. Of these, Kir is believed to be of critical importance in the modulation of extracellular [K+] in the CNS. Using the whole‐cell patch‐clamp technique, we analyzed modulation of Kir currents by β‐adrenergic receptor activation. The selective β‐adrenergic agonist isoproterenol (1–100 µM) and epinephrine (1–100 µM) each reduced peak Kir current amplitudes to 52.7 ± 12.5 and 63.6 ± 7.0%, respectively, at 100 µM. Forskolin (KD of ∼25 µM), an activator of adenylate cyclase (AC), and dibutyryl‐cyclic AMP (1 mM), a membrane‐permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]i, the product of AC, and resulted in similar reductions of Kir currents. By contrast, 1,9‐dideoxyforskolin (1–50 µM), a forskolin analogue that does not activate AC, did not affect Kir currents, indicating that AC activity is a required element for Kir modulation. Three inhibitors of PKA—Rp‐adenosine 3′,5′‐cyclic monophosphothioate, H‐7, and adenosine 3′,5′‐cyclic monophosphate‐dependent protein kinase inhibitor—failed to inhibit Kir current reduction by β‐adrenergic agonists. These results indicate that β‐adrenergic receptor ligands can modulate Kir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial Kir currents and thereby may affect glial K+“spatial buffering” in the CNS.
Functional Wild‐Type and Carboxy‐Terminal‐Tagged Rat Substance P Receptors Expressed in Baculovirus‐Infected Insect Sf9 Cells
Authors: Schreurs J. , Yamamoto R. , Lyons J. , Munemitsu S. , Conroy L. , Clark R. , Takeda Y. , Krause J. E. , Innis M.
Published: July 2010

Abstract: The rat substance P (SP) receptor (SPR) was expressed in insect Sf9 cells by infection with recombinant baculovirus. The receptor bound SP with high affinity (KD = 360 pM) and had a rank order of affinity of SP > neurokinin A > neurokinin B. Ligand activation of the receptor resulted in an increase in both inositol lipid hydrolysis and intracellular Ca2+ concentration ([Ca2+]i). However, high‐level expression of the receptor, in the absence of ligand, was correlated with increased basal turnover of inositol lipids and an elevated rate of Ca2+ influx. These results demonstrate that the Sf9 cells provide a suitable environment for the high‐level expression of a functionally active SPR. Two carboxy‐terminal epitope‐tagged receptors (SPR‐KT3 = SPR‐TPPPEPET, COOH; SPR‐Glu = SPR‐EEEEYMPME, COOH) were also expressed. The affinity of the KT3‐tagged receptor for ligand was similar to that of the wild‐type receptor (KD = 405 pM), and that of the Glu‐tagged receptor was slightly lower (KD = 1,082 pM). The high‐affinity SP binding site of all three receptors was sensitive to guanosine 5′‐O‐(3‐thiotriphosphate) pretreatment. The maximal signal‐transducing ability of the epitope‐tagged receptors was comparable to that of the wild‐type receptor ([Ca2+]i rise as a percentage of wild‐type: SPR‐KT3, 80–100%; SPR‐Glu, 88–100%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild‐type and tagged receptors.
Prostaglandin H Synthetase‐Mediated Metabolism of Dopamine: Implication for Parkinson's Disease
Authors: Mattammal Michael B. , Strong Randy , Lakshmi Vijaya M. , Chung Hyung D. , Stephenson Alan H.
Published: July 2010

Abstract: Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age‐ and post‐mortem interval‐matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E2 (PGE2, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE2 synthesis. Dopamine stimulated PHS synthesis of PGE2. [3H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H2O2‐supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA‐mediated cooxidation of dopamine but not H2O2‐mediated metabolism. PHS‐mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. 32P‐postlabeling was used to detect dopamine‐DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine‐DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/µg DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.
Neuropathy Target Esterase of Hen Brain: Active Site Reactions with 2‐[octyl‐3H]Octyl‐4H‐1,3,2‐Benzodioxaphosphorin 2‐Oxide and 2‐Octyl‐4H‐1,3,2‐[aryl‐3H] Benzodioxaphosphorin 2‐Oxide
Authors: Yoshida Minoru , Tomizawa Motohiro , Wu Shao‐Yong , Quistad Gary B. , Casida John E.
Published: July 2010

