bluuebunny/crossref_metadata_2025 huggingface dataset:
The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates
Authors: Watson Michael A. , Yamada Mitsuhiko , Yamada Misa , Cusack Bernadette , Veverka Karen , Bolden‐Watson Carolyn , Richelson Elliott
Published: October 2006

Abstract: To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA 1 neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)‐K1 cells. Colony clones, selected for resistance to antibiotic G‐418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO‐rNTR‐10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/106 cells (sim4.4 × 105 sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E‐115, the Bmax exceeded previously reported values. Incubation of intact CHO‐rNTR‐10 cells with neurotensin caused the release of inositol phosphates in a dose‐dependent manner (EC50= 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8‐13) was more potent than neurotensin. Neurotensin‐mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.
In Vivo Activation of Kainate Receptors Induces Dephosphorylation of the Heavy Neurofilament Subunit
Authors: Wang Shu , Hamberger Anders , Ding Mei , Haglid Kenneth G.
Published: October 2006

Abstract: Injection of kainic acid (KA) into the rat hippocampus reduced the phosphorylation‐related immunoreactivity of the heavy subunit of neurofilament proteins (NF‐H). The effect was demonstrated quantitatively with a dot‐immunobinding assay and qualitatively by immunoblotting with monoclonal antibodies against phosphorylation‐dependent and nonphosphorylation‐related epitopes of NF‐H. The KA‐induced reduction affected 50% of the phosphorylated NF‐H in half of the hippocampus after 48 h. At the same time, the nonphosphorylation‐related NF‐H immunoreactivity increased as revealed by immunoblotting, indicating a shift from phosphorylated to nonphosphorylated NF‐H. The effects on NF‐H preceeded a decrease in content of the neuron‐specific enolase, a soluble neuronal cytoplasmic protein. No alterations of the light subunit of neurofilament proteins occurred, suggesting that KA has a preferential effect on NF‐H phosphorylation. N‐Methyl‐D‐aspartate administered similarly did not lead to a rapid dephosphorylation of NF‐H. We propose that kainate receptor‐mediated dephosphorylation in NF‐H is involved in the signal transduction of excitatory amino acids with consequences for neuronal functions dependent on intermediary filament phosphorylation.
Different Glycosylation in Acetylcholinesterases from Mammalian Brain and Erythrocytes
Authors: Liao Jian , Heider Harald , Sun Man‐Chi , Brodbeck Urs
Published: October 2006

Abstract: Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine‐linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate‐polyacrylamide gel before and after deglycosylation with N‐glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent‐soluble AChE from electric organs of Torpedo nacline timilei as well as Elec‐39 raised against AChE from Electrophorus electricus cross‐reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N‐glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N‐linked carbohydrates. Analysis of N‐acetylglucosamine sugars revealed the presence of N‐acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N‐linked glycosylation. On the other hand, N‐acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O‐linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.
Activation of Protein Kinase C Suppresses the γ‐Aminobutyric AcidB Receptor‐Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine
Authors: Taniyama Kohtaro , Niwa Masami , Kataoka Yasufumi , Yamashita Kimihiro
Published: October 2006

Abstract: Modulation of the γ‐aminobutyric acidB (GABAB) receptor‐mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation‐evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) potentiated the high K+‐evoked Ca2+‐dependent release of NA and ACh, but not the ouabain‐evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic‐vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K+‐evoked release of NA and ACh, but not the ouabain‐evoked Ca2+‐independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor‐mediated inhibition of high K+‐evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation‐evoked Ca2+‐dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor‐mediated response.
Dopamine Stimulates [3H]Phorbol 12,13‐Dibutyrate Binding in Cultured Striatal Cells
Authors: McMillian M. K. , He X. P. , Hong J. S. , Pennypacker K. R.
Published: