bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Analysis and Tissue‐Specific Expression of Drosophila Na+,K+‐ATPase Subunits
Authors: Sun Banghua , Wang Weiya , Salvaterra Paul M.
Published: July 2010

Abstract: We have previously purified and characterized a nervous system‐specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the β subunits of Na+,K+‐ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+‐ATPase α subunit cDNA clone (PSα; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+‐ATPase activity when PSα mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+‐ATPase subunits in a tissue‐ and cell type‐specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+‐ATPase expression in various cell and tissue types.
Neurotrophic Factors Prevent Ceramide‐Induced Apoptosis Downstream of c‐Jun N‐Terminal Kinase Activation in PC12 Cells
Authors: Hartfield Perry J. , Bilney Andrea J. , Murray Andrew W.
Published: July 2010

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum‐free medium. The present study determines whether neurotrophic factors can prevent ceramide‐induced apoptosis in PC12 cells and investigates the role that c‐Jun N‐terminal kinase (JNK) activation may play in this system. Ceramide‐induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase‐activating peptide, 4‐(8‐chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐dl‐Asp fluoromethyl ketone (zVAD‐FMK). It was surprising that inhibition of extracellular signal‐regulated kinase and/or phosphatidylinositol 3‐kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide‐induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time‐dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD‐FMK attenuated ceramide‐stimulated JNK activation. Further experiments indicated that ceramide‐induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD‐FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide‐induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide‐induced apoptosis by affecting signaling events downstream of JNK activation.
Endothelin‐Stimulated Capacitative Calcium Entry in Enteric Glial Cells: Synergistic Effects of Protein Kinase C Activity and Nitric Oxide
Authors: Zhang Weizhen , Sarosi George A. , Barnhart Douglas C. , Mulholland Michael W.
Published: July 2010

Abstract: Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 nM) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni2+ (89 ± 2%) and by La3+ (78 ± 2%) but was not affected by L‐, N‐, or P‐type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose‐dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor NG‐nitro‐l‐arginine decreased calcium influx in a dose‐dependent manner. The combination of chelerythrine and NG‐nitro‐l‐arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum‐inhibitable channels through a process regulated by protein kinase C and nitric oxide.
Tissue Transglutaminase Is an In Situ Substrate of Calpain: Regulation of Activity
Authors: Zhang Jianwen , Guttmann Rodney P. , Johnson Gail V. W.
Published: July 2010

Abstract: Tissue transglutaminase (tTG) is a calcium‐dependent enzyme that catalyzes the transamidation of specific polypeptide‐bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium‐activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine‐5′‐O‐(3‐thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH‐SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an ∼50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.
The Peptide Transporter PepT2 Mediates the Uptake of the Glutathione Precursor CysGly in Astroglia‐Rich Primary Cultures
Authors: Dringen Ralf , Hamprecht Bernd , Bröer Stefan
Published: July 2010

Abstract: The intracellular content of glutathione in astroglia‐rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24‐h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200‐fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200‐fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia‐rich primary cultures was demonstrated by application of RT‐PCR. These results demonstrate that PepT2 is expressed in astroglia‐rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.
Dynamic Regulation of the Activated, Autophosphorylated State of Ca2+/Calmodulin‐Dependent Protein Kinase II by Acute Neuronal Excitation In Vivo
Authors: Yamagata Yoko , Obata Kunihiko
Published: July 2010