Abstract: 2‐Octyl‐4H‐1,3,2‐benzodioxaphosphorin 2‐oxide (octyl‐BDPO) is one of the most potent inhibitors known for neuropathy target esterase (NTE) of hen brain with 50% inhibition at 0.2 nM. Two NTE‐like proteins, i.e., resistant to paraoxon and sensitive to mipafox, of ∼155 and ∼119 kDa (designated NTE‐155 and NTE‐119, respectively) are labeled by [octyl‐3H]octyl‐BDPO and separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Labeling with [aryl‐3H]octyl‐BDPO is only ∼15% of that with [octyl‐3H]octyl‐BDPO, indicating that the majority of the phosphorylated NTE undergoes aging with only a small proportion of nonaged target or intramolecular group transfer (“alkylation”). NTE‐155 and NTE‐119 have the same kinetic constants and maximal number of phosphorylation sites, equivalent for each of them to 26 fmol/mg of protein and totalling at least 0.44–1.2 µg of NTE protein/g of brain. Structure‐activity investigations involving 17 combinations of organophosphorus (OP) compounds of varied chemical type, stereo‐chemistry, and concentration establish an excellent correlation (r = 0.95) between inhibition of NTE activity and protein labeling and thereby the toxicological relevance of these assays, which equally implicate NTE‐155 and NTE‐119 (probably an autolysis product of NTE‐155) as targets in OP‐induced delayed neuropathy. [octyl‐3H]‐Octyl‐BDPO is an improved probe for NTE in terms of its potency, reactivity, selectivity, and the formation of 3H‐labeled NTE with a stable phosphorus‐carbon bond.
Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade
Authors: Grimaldi Maurizio , Arcone Rosaria , Ciliberto Gennaro , Schettini Gennaro
Published: July 2010

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short‐term phorbol 12‐myristate 13‐acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12‐myristate 13‐acetate enhanced concentration‐ and time‐dependently interleukin 6 release. This stimulatory action of phorbol 12‐myristate 13‐acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration‐related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12‐myristate 13‐acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12‐myristate 13‐acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.
Induction of Functional Interleukin‐2 Receptor in Mouse Microglia
Authors: Sawada Makoto , Suzumura Akio , Marunouchi Tohru
Published: July 2010

Abstract: Interleukin (IL)‐2, initially discovered for its mitogenic activity on T cells, also acts on monocytes, resulting in the activation of cytokine production, superoxide production, and tumoricidal activity. Because severe brain damage was observed in IL‐2‐transgenic mice, this cytokine may have some influence(s) on the cells of the CNS. We investigated IL‐2 receptor‐bearing cells in the CNS and found that activated microglia expressed α‐chain mRNA and immunoreactive IL‐2 receptor β‐chain protein in culture. Although microglia did not express IL‐2 receptors under normal culture conditions, they were induced to express these receptors by lipopolysaccharide (LPS) in a time‐dependent manner. The IL‐2 receptors were found to be functional because the viability and growth activity of LPS‐treated microglia, but not untreated controls, increased in response to recombinant mouse IL‐2 as determined by a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide colorimetric assay and bromodeoxyuridine uptake experiment, respectively. These effects of recombinant IL‐2 were blocked by pretreatment with anti‐mouse IL‐2 receptor β‐chain antibody. Our findings suggest that activated microglia in the CNS can respond to this T cell‐derived factor regulating their growth, which may be an important mechanism of communication between nervous and immune systems in physiological and pathological conditions.
Molecular Characterization of a Neuronal‐Specific Protein that Stimulates the Activity of Cdk5
Authors: Shetty K. T. , Kaech S. , Link W. T. , Jaffe H. , Flores C. M. , Wray S. , Pant H. C. , Beushausen S.
Published: July 2010

Abstract: Cyclin‐dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and τ protein phosphorylation. This report describes the characterization of a 62‐kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full‐length cDNA was characterized coding for a 67.5‐kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose‐dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino‐terminal peptide‐specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.