bluuebunny/crossref_metadata_2025 huggingface dataset:
γ‐Hydroxybutyric Acid Increases Intracellular Ca2+ Concentration and Nuclear Cyclic AMP‐Responsive Element‐and Activator Protein 1 DNA‐Binding Activities Through GABAB Receptor in Cultured Cerebellar Granule Cells
Authors: Ito Yoshihisa , Ishige Kumiko , Zaitsu Eiko , Anzai Kaijiro , Fukuda Hideomi
Published: July 2010

Abstract: In primary cultures of mouse cerebellar granule cells, a brief stimulation by γ‐hydroxybutyric acid (GHB, 0.1–3 mM) significantly increased the intracellular Ca2+ concentration ([Ca2+]i) in a concentration‐dependent manner. In addition, gel mobility assay showed that exposure of the cells to GHB also increased nuclear DNA‐binding activity specific for the cyclic AMP‐responsive element (CRE) and activator protein 1 (AP‐1) transcriptional element in a concentration‐dependent manner. The concentration range of GHB that increased the DNA‐binding activity was essentially the same as the concentration range that elicited the increase in [Ca2+]i. The GHB‐induced increases in [Ca2+]i and nuclear DNA‐binding activity were antagonized by specific GABAB antagonists such as p‐[3‐aminopropyl]‐p‐diethoxymethylphosphinic acid (CGP 35 348) and 3‐N‐[1‐(S)‐(3,4‐dichlorobenzyl)ethanol‐2‐(S)‐hydroxy‐P‐benzylphosphinic acid (CGP 55 845). In addition, the GHB‐induced increase in [Ca2+]i was abolished by pretreatment of the cells with islet‐activating protein. Furthermore, treatment of the cells with 1,2‐bis(2′‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (BAPTA‐AM) and thapsigargin blocked the GHB‐induced increase in nuclear DNA‐binding activity. GHB inhibited [3H]baclofen binding to cultured cerebellar granule cells and mouse cerebellar membranes. These results suggest that stimulation of GABAB receptors by GHB activates intracellular Ca2+ stores and that the increased [Ca2+]i resulting from release of stored Ca2+ plays an important role in increasing the CRE‐ and AP‐1 DNA‐binding activities in cultured cerebellar granule cells.
Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide‐Generating Compounds in Intact Neuronal Cells
Authors: Hu Jingru , El‐Fakahany Esam E.
Published: July 2010

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO‐generating compounds S‐nitroso‐N‐acetylpenicillamine (SNAP), 3‐morpholinosydnonimine (SIN‐1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E‐115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l‐[3H]arginine into l‐[3H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN‐1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN‐1 and SNP markedly stimulated l‐[3H]citrulline formation in a time‐ and concentration‐dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E‐115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN‐1 or SNP in intact cells was dependent on the presence of extracellular Ca2+, suggesting that it might be due to increased Ca2+ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN‐1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken‐cell preparations and in intact cells. Thus, modulation of NOS activity by NO‐releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long‐term use of NO‐generating compounds as therapeutic agents.
Effects of Chronic Lithium Treatment on Agonist‐Enhanced Extracellular Concentrations of Cyclic AMP in the Dorsal Hippocampus of Freely Moving Rats
Authors: Mørk Arne , Geisler Arne
Published: July 2010

Abstract: Studies on brain slices and homogenates suggest that chronic lithium treatment affects the activity of adenylate cyclases in the brain. To investigate whether chronic lithium administration influences the cyclic AMP (cAMP) synthesis in vivo, we have used microdialysis to assess lithium‐induced alterations in extracellular concentrations of cAMP in the dorsal hippocampus of freely moving rats. Local infusion of noradrenaline or forskolin through the microdialysis probes produced rapid increases in the extracellular concentrations of cAMP in the dorsal hippocampus. Lithium administration for 4 weeks (serum lithium concentration of 0.8 ± 0.11 mmol/L) did not affect the baseline levels of cAMP. However, in rats fed a lithium‐supplemented diet, noradrenaline‐ and forskolin‐induced enhancement of cAMP levels was decreased in the dorsal hippocampus. The rats were videotaped 18 min before and 27 min after initiating the introduction of noradrenaline and forskolin into the dorsal hippocampus. The infusion of agonists induced a moderate behavioral excitation. Rats treated with lithium were less active compared with the control rats. Taken together, these data confirm that chronic lithium administration affects the cAMP signaling system in the brain of living animals, presumably by interfering with a site beyond the receptor level.
Demonstration of κ3‐Opioid Receptors in the SH‐SY5Y Human Neuroblastoma Cell Line
Authors: Cheng Jie , Standifer Kelly M. , Tublin Pamela R. , Su Wendy , Pasternak Gavril W.
Published: July 2010

Abstract: In addition to the μ‐ and δ‐opioid receptors previously reported, the SH‐SY5Y human neuroblastoma cell line has high levels of κ3 receptors, accounting for 40% of total opioid binding, as measured with [3H]‐diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in κ3 binding. Fully 75% of the increased binding corresponds to κ3 sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin‐stimulated cyclic AMP accumulation, and this effect is readily blocked by the μ antagonist d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP). Naloxone benzoylhydrazone, a κ3 agonist, inhibits forskolin‐stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH‐SY5Y cells contain high levels of functional κ3 receptors.
In Vivo Assessment of Dopamine Uptake in Rat Medial Prefrontal Cortex: Comparison with Dorsal Striatum and Nucleus Accumbens
Authors: Cass Wayne A. , Gerhardt Greg A.
Published: July 2010

Abstract: In vivo electrochemistry was used to characterize dopamine clearance in the medial prefrontal cortex and to compare it with clearance in the dorsal striatum and nucleus accumbens. When calibrated amounts of dopamine were pressure‐ejected into the cortex from micropipettes adjacent to the recording electrodes, transient and reproducible dopamine signals were detected. The local application of the selective uptake inhibitors GBR‐12909, desipramine, and fluoxetine before the application of dopamine indicated that at the lower recording depths examined (2.5–5.0 mm below the brain surface), locally applied dopamine was cleared from the extracellular space primarily by the dopamine transporter. The norepinephrine transporter played a greater role at the more superficial recording sites (0.5–2.25 mm below the brain surface). To compare clearance of dopamine in the medial prefrontal cortex (deeper sites only), striatum, and nucleus accumbens, varying amounts of dopamine were locally applied in all three regions of individual animals. The signals recorded from the cortex were of greater amplitude and longer time course than those recorded from the striatum or accumbens (per picomole of dopamine applied), indicating less efficient dopamine uptake in the medial prefrontal cortex. The fewer number of transporters in the medial prefrontal cortex may be responsible, in part, for this difference, although other factors may also be involved. These results are consistent with the hypothesis that regulation of dopaminergic function is unique in the medial prefrontal cortex.
Overexpression of the Neurotrophic Cytokine S100β in Human Temporal Lobe Epilepsy
Authors: Griffin W. S. T. , Yeralan O. , Sheng J. G. , Boop F. A. , Mrak R. E. , Rovnaghi C. R. , Burnett B. A. , Feoktistova A. , Van Eldik L. J.
Published: July 2010

Abstract: Neuritic sprouting and disturbances of calcium homeostasis are well described in epilepsy. S100β is an astrocyte‐derived cytokine that promotes neurite growth and induces increases in levels of intracellular calcium in neurons. In sections of neocortex of surgically resected temporal lobe tissue from patients with intractable epilepsy, we found that the number of S100β‐immunoreactive astrocytes was approximately threefold higher than that found in control patients (p < 0.001). These astrocytes were activated, i.e., enlarged, and had prominent processes. Temporal lobe tissue levels of S100β were shown by ELISA to be fivefold higher in 21 epileptics than in 12 controls (p < 0.001). The expression of the astrocyte intermediate filament protein, glial fibrillary acidic protein, was not significantly elevated in epileptics, suggesting a selective up‐regulation of S100β expression. Our findings, together with established functions of S100β, suggest that this neurotrophic cytokine may be involved in the pathophysiology of epilepsy.
Apoptotic Cell Death Induced by β‐Amyloid1–42 Peptide Is Cell Type Dependent
Authors: Gschwind Martin , Huber Gerda
Published: July 2010

Abstract: β‐Amyloid peptide (Aβ), a proteolytic fragment of the β‐amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB2a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ‐induced cell death (necrosis or apoptosis) may depend on the cell type.
Stimulation of Cyclic GMP Production via a Nitrosyl Factor in Sensory Neuronal Cultures by Algesic or Inflammatory Agents
Authors: Bauer M. B. , Murphy S. , Gebhart G. F.
Published: July 2010

Abstract: Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E2, substance P, and calcitonin gene‐related peptide did not affect basal, capsaicin‐stimulated, or bradykinin‐stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by l‐arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin‐sensitive (small diameter) afferent neurons, attenuated capsaicin‐ and bradykinin‐stimulated cyclic GMP production but did not affect basal or sodium nitroprusside‐stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin‐sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non‐neuronal cells.
Iron Regulation in the Developing Rat Brain: Effect of In Utero Ethanol Exposure
Authors: Miller Michael W. , Roskams A. Jane I. , Connor James R.
Published: July 2010

Abstract: Fetal alcohol syndrome produces defects that parallel abnormalities associated with early iron deficiency. Hence, we examined the effects of prenatal exposure to ethanol on iron, transferrin, and ferritin concentrations. The subjects were the offspring of pregnant rats fed an ethanol‐containing diet (Et), pair‐fed an isocaloric control diet (Ct), or fed chow and water. The amounts of iron, transferrin, and ferritin were assessed in three CNS regions (cerebral cortex, subcortical forebrain, and brain‐stem). In all three segments of the control rats, iron, transferrin, and ferritin levels decreased during the first 2 postnatal weeks, reached a minimum during week 3, and then rose to adult levels. This pattern was delayed by ethanol treatment, e.g., the minimal concentrations in iron, transferrin, and ferritin in the Et‐treated rats were achieved later (3 days, 7 days, and 2 weeks, respectively) than they were in the Ct‐treated rats. Ethanol‐induced alterations in iron homeostasis persisted into adulthood; iron concentration was reduced, transferrin concentration was unaffected, and ferritin concentration was increased. The net result was that the timely delivery and bioavailability of iron were compromised by ethanol exposure. The defects in iron regulation are permanent and may underlie ethanol‐induced abnormalities in iron‐dependent growth processes such as myelination.
Degradation of Dynorphin‐Related Peptides by the Puromycin‐Sensitive Aminopeptidase and Aminopeptidase M
Authors: Safavi Afshin , Hersh Louis B.
Published: July 2010

Abstract: The degradation of dynorphin‐related peptides by the puromycin‐sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. Ki determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin‐related peptides, i.e., Ki for dynorphin A‐17 = 23–30 nM with the Ki increasing to 25–50 µM for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, kcat remains relatively constant; however, as the peptide size increases beyond a decapeptide, kcat decreases significantly. With the puromycin‐sensitive aminopeptidase, similar results were obtained except that kcat was greatest for the pentapeptide. Thus, if one considers kcat/Km as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin‐sensitive aminopeptidase in the degradation of extended dynorphin‐related peptides.
Internalization of Voltage‐Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis
Authors: Dargent Bénédicte , Jullien Florence , Couraud François
Published: July 2010

Abstract: In fetal rat brain neurons, activation of voltage‐dependent Na+ channels induced their own internalization, probably triggered by an increase in intracellular Na+ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP‐ and cyclic GMP‐dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na+ channel activation. An increase in intracellular Ca2+ concentration induced either by thapsigargin, a Ca2+‐ATPase blocker, or by A23187, a Ca2+ ionophore, was unable to provoke Na+ channel internalization. However, Ca2+ seems to be necessary because both neurotoxin‐ and amphotericin B‐induced Na+ channel internalizations were partially inhibited by BAPTA‐AM. The selective inhibitor of Ca2+/calmodulin‐dependent protein kinase II, KN‐62, caused a dose‐dependent inhibition of neurotoxin‐induced internalization due to a blockade of channel activity but did not prevent amphotericin B‐induced internalization. The rate of increase in Na+ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na+ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na+ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na+ channel internalization in neurons.
Functional Comparison of d‐Serine and Glycine in Rodents: The Effect on Cloned NMDA Receptors and the Extracellular Concentration
Authors: Matsui Taka‐aki , Sekiguchi Masayuki , Hashimoto Atsushi , Tomita Urara , Nishikawa Toru , Wada Keiji
Published: July 2010

Abstract: We compared the activity of free d‐Ser on the potentiation of cloned NMDA receptors with that of Gly by using a Xenopus oocyte expression system. The extracellular concentration of free d‐Ser and Gly was further studied by means of microdialysis. The ED50 values of d‐Ser were three to four times lower than those of Gly in any combination of ε1, ε2, ε3, or ε4 and ζ1. Site‐directed mutagenesis of ζ1 subunits revealed that some aromatic residues necessary for the action of Gly affected the ED50 value of d‐Ser. This result showed that the residues play crucial roles in the action of d‐Ser. In vivo microdialysis of rodent brain revealed that the extracellular concentration of free d‐Ser in the frontal cortex (6.5 µM) was high enough to saturate the Gly site on the NMDA receptor, but that in the cerebellum was not. These findings suggest that d‐Ser is a candidate of the endogenous potentiator of the NMDA receptor in the rodent frontal cortex.
Pentylenetetrazole‐Induced Seizure Up‐Regulates Levels of Microtubule‐Associated Protein 1B mRNA and Protein in the Hippocampus of the Rat
Authors: Fischer Barbara , Retchkiman Igor , Bauer Joachim , Platt Dieter , Popa‐Wagner Aurel
Published: July 2010

Abstract: Stimuli that evoke seizure are capable of inducing structural changes in the hippocampus. However, late‐acting genes related to these changes have not been described. Administration of pentylenetetrazole (PTZ; 50 mg/kg) to rats of various ages evoked tonic‐clonic seizures. Using RNA gel blot analysis we found that the level of the mRNA for microtubule‐associated protein 1B (MAP1B) was robustly increased in the hippocampus of 3‐month‐old rats. The levels of MAP1B mRNA in hippocampus peaked at 40 h and began to decline by 72 h following PTZ treatment. Immunoblotting with anti‐MAP1B antibody demonstrates the increase in content of immunoreactive proteins 40–72 h after seizure onset in the hippocampus of PTZ‐treated rats. These results indicate that MAP1B is a sensitive indicator of hippocampal structural changes occurring in response to PTZ‐induced seizure activity.
Drug‐Induced Up‐Regulation of Dopamine D2 Receptors on Cultured Cells
Authors: Starr Stephanie , Kozell Laura B. , Neve Kim A.
Published: July 2010

Abstract: Ligand‐induced up‐regulation of recombinant dopamine D2 receptors was assessed using C6 glioma cells stably expressing the short (415‐amino‐acid; D2S) and long (444‐amino‐acid; D2L) forms of the receptor. Overnight treatment of C6‐D2L cells with N‐propylnorapomorphine (NPA) caused a time‐ and concentration‐dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 µM NPA was maximal after 10 h, at which time the density of D2L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2L receptors. The receptor up‐regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30–60%) increases in receptor density. Prolonged treatment with 10 µM NPA desensitized D2L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA‐induced receptor up‐regulation in each of four clonal lines of C6‐D2L cells (mean increase, 80%) was greater than in all four lines of C6‐D2S cells (33%). Inactivation of pertussis toxin‐sensitive G proteins had no effect on the basal density of D2L receptors or on the NPA‐induced receptor up‐regulation. Treatment with 5 µg/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA‐induced increase. Chimeric D1/D2 receptors were used to identify structural determinants of dopamine receptor regulation. Treatment with the D1/D2 agonist NPA decreased the density of D1 and chimeric CH4 and CH3 receptors. The latter two receptors have D1 sequence from the amino‐terminus to the amino‐terminal end of transmembrane region (TM) VII and VI, respectively. CH2, with D1 sequence up to the amino‐terminal end of TM V, and thus the third cytoplasmic loop of the D2 receptor, was up‐regulated by NPA or the D2‐selective agonist quinpirole. Quinpirole treatment decreased the density of CH3 and had no effect on CH4 or D1 receptors. The different responses of CH2 and CH3 to agonist treatment suggest a role for TM V and the third cytoplasmic loop in the direction of receptor regulation.
Protein Kinase C‐Mediated Suppression of the Presynaptic Adenosine A1 Receptor by a Facilitatory Metabotropic Glutamate Receptor
Authors: Budd David C. , Nicholls David G.
Published: July 2010

Abstract: KCl‐evoked glutamate exocytosis from cerebrocortical synaptosomes can be inhibited by the adenosine A1 receptor agonist cyclohexyladenosine (CHA). Inhibition is associated with a decreased KCl‐evoked Ca2+ level elevation, and the effect of the agonist is occluded by prior incubation with the Agelenopsis aperta neurotoxin ω‐agatoxin‐IVA at 250 nM. The inhibition is suppressed in the presence of 3 nM phorbol dibutyrate (PDBu) or by activation of the protein kinase C (PKC)‐coupled metabotropic glutamate receptor by 100 µM (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate [(1S,3R)ACPD]. A tonic inhibition of release by leaked exogenous adenosine can be reversed by adenosine deaminase or by PDBu addition. The CHA‐induced inhibition can be enhanced by the PKC inhibitor Ro 31‐8220. The mechanism for the suppression of the adenosine A1 receptor‐mediated inhibition is distinct from that previously described for the (1S,3R)ACPD‐evoked, PKC‐mediated, facilitatory pathway, which enhances phosphorylation of the MARCKS protein, 4‐aminopyridine‐induced action potentials, and release of glutamate because the latter requires at least 100 nM PDBu [or the combination of (1S,3R)ACPD and arachidonic acid] and is not seen following KCl depolarization. Both PKC‐mediated pathways may be involved in the presynaptic events associated with the establishment of synaptic plasticity.
Polyamines in Human Brain: Regional Distribution and Influence of Aging
Authors: Morrison Lesley D. , Becker Laurence , Ang Lee C. , Kish Stephen J.
Published: July 2010

Abstract: Although much evidence has implicated polyamines in brain development and function, little information is available on these substances in human brain. We examined the influence of regional distribution and aging on putrescine, spermidine, and spermine levels in autopsied human brain. In the adult brain, concentrations of spermidine were the highest, followed by spermine and putrescine. All three polyamines showed a distinct and uneven distribution profile among the 10 examined brain areas. Spermidine levels were especially high in white matter and thalamus (20 and 9.3 nmol/mg of protein, respectively), whereas spermine concentrations were highest in cerebellar cortex (3.4 nmol/mg of protein). High levels of putrescine were observed in cerebral cortices, putamen, and hippocampus (0.7–1.2 nmol/mg of protein), with lowest levels in cerebellum and thalamus (0.3–0.5 nmol/mg of protein). No statistically significant influence of aging (1 day to 103 years; n = 57) on either putrescine or spermine levels in occipital cortex was observed. In contrast, spermidine levels increased markedly from birth, reaching maximal levels at ∼40 years of age (+228% increase in the mean 41‐year‐old group vs. 6‐week‐old group), which were maintained up to senescence. These observations in human brain thus differ from those reported in the rodent, in which levels of all three polyamines show a pronounced postnatal reduction. Our data support the notion that polyamines may have roles in both postnatal brain development and in mature brain function.
Increased Nitric Oxide Synthase Activities and l‐[3H]Arginine Uptake in Brain Following Portacaval Anastomosis
Authors: Raghavendra Rao V. L. , Audet R. M. , Butterworth R. F.
Published: July 2010

Abstract: Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal‐systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval‐shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS), in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham‐operated controls. As l‐arginine transport is a prerequisite for nitric oxide production, we also studied l‐[3H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval‐shunted and sham‐operated rats. l‐[3H]Arginine uptake was significantly increased (by ∼50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.
Mechanistic Positron Emission Tomography Studies of 6‐[18F]Fluorodopamine in Living Baboon Heart: Selective Imaging and Control of Radiotracer Metabolism Using the Deuterium Isotope Effect
Authors: Ding Yu‐Shin , Fowler Joanna S. , Gatley S. John , Logan Jean , Volkow Nora D. , Shea Colleen
Published: July 2010

Abstract: Mechanistic positron emission tomography (PET) studies using the deuterium isotope effect and specific pharmacological intervention were undertaken to examine the behavior of 6‐[18F]fluorodopamine (6‐[18F]FDA; 1) and (−)‐6‐[18F]fluoronorepinephrine {(−)‐6‐[18F]FNE; 2} in the baboon heart. Two regiospecifically deuterated derivatives of 6‐[18F]FDA [α,α‐D2(3) and β,β‐D2 (4)] were used to assess the contributions of monoamine oxidase (MAO) and dopamine β‐hydroxylase, respectively, to the clearance kinetics of 6‐[18F]FDA. Compound 3 showed a reduced rate of clearance, consistent with MAO‐catalyzed cleavage of the α C‐D bond, whereas compound 4 showed no change, indicating that cleavage of the β C‐D bond is not a rate‐limiting step. Pretreatment with pargyline, an MAO inhibitor, also decreased the rate of clearance. Desipramine and tomoxetine [norepinephrine (NE) uptake inhibitors], but not GBR‐12909 (a dopamine uptake inhibitor), blocked the uptake of both (−)‐6‐[18F]FNE and 6‐[18F]FDA, with (−)‐6‐[18F]FNE showing a higher degree of blockade. Chiral HPLC demonstrated that 6‐[18F]FDA is stereoselectively converted to (−)‐6‐[18F]FNE in vivo in the rat heart. These studies demonstrate that (a) the more rapid clearance of 6‐[18F]FDA relative to (−)‐6‐[18F]FNE can be largely accounted for by metabolism by MAO; (b) selective deuterium substitution can be used to protect a radiotracer from metabolism in vivo and to favor a particular pathway; (c) 6‐[18F]FDA and (−)‐6‐[18F]FNE share the NE transporter; (d) 6‐[18F]FDA is stereoselectively converted to (−)‐6‐[18F]FNE in vivo; and (e) the profile of radioactivity in the heart for 6‐[18F]FDA is complex, probably including labeled metabolites as well as neuronal and nonneuronal uptake.
Ascorbate and Glutamate Release in the Rat Hippocampus After Perforant Path Stimulation: A “Dialysis Electrode” Study
Authors: Walker M. C. , Galley P. T. , Errington M. L. , Shorvon S. D. , Jefferys J. G. R.
Published: July 2010

Abstract: Excitatory amino acids have been proposed to play a critical role in the development and maintenance of epileptic seizures and in the development of neuronal damage. Previous animal studies of glutamate during seizures, however, have often failed to measure any rise in glutamate. We have overcome many of the problems of these studies by using an animal model in which epileptic afterdischarges are induced by stimulation of the perforant path, and glutamate and ascorbate are measured using a newly developed microdialysis electrode that combines the advantages of microdialysis and in vivo electrochemistry. We have successfully shown (1) a rise in glutamate after an epileptic afterdischarge, (2) a concomitant initial fall and then a later rise in ascorbate, and (3) progressive dwindling of this effect when afterdischarges are repeated within minutes, despite similar electroencephalographic responses. The possible mechanisms of these effects are discussed and include ascorbate/glutamate heteroexchange, reversal of the glutamate uptake mechanism, and augmentation of glutamate uptake after a seizure.
Purification and Characterization of Guanine Nucleotide‐Exchange Factor, eIF‐2B, and p37 Calmodulin‐Binding Protein from Calf Brain
Authors: Alcázar A. , Martín M. E. , Soria E. , Rodríguez S. , Fando J. L. , Salinas M.
Published: July 2010

Abstract: Eukaryotic initiation factor 2B, or guanine nucleotide‐exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three‐part chromatographic step in heparin‐Sepharose and in SP‐5PW and diethylaminoethyl‐5PW ion‐exchange high‐performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide‐exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl‐tRNA and GTP. The method yields guanine nucleotide‐exchange factor (75%) and highly purified initiation factor 2 (>95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N‐ethylmaleimide. A 37‐kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin‐binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the α subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.
Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues
Authors: Chambliss Ken L. , Zhang Yu‐An , Rossier Eva , Vollmer Brigitte , Gibson K. Michael
Published: July 2010

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.
Tryptophan Hydroxylase Is Phosphorylated by Protein Kinase A
Authors: Johansen Patricia A. , Jennings Ian , Cotton Richard G. H. , Kuhn Donald M.
Published: July 2010

Abstract: The effect of protein kinase A on the catalytic activity and phosphorylation of brain tryptophan hydroxylase was examined. Stimulation of endogenous protein kinase A by cyclic AMP or its analogues, dibutyryl‐cyclic AMP and 8‐thiomethyl‐cyclic AMP, failed to activate tryptophan hydroxylase. The activation of tryptophan hydroxylase by calcium/calmodulin‐phosphorylating conditions was not modified by cyclic AMP. Endogenous protein kinase A phosphorylated a large number of proteins and tryptophan hydroxylase could be identified as one substrate by sucrose gradient centrifugation, immunoprecipitation, and immunoblotting. These results indicate that tryptophan hydroxylase is phosphorylated by protein kinase A in brain and question whether this protein kinase exerts direct regulatory influence over tryptophan hydroxylase activity via phosphorylation.
Amyloid β‐Protein Induces Its Own Production in Cultured Degenerating Cerebrovascular Smooth Muscle Cells
Authors: Davis‐Salinas Judianne , Saporito‐Irwin Susan M. , Cotman Carl W. , Van Nostrand William E.
Published: July 2010

Abstract: The progression of Alzheimer's disease and related disorders involves amyloid β‐protein (Aβ) deposition and pathologic changes in the parenchyma as well as cerebral blood vessels. The cerebrovascular Aβ deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall, which have been shown to express the Aβ precursor (AβPP) and Aβ. Here, we show that Aβ1–42, an abundant cerebrovascular form of Aβ, causes cellular degeneration in cultured human cerebrovascular smooth muscle cells. This stress response is accompanied by a striking increase in the levels of cellular AβPP and soluble Aβ peptide produced in these degenerating cells. These data provide the first experimental evidence that Aβ can potentially contribute to the onset and progression of the cerebrovascular pathology. The present findings suggest that this mechanism may involve a molecular cascade with a novel product‐precursor relationship that results in the adverse production and subsequent accumulation of Aβ.
Influence of Selective Inhibition of Monoamine Oxidase A or B on Striatal Metabolism of l‐DOPA in Hemiparkinsonian Rats
Authors: Finberg J. P. M. , Wang J. , Goldstein D. S. , Kopin I. J. , Bankiewicz K. S.
Published: July 2010

Abstract: The effect of selective inhibition of monoamine oxidase (MAO) subtypes A and B on striatal metabolism of DOPA to dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and 4‐hydroxy‐3‐methoxyphenylacetic acid (homovanillic acid; HVA) was studied in halothane‐anesthetized rats 3 weeks after unilateral 6‐hydroxydopamine lesion of the substantia nigra. Implantation of bilateral microdialysis probes allowed simultaneous quantitation of metabolite production on lesioned and control sides. The DOPA was administered as a 15‐min bolus of 1 mM solution in the striatal microdialysate. Rats were pretreated with the selective MAO‐A inhibitor clorgyline, or the selective MAO‐B inhibitors deprenyl or TVP‐101 [2,3‐dihydro‐N‐2‐propynyl‐1H‐inden‐1‐amine‐(1R)‐hydrochloride]. Intrastriatal infusion of DOPA caused an increased efflux of DA, DOPAC, and HVA, which was greater on the intact side. Clorgyline, but not deprenyl or TVP‐101, increased post‐DOPA DA efflux on both intact and lesioned sides. Clorgyline also caused a marked suppression of post‐DOPA DOPAC and HVA effluxes, whereas only mild effects were produced by the MAO‐B inhibitors. There was no evidence for a differential effect of MAO‐B inhibition on efflux of DA or metabolites in the lesioned as compared with the control striatum. The results indicate a major role for MAO‐A in DA metabolism both intra‐ and extraneuronally in the rat striatum.
Effect of Amygdaloid Kindling on the Content and Release of Amino Acids from the Amygdaloid Complex: In Vivo and In Vitro Studies
Authors: Kaura S. , Bradford H. F. , Young A. M. J. , Croucher M. J. , Hughes P. D.
Published: July 2010

Abstract: The tissue content and the interstitial fluid levels of glutamate, aspartate, GABA, glutamine, glycine, and serine were studied in amygdaloid‐kindled rat brain. Interstitial levels were studied in vivo before and during stage 5 full limbic seizures using microdialysis. Slices of amygdala from kindled and sham‐operated animals were used to study baseline and KCl‐evoked release in vitro. The contents of these amino acids were measured in slices of amygdala, hippocampus, and cerebral cortex from kindled and sham‐operated animals. Kindled brains showed two‐ to threefold higher levels of glutamate, aspartate, and GABA and 12‐fold higher levels of glutamine than sham‐operated controls. Correlating with this, interstitial fluid levels of glutamate were two‐ to threefold higher from kindled amygdala than from control both in vivo (microdialysis) and in vitro (superfusion). GABA levels in interstitial fluid from kindled amygdala were reduced by 67% compared with control amygdala.
Carbohydrate Analysis of the B2 Bradykinin Receptor from Rat Uterus
Authors: Yaqoob Mohammed , Snell Christopher R. , Burgess Gillian M.
Published: July 2010

Abstract: The B2 bradykinin receptor purified from rat uterus has an apparent molecular mass of 81 kDa. This is higher than the value of 42 kDa estimated from the sequence data of rat and human B2 receptors. Carbohydrate analysis of the rat B2 bradykinin receptor indicated that it was a sialoglycoprotein with three N‐linked complex oligosaccharide side chains. This was consistent with the sequence, which has three potential glycosylation sites. The receptor did not appear to possess O‐linked carbohydrate side chains. Removal of the N‐linked carbohydrates with endo‐β‐N‐acetylglucosaminidase yielded a core protein of 42–44 kDa. The presence of these N‐linked carbohydrates explains the discrepancy between the molecular size of the purified receptor protein and that estimated from the sequence. The sequence of the rat receptor suggests an isoelectric point of about pH 7.0, but the purified receptor had an isoelectric point of pH 4.5–4.7. Sialic acid residues on the N‐linked side chains are likely to be responsible for the acidic nature of the rat receptor. Carbohydrate does not appear to play a role in ligand‐receptor interactions, as deglycosylation did not alter the affinity of the B2 bradykinin receptor for bradykinin or the B2‐selective antagonist HOE‐140.
Activation of Protein Kinase C by the Capsaicin Analogue Resiniferatoxin in Sensory Neurones
Authors: Harvey J. S. , Davis C. , James I. F. , Burgess G. M.
Published: July 2010

Abstract: Resiniferatoxin and capsaicin are sensory neurone‐specific excitotoxins that operate a common cation channel in nociceptors. Resiniferatoxin is structurally similar to capsaicin and to phorbol esters. Specific [3H]‐resiniferatoxin binding, which was detected in the membrane (KD value 1.8 ± 0.2 nM) but not cytosolic fraction of rat dorsal root ganglia, could not be displaced by phorbol 12,13‐dibutyrate. Conversely, resiniferatoxin did not displace [3H]phorbol 12,13‐dibutyrate binding in either the cytosolic or membrane fraction. Resiniferatoxin and capsaicin both caused translocation of protein kinase C in dorsal root ganglion neurones (EC50 value 18 ± 3 nM). This translocation was greatly reduced but not abolished, in the absence of external Ca2+, suggesting that it was secondary to Ca2+ entry. Resiniferatoxin also caused direct activation of a Ca2+‐ and lipid‐dependent kinase (or kinases) in the cytosolic fraction of dorsal root ganglia, at concentrations (100 nM to 10 µM) higher than required for displacement of [3H]resiniferatoxin binding or translocation of protein kinase C. Capsaicin (up to 10 µM) was unable to mimic this effect. These data imply that although resiniferatoxin‐induced translocation of protein kinase C in dorsal root ganglion neurones was mainly indirect, it also caused direct activation of a protein kinase C‐like kinase in these cells.
Functional Importance of the Carboxyl Tail Cysteine Residues in the Human D1 Dopamine Receptor
Authors: Jensen Anders A. , Pedersen Ulla B. , Kiemer Anita , Din Nanni , Andersen Peter H.
Published: July 2010