Abstract: Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has been implicated in various neuronal functions, including synaptic plasticity. To examine the physiological regulation of its activated, autophosphorylated state in relation to acute neuronal excitation in vivo, we studied the effect of electroconvulsive treatment in rats on CaMKII activity and in situ autophosphorylation levels. As early as 30 s after the electrical stimulation, a profound but transient decrease in its Ca2+/calmodulin‐independent activity, as well as in the level of its autophosphorylation at Thr286 (α)/Thr287 (β) measured by using phosphorylation state‐specific antibodies, was observed in homogenate from hippocampus and parietal cortex, which was reversible in 5 min. In the later time course, a moderate, reversible increase, which peaked at around 60 min after the electrical stimulation, was observed in parietal cortex but not in hippocampus. The early‐phase decrease was found to occur exclusively in the soluble fraction. In addition, partial translocation of CaMKII from the soluble to the particulate fraction seems to have occurred in this early phase. Thus, the activated, autophosphorylated state of CaMKII is under dynamic and precise regulation in vivo, and its regulatory mechanisms seem to have regional specificity.
Evidence for the Lack of Involvement of Sphingomyelin Hydrolysis in the Tumor Necrosis Factor‐Induced Secretion of Nerve Growth Factor in Primary Astrocyte Cultures
Authors: Galve‐Roperh Ismael , Sánchez Cristina , Ségui Bruno , Haro Amador , Díaz‐Laviada Inés , Levade Thierry
Published: July 2010

Abstract: The signal mechanism underlying tumor necrosis factor α (TNFα) up‐regulation of nerve growth factor (NGF) production was studied in primary rat astrocyte cultures. Because ceramide is also able to induce NGF secretion and because TNFα is a known agonist of the sphingomyelin (SPM)‐ceramide pathway, we investigated whether the TNFα‐induced NGF secretion by primary astrocytes is mediated by ceramide. TNFα stimulation of NGF secretion was shown to be independent of protein kinase C, abrogated by the tyrosine phosphoprotein phosphatase inhibitor phenylarsine oxide (PAO), and independent of the activation of the mitogen‐activated protein kinase (MAPK) cascade. In marked contrast, inhibition of MAPK counteracted the NGF secretion induced by ceramide. TNFα stimulation of the nuclear transcription factor NF‐κB was prevented by cell pretreatment with PAO, whereas ceramide and sphingomyelinase had a marginal effect on NF‐κB activation. Moreover, TNFα failed to activate the SPM pathway, as indicated by the lack of SPM degradation and the absence of ceramide generation. To clarify further the role of NF‐κB in NGF synthesis, electrophoretic mobility shift assays were performed with an NF‐κB site from the NGF promoter. The absence of significant binding of NF‐κB to the NGF gene promoter indicates the existence of an indirect role of NF‐κB in the regulation of NGF synthesis. Altogether, our data strongly suggest that TNFα‐mediated up‐regulation of NGF occurs independently of ceramide generation.
Inhibition of Dopamine Release by Prostaglandin EP3 Receptor via Pertussis Toxin‐Sensitive and ‐Insensitive Pathways in PC12 Cells
Authors: Nakamura Kazuhiro , Katoh Hironori , Ichikawa Atsushi , Negishi Manabu
Published: July 2010

Abstract: Prostaglandin EP3 receptor is involved in the inhibition of neurotransmitter release from presynaptic nerve terminals in various tissues. We have examined the regulation of neurotransmitter release by the EP3 receptor using a PC12 cell line that stably expresses the EP3B receptor isolated from bovine adrenal medulla. In the cells, M&B28767, an EP3 agonist, inhibited the 50 mM KCl‐ or 10 nM bradykinin‐induced [3H]dopamine release in a concentration‐dependent manner (10 pM to 0.1 µM). This inhibition was partially reversed by pretreatment with pertussis toxin, whereas under the same condition, the agonist‐induced inhibition of forskolin‐stimulated cyclic AMP accumulation was suppressed completely. In contrast, M&B28767 did not affect the high K+‐ or bradykinin‐induced increase in intracellular Ca2+ concentration. Moreover, M&B28767 also inhibited the [3H]dopamine release induced by the Ca2+ ionophore ionomycin, and this inhibition was also partially reversed by pretreatment with pertussis toxin. These results indicate that the EP3 receptor is coupled to dual pathways, pertussis toxin‐sensitive and ‐insensitive G‐protein pathways, to regulate neurotransmitter release without changing Ca2+ influx in neuronal cells.
Coupled Effects of Mass Transfer and Uptake Kinetics on In Vivo Microdialysis of Dopamine
Authors: Yang Hua , Peters Jennifer L. , Michael Adrian C.
Published: July 2010

Abstract: Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug‐naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high‐affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.
Mrp1 Multidrug Resistance‐Associated Protein and P‐Glycoprotein Expression in Rat Brain Microvessel Endothelial Cells
Authors: Regina Anthony , Koman Ahmet , Piciotti Michèle , El Hafny Bouchra , Center Melvin S. , Bergmann Ralf , Couraud Pierre‐Olivier , Roux Françoise
Published: July 2010

Abstract: Two membrane glycoproteins acting as energy‐dependent efflux pumps, mdr‐encoded P‐glycoprotein (P‐gp) and the more recently described multidrug resistance‐associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P‐gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood‐brain barrier, but localization of MRP has not been well characterized yet. Using RT‐PCR and immunoblot analysis, we have compared the expression of P‐gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P‐gp‐encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P‐gp, is not predominantly expressed in the blood‐brain barrier endothelial cells and that Mrp1 and the mdr1b P‐gp isoform may be present in other cerebral cells.
Acetylcholinesterase Is Increased in the Brains of Transgenic Mice Expressing the C‐Terminal Fragment (CT100) of the β‐Amyloid Protein Precursor of Alzheimer's Disease
Authors: Sberna Gian , Sáez‐Valero Javier , Li Qiao‐Xin , Czech Christian , Beyreuther Konrad , Masters Colin L. , McLean Catriona A. , Small David H.
Published: July 2010

Abstract: Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid β protein (Aβ). The aim of this study was to investigate AChE expression using a transgenic mouse model of Aβ overproduction. The β‐actin promoter was used to drive expression of a transgene encoding the 100‐amino acid C‐terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full‐length human APP CT100 and Aβ were overexpressed in the brain. Levels of salt‐extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (GA1) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of GA1 AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.
Potentiation of Astroglial Nitric Oxide Synthase Type‐2 Expression by Lithium Chloride
Authors: Feinstein Douglas L.
Published: July 2010

Abstract: The mechanisms underlying the antimanic effects of lithium are largely unknown but may involve long‐term changes in brain gene expression. To determine if lithium could modify gene expression in astrocytes, the predominant cell type in brain, we tested the effects of LiCl on expression of nitric oxide synthase type 2 (NOS‐2) in cultured glial cells. Incubation of primary rat astrocytes with endotoxin [lipopolysaccharide (LPS)] and proinflammatory cytokines induced NOS‐2 gene and protein expression, as assessed by nitrite production and measurement of l‐citrulline synthesis in whole cell lysates. Incubation with LiCl, but not KCl, increased NOS‐2 activity up to 1.6‐fold. LiCl also potentiated (up to 2.7‐fold) the induction of NOS‐2 expression by LPS plus interferon‐γ in C6 glioma cells but had little effect on LPS‐induced nitrite accumulation from mouse RAW 264.7 macrophages. LiCl increased NOS‐2 mRNA steady‐state levels, suggesting an effect on mRNA stability and/or NOS‐2 gene transcription. These results demonstrate that LiCl can modify astroglial gene expression and suggest that chronic treatment with lithium could exacerbate inflammatory responses in brain glial cells.
D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin‐Sensitive G Proteins and Ras‐Involved ERK and SAP/JNK Pathways in Rat C6‐D2L Glioma Cells
Authors: Luo Yongquan , Kokkonen Gertrude C. , Wang Xiantao , Neve Kim A. , Roth George S.
Published: July 2010

Abstract: Dopamine D2 receptors are members of the G protein‐coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) pathways through a mechanism involving D2 receptor‐G protein complexes and the Ras GTP‐binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA‐induced kinase activation was prevented by either (+)‐butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (−)‐butaclamol, the inactive isomer of (+)‐butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA‐induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA‐induced JNK activation and subsequent c‐Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras‐dependent mitogen‐activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA‐induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c‐Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor‐stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.
Plasticity of Agonist Binding Sites in Hetero‐Oligomers of the Unitary Glutamate Receptor Subunit XenU1
Authors: Soloviev Mikhail M. , Abutidze Ketevan , Mellor Ian , Streit Peter , Grishin Eugene V. , Usherwood Peter N. , Barnard Eric A.
Published: July 2010

Abstract: Two subunits from Xenopus, XenNR1G and the “short” subunit XenU1, have previously been coexpressed to form a unitary (NMDA/non‐NMDA type) glutamate receptor. We now show that an antibody to XenNR1G or an antibody to XenU1 precipitates the binding sites of both XenNR1G and XenU1, with the recombinant subunits or with solubilised Xenopus brain membranes, i.e., the combination occurs in vivo. The expressed XenU1 subunits are in the cell membrane and oriented correctly. XenU1 binds not only kainate with high affinity (KD 1.2 nM at 25°C), but also the glycine site antagonist 5,7‐dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPγS displaces competitively all of the bound [3H]kainate, but glycine has no effect. The results suggest that a common binding site for kainate, DCKA, and GTP can exist on XenU1. In the XenNR1G/XenU1 complex, the kainate affinity is lowered eightfold, whereas the DCKA affinity is considerably increased (KD 147 nM). Only 18% of the binding to the complex has the properties of the NMDA receptor glycine site, the rest being due to switching of the high‐affinity kainate site of XenU1 (low‐affinity DCKA) to a high‐affinity DCKA (low‐affinity kainate) conformation. Surprisingly, a mammalian NR2 subunit can also combine with XenU1, and this introduces similar reciprocal changes in the binding of kainate and DCKA. The combined evidence suggests a common basic mode of agonist site formation in different subunit types of the ionotropic glutamate receptors.
Nociceptin (ORL‐1) and μ‐Opioid Receptors Mediate Mitogen‐Activated Protein Kinase Activation in CHO Cells Through a Gi‐Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist‐Mediated Desensitization
Authors: Hawes Brian E. , Fried Steven , Yao Xiaorui , Weig Blair , Graziano Michael P.
Published: July 2010

Abstract: The recently identified 17‐amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor‐like‐1 (ORL‐1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL‐1 receptor (OFQR) may be to modulate opioid‐induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL‐1 (OFQR) modify opioid‐stimulated effects, however, is unclear. Both ORL‐1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)‐sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen‐activated protein (MAP) kinase in CHO cells expressing ORL‐1 receptor (OFQR). Nociceptin (OFQ)‐stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of β‐adrenergic receptor kinase (βARKct), which specifically blocks Gβγ‐mediated signaling. Expression of the proline‐rich domain of SOS (SOS‐PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)‐stimulated MAP kinase activation. The phosphatidylinositol 3‐kinase (PI‐3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)‐stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing μ‐opioid receptor, [d‐Ala2,N‐Me‐Phe4,Gly‐ol]‐enkephalin (DAMGO; a μ‐opioid receptor‐selective agonist) stimulated PTX‐sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS‐PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL‐1 (OFQR) and μ‐opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ‐subunit of Gi, a PI‐3K, and SOS, independent of PKC activity. In cells expressing both ORL‐1 (OFQR) and μ‐opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)‐ or DAMGO‐stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO‐stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)‐stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL‐1 (OFQR) can modulate μ‐opioid receptor signaling in a cellular system.
Inhibition of Neuronal Apoptosis by a Metalloporphyrin Superoxide Dismutase Mimic
Authors: Patel Manisha
Published: July 2010

Abstract: The objective of this study was to determine whether free radicals play a pathogenic role in neuronal apoptosis. The ability of Mn(III) tetrakis(benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimic, to inhibit staurosporine‐induced neuronal apoptosis was tested in mixed cerebrocortical cultures. Staurosporine produced concentration‐dependent cell death that was markedly inhibited by MnTBAP. Immunocytochemical analyses of cultures for neuron‐ and astrocyte‐specific markers revealed that high concentrations of staurosporine induced the death of both neurons and astrocytes; both cell types were protected by MnTBAP. A less active congener of MnTBAP failed to protect cells against staurosporine‐induced apoptosis. MnTBAP also protected cortical cultures against ceramide‐induced apoptosis. These results support a role for oxidative stress in neuronal apoptosis.
Lack of Correlation Between the Effects of Transient Exposure to Glutamate and Those of Hypoxia/Reoxygenation in Immature Neurons In Vitro
Authors: Chihab Rifki , Bossenmeyer Carine , Oillet Jean , Daval Jean‐Luc
Published: July 2010

Abstract: To assess the influence of brain immaturity on the effects of oxygen deprivation and the participation of excitotoxicity, the consequences of a 6‐h exposure to either hypoxia (95% N2/5% CO2) or 100 µM glutamate were studied in cultured fetal rat forebrain neurons taken at two maturational stages, i.e., 6 and 13 days in vitro. Cells were examined for their morphology, viability, energy metabolism reflected by 2‐d‐[3H]deoxyglucose uptake, and protein synthesis assessed by [3H]leucine incorporation. Apoptosis and necrosis were scored using the fluorescent dye 4,6‐diamidino‐2‐phenylindole. Whereas 6‐day‐old neurons responded to a 6‐h hypoxia by transient hypermetabolism, biphasic increase in protein synthesis, and cycloheximide‐sensitive apoptotic death within 72 h postexposure, glutamate did not affect cell characteristics by the same time. In 13‐day‐old neurons, hypoxia induced both apoptosis (8.2%) and necrosis (22.3%). At this age, glutamate definitely reduced energy metabolism (26%) and protein synthesis (17%) by the end of exposure. The percentage of necrotic neurons reached 40.7%, but the rate of apoptosis was unchanged compared with controls. Therefore, excitotoxicity cannot account for hypoxia‐induced injury in immature neurons, but its participation is suggested in older cells by the suppression of the necrotic component of hypoxia by glutamate receptor antagonists at 13 days.
Binding of Protein Kinase C Isoforms to Actin in Aplysia
Authors: Nakhost Arash , Forscher Paul , Sossin Wayne S.
Published: July 2010

Abstract: Recently, two of the 10 vertebrate protein kinase C (PKC) isoforms, PKCβII and PKCε, have been shown to bind specifically to actin filaments, suggesting that these kinases may regulate cytoskeletal dynamics. Here, we present evidence that two PKCs from the marine mollusk Aplysia californica, PKC Apl I, a Ca2+‐activated PKC, and PKC Apl II, a Ca2+‐independent PKC most similar to PKCε and η, also bind F‐actin. First, they both cosedimented with purified actin filaments in a phorbol ester‐dependent manner. Second, they both translocated to the Triton‐insoluble fraction of the nervous system after phorbol ester treatment. PKC Apl II could also partially translocate to actin filaments and associate with the Triton‐insoluble fraction in the absence of phorbol esters. Translocation to purified actin filaments was increased in the presence of a PKC inhibitor, suggesting that PKC phosphorylation reduces PKC bound to actin. Although both kinases bound F‐actin, actin was not sufficient to activate the kinases. In support of a physiological role for actin‐PKC interactions, immunochemical localization of PKC Apl II in neuronal growth cones revealed a striking colocalization with F‐actin. Our results are consistent with a role for actin‐PKC interactions in regulating cytoskeletal dynamics in Aplysia.
γ‐Aminobutyric AcidA Receptor Function Is Desensitised in Rat Cultured Cerebellar Granule Cells Following Chronic Flunitrazepam Treatment
Authors: Brown Maria J. , Wood Martyn D. , Coldwell Martyn C. , Bristow David R.
Published: July 2010

Abstract: This study examined γ‐aminobutyric acidA (GABAA) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced (p < 0.05) the maximal GABA‐stimulated increase in extracellular acidification rate (Emax) (16.5 ± 1.2% and 11.3 ± 1.0%, 2‐day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7‐day control and treated cells, respectively; best‐fit Emax± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 ± 1.7 and 2.3 ± 1.8 µM, 2‐day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µM, 7‐day control and treated cells, respectively; best‐fit EC50± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABAA receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle‐treated cells. The results suggest that changes in GABAA receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam‐mediated desensitisation of GABAA receptor function.
IMP‐GMP 5′‐Nucleotidase from Rat Brain: Activation by Polyphosphates
Authors: Marques Agostinho F. P. , Teixeira Natércia A. , Gambaretto Claudio , Sillero Antonio , Günther Sillero Maria A.
Published: July 2010

Abstract: IMP‐GMP 5′‐nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 ± 1,509) in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.5–6.8; relative velocity measured in the presence of MgCl2, MnCl2, CoCl2, and NiCl2 (2 mM), 100, 60, 11, and <1, respectively; preferred substrates, IMP and GMP; and activation constant (Ka) found for Ap4A, Ap5A, and Ap6A, 83 ± 38, 77 ± 32, and 57 ± 12 µM, respectively. Under assay conditions where activation by Ap4A was fivefold, the activation produced by dinucleotides was as follows: Ap4G (4.0), Ap4I (2.9), Ap4X (3.3), Ap4C (0.7), Ap4U (1.1), Ap4εA (1.5), Ap4ddA (1.7), Gp4G (2.2), Ap3A (1.1), and Ap2A (1.2). Polyphosphates P18, P19, P20, and P35 were activators of the reaction with calculated Ka values of 3.5 ± 0.5, 0.9 ± 0.2, 0.6 ± 0.2, and 1.3 ± 0.5 µM, respectively. The following compounds, at 0.1 mM, were effectors of the phosphotransferase reaction producing the fold activation indicated: Ap4A (8.3), Ap5A (10.2), Ap6A (10.1), Ap4G (7.7), Ap4X (7.6), Ap4U (2.1), glycerate 2,3‐bisphosphate (3.9), and unpurified P15 (7.6). Two enzyme forms of IMP‐GMP 5′‐nucleotidase were detected when the extracts from rat tissues or from the crustacean Artemia were subjected to chromatography on a Dyematrex Green A column. The ratio of the hydrolytic activities under both peaks (peak I/peak II) was as follows: brain (1.5), heart (1.9), liver (1.6), lung (2.0), testis (3.8), and Artemia cysts (2.0).
Carrier‐Mediated Release, Transport Rates, and Charge Transfer Induced by Amphetamine, Tyramine, and Dopamine in Mammalian Cells Transfected with the Human Dopamine Transporter
Authors: Sitte H. H. , Huck S. , Reither H. , Boehm S. , Singer E. A. , Pifl C.
Published: July 2010

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d‐ and l‐amphetamine as well as substrate‐induced release of preloaded N‐methyl‐4‐[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium‐dependent and was inhibited by ouabain and cocaine, which also prevented substrate‐induced release of MPP+. Patch‐clamp recordings revealed that all four substrates elicited voltage‐dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch‐clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d‐amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate‐induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.
GABABR1a/R1b‐Type Receptor Antisense Deoxynucleotide Treatment of Melanotropes Blocks Chronic GABAB Receptor Inhibition of High Voltage‐Activated Ca2+ Channels
Authors: Morris Stephen J. , Beatty Diane M. , Chronwall Bibie M.
Published: July 2010

Abstract: GABAB and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage‐activated Ca2+ channels (HVA‐CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA‐CC activity by down‐regulation. Here we report that chronic GABAB, but not GABAA, agonist treatment also resulted in HVA‐CC inhibition. Two GABAB receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK‐293 cells. We have constructed an antisense deoxynucleotide knockdown‐type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABAB antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA‐CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABAB receptor, which, like the D2 receptor, is coupled to chronic HVA‐CC inhibition in melanotropes.
Cloning and Characterization of a Novel Form of Tyrosine Hydroxylase from the Human Parasite, Schistosoma mansoni
Authors: Hamdan Fadi F. , Ribeiro Paula
Published: July 2010

Abstract: Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate‐limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced‐leader (SL) sequence at its 5′ end, whereas the other does not appear to be trans‐spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N‐terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified ∼52‐fold to apparent homogeneity and had a final specific activity of 0.78 µmol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.
Glutamatergic Regulation of Basal and Stimulus‐Activated Dopamine Release in the Prefrontal Cortex
Authors: Takahata Ryuichi , Moghaddam Bita
Published: July 2010

Abstract: The present study was undertaken to determine whether basal and stimulus‐activated dopamine release in the prefrontal cortex (PFC) is regulated by glutamatergic afferents to the PFC or the ventral tegmental area (VTA), the primary source of dopamine neurons that innervate the rodent PFC. In awake rats, blockade of NMDA or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors in the VTA, or blockade of AMPA receptors in the PFC, profoundly reduced dopamine release in the PFC, suggesting that the basal output of dopamine neurons projecting to the PFC is under a tonic excitatory control of NMDA and AMPA receptors in the VTA, and AMPA receptors in the PFC. Consistent with previous reports, blockade of cortical NMDA receptors increased dopamine release, suggesting that NMDA receptors in the PFC exert a tonic inhibitory control on dopamine release. Blockade of NMDA or AMPA receptors in the VTA as well as blockade of AMPA receptors in the PFC reduced the dopaminergic response to mild handling, suggesting that activation of glutamate neurotransmission also regulates stimulus‐induced increase of dopamine release in the PFC. In the context of brain disorders that may involve cortical dopamine dysfunction, the present findings suggest that abnormal basal or stimulus‐activated dopamine neurotransmission in the PFC may be secondary to glutamatergic dysregulation.
Hypoxic Cell Death in Human NT2‐N Neurons: Involvement of NMDA and Non‐NMDA Glutamate Receptors
Authors: Rootwelt Terje , Dunn Michelle , Yudkoff Marc , Itoh Takayuki , Almaas Runar , Pleasure David
Published: July 2010

Abstract: Human NTera2 teratocarcinoma cells were differentiated into postmitotic NT2‐N neurons and exposed to hypoxia for 6 h. The cultures were evaluated microscopically, and percent lactate dehydrogenase (LDH) release after 24 and 48 h was used as an assay for cell death. After 48 h LDH release was 24.3 ± 5.6% versus 13.8 ± 3.7% in controls (p < 0.001). Cell death was greatly diminished by MK‐801 pretreatment (15.4 ± 5.1%, p < 0.001). If glutamate was omitted from the medium, glutamate levels after 6 h of hypoxia were reduced from 101 ± 63 to 2.3 ± 0.3 µM, and cell death at 48 h was also markedly reduced (15.4 ± 4.5%, p < 0.001). The α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (18.7 ± 5.1%, p < 0.001) and mild hypothermia (33.5–34°C) during hypoxia (19.5 ± 2.75, p < 0.05) were moderately protective. Basic fibroblast growth factor (24.1 ± 3.2%), the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester (22.8 ± 8.1%), the antioxidant N‐tert‐butyl‐o‐phenylnitrone (18.9 ± 5.9%), and the 21‐aminosteroid U74389G (24.0 ± 3.4%) did not protect the cells. N‐Acetyl‐l‐cysteine even tended to increase cell death (30.1 ± 2.5%, p = 0.06). Treatment with MK‐801 at the end of hypoxia did not reduce cell death (23.3 ± 2.3%). In separate experiments, a 15‐min exposure to 1 mM glutamate without hypoxia did not result in significant cell death (14.7 ± 2.4 vs. 12.2 ± 2.1%,p = 0.07). We conclude that, although somewhat resistant to glutamate toxicity when normoxic, NT2‐N neurons die via an ionotropic glutamate receptor‐mediated mechanism when exposed to hypoxia in the presence of glutamate. As far as we know, this is the first reported analysis of the mechanism of hypoxic cell death in cultured human neuronlike cells.
Proton‐Decoupled 31P Magnetic Resonance Spectroscopy Reveals Osmotic and Metabolic Disturbances in Human Hepatic Encephalopathy
Authors: Bluml Stefan , Zuckerman Eli , Tan Jeannie , Ross Brian D.
Published: July 2010

Abstract: Quantitative proton and quantitative proton‐decoupled 31P magnetic resonance spectroscopy (MRS) of the brain was performed in 16 patients with liver disease (10 with and six without chronic hepatic encephalopathy) and four patients with hyponatremia, as well as 20 age‐matched normal subjects. Patients with hepatic encephalopathy were distinguished from controls by significant reduction in levels of cerebral nucleoside triphosphate (2.45 ± 0.20 vs. 2.91 ± 0.21 mmol/kg of brain; p < 0.0003), inorganic phosphate (p < 0.03), and phosphocreatine (p < 0.04). In addition of increased levels of cerebral glutamate plus glutamine and decreased concentrations of myo‐inositol, patients with hepatic encephalopathy showed a reduction of total visible choline and of glycerophosphoryl‐choline (0.67 ± 0.13 vs. 0.92 ± 0.20 mmol/kg of brain in controls; p < 0.005) in 1H MRS, and of glycerophosphoryl‐ethanolamine (0.40 ± 0.12 vs. 0.68 ± 0.12 mmol/kg of brain in controls; p < 0.0003) in proton‐decoupled 31P MRS. Of the reduction of “total choline,” 61% was accounted for by glycerophosphorylcholine, a cerebral osmolyte. Similar metabolic abnormalities were seen in hyponatremic patients. The results are consistent with disturbances of cerebral osmoregulation and energy metabolism in patients with chronic hepatic encephalopathy.
Activation of JNK Pathway and Induction of Apoptosis by Manganese in PC12 Cells
Authors: Hirata Yoko , Adachi Kayo , Kiuchi Kazutoshi
Published: July 2010

Abstract: Manganese is known to induce neurological disorders similar to parkinsonisms. A dopamine deficiency has been demonstrated in Parkinson's disease and in chronic manganese poisoning, suggesting that the mechanisms underlying the neurotoxic effects of the metal ion are related to a functional abnormality of the extrapyramidal system. However, the details have yet to be elucidated. Here we report that manganese causes characteristic internucleosomal DNA fragmentation, a biochemical hallmark of apoptosis, in PC12 cells. It was transcription dependent, relatively specific for manganese, and blocked in Bcl‐2‐overexpressed PC12 cells. The results indicate that apoptosis may play a role in the dopaminergic neurotoxicity associated with manganese, the first metal to be reported to induce this form of cell death. The early biochemical events show the impairment of energy metabolism, and the process may require new synthesis of proteins such as c‐Fos and c‐Jun. In addition, manganese induces phosphorylation of c‐Jun at Ser63 and Ser73 and SEK1/MKK4 (c‐Jun N‐terminal kinase kinase) at Thr258 and tyrosine phosphorylation of several proteins. These results indicate that manganese activates specific signal cascades including the c‐Jun N‐terminal kinase pathway.
In Vivo Aggregation of β‐Amyloid Peptide Variants
Authors: Fay David S. , Fluet Amy , Johnson Carolyn J. , Link Christopher D.
Published: July 2010

Abstract: Transgenic Caenorhabditis elegans animals have been engineered to express wild‐type and single‐amino acid variants of a long form of human β‐amyloid peptide (Aβ 1–42). These animals express high levels (∼300 ng of Aβ/mg of total protein) of apparently full‐length peptide, as determined by quantitative immunoblot. Expression of wild‐type Aβ in these animals leads to rapid production of amyloid deposits reactive with Congo red and thioflavin S. This model system has been used to examine the effect of Leu17Pro, Leu17Val, Ala30‐Pro, Met35Cys, and Met35Leu substitutions on the in vivo production of amyloid deposits. We find that the Leu17Pro and Met35Cys substitutions completely block the formation of thioflavin S‐reactive deposits, implicating these as key residues for in vivo amyloid formation. We have also constructed transgenic strains expressing a novel Aβ variant, the single‐chain dimer. Animals expressing high levels of this variant also fail to produce thioflavin S‐reactive deposits.
Increased Levels of Apolipoprotein D in Cerebrospinal Fluid and Hippocampus of Alzheimer's Patients
Authors: Terrisse L. , Poirier J. , Bertrand P. , Merched A. , Visvikis S. , Siest G. , Milne R. , Rassart É.
Published: July 2010

Abstract: Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimer's disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 ± 1.58 µg/ml) and patients with other pathologies (3.29 ± 1.35 µg/ml) compared with those in the CSF of normal subjects (1.15 ± 0.71 µg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the ε4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.