Abstract: To assess the importance of the cysteine residues Cys347 and Cys351 in the carboxylic tail in the human D1 dopamine receptor, seven mutant receptors were constructed by PCR. The pharmacological and functional properties of the wild‐type and mutant receptors were assessed following transient expression in COS‐7 cells. Affinities for [3H]SCH 23390 of mutant S347 (Cys347→ Gly), T348 (Tyr348→ stop), S351 (Cys351→ Gly), T351 (Cys351→ stop), T352 (Pro352→ stop), and S347/S351 (Cys347→ Gly and Cys351→ Gly) were similar to that of wild‐type receptor, whereas the expression levels were reduced up to 80%. The potency of dopaminergic antagonists for these mutant receptors was very similar to that of the wild‐type receptor. However, mutant T347 (Cys347→ stop) showed a 15–25‐fold reduced affinity for the antagonists SCH 23390, (+)‐butaclamol, and cis‐flupentixol, thus not allowing radioligand analysis. Wild‐type and mutant receptors responded dose‐dependently with similar potency to dopamine and SKF 38393 with an increased adenylyl cyclase activity. However, mutant receptors with the Cys347 residue changed or removed displayed a diminished ability to activate adenylyl cyclase. Dopamine preexposure desensitized wild‐type and mutant S351 receptors. However, mutant receptors with Cys347 replaced or the distal part of the carboxyl tail removed were unable to desensitize. Thus, Cys347 in the cytoplasmic tail of the human D1 dopamine receptor is important for the receptor in maintaining the conformation for antagonist binding, to play a crucial role in activation of adenylyl cyclase, and to be essential for agonist‐induced desensitization.
Spinal Cord Dynorphin Precursor Intermediates Decline During Late Gestation
Authors: Medina Victor M. , Gupta Daya , Gintzler Alan R.
Published: July 2010

Abstract: This laboratory has previously reported that the maternal opioid analgesia associated with pregnancy and parturition is mediated, at least in part, by a maternal spinal cord dynorphin/κ opioid system. This analgesia is accompanied by an increase in dynorphin peptides (1–17 and 1–8) in the lumbar spinal cord. Levels of trypsin‐generated arginine6‐leucine‐enkephalin (Leu‐Enk‐Arg)‐immunoreactive determinants were also determined and used to reflect the content of dynorphin precursor intermediates. In spinal tissue, the amount of dynorphin A (1–17) contained in the form of precursor is, at a minimum, 10‐fold higher than the content of mature dynorphin A (1–17) or dynorphin (1–8). During gestational day 22, the content of dynorphin precursor is reduced significantly (∼50%). The decline in the magnitude of dynorphin precursor intermediates in the spinal cord of pregnant rats vastly exceeds the magnitude of increase in the content of dynorphin peptides (1–17 and 1–8). This difference can best be explained by postulating a corresponding increase in the rate of release of spinal cord dynorphin (1–17). It is suggested that enhanced processing of dynorphin precursor intermediates represents the initial biochemical level of adaptation of spinal dynorphin neurons to increased demands of pregnancy.
Poly(ADP‐Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage
Authors: Zhang Jie , Pieper Andrew , Snyder Solomon H.
Published: July 2010

Abstract: DNA damage activates a nuclear enzyme poly(ADP‐ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP‐ribose groups to nuclear proteins such as histones and PARS itself. N‐Methyl‐d‐aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β‐protein, hydrogen peroxide, N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and its active metabolite N‐methyl‐4‐phenylpyridine (MPP+). With MPP+, PARS activity is increased fivefold in 1 h and 20‐fold by 3 h. By contrast, direct measurement of DNA damage by the terminal‐deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.
GM1 Ganglioside in the Nuclear Membrane Modulates Nuclear Calcium Homeostasis During Neurite Outgrowth
Authors: Wu Gusheng , Lu Zi‐Hua , Ledeen Robert W.
Published: July 2010

Abstract: GM1 in the nuclear membrane, previously shown to be up‐regulated during neurite outgrowth, has been found to influence nuclear Ca2+ flux during differentiation of Neuro‐2a cells. Nuclei were isolated from cultured Neuro‐2a cells before and after neuraminidase‐induced neuritogenesis and incubated with 45Ca2+ for varying periods to determine uptake/efflux of Ca2+. At 5, 10, and 15 min 45Ca2+ levels in nuclei from differentiated cells were significantly lower than those in nuclei from untreated cells. The same result was obtained when the GM1 level was elevated artificially by preincubation of the nuclei in 10 µM GM1. In experiments designed to measure efflux specifically, isolated nuclei preincubated in GM1 released 45Ca2+ more rapidly than untreated nuclei. We conclude that one role of GM1 in the nuclear membrane is to alter Ca2+ regulatory mechanisms in the nucleus following onset of neuronal process outgrowth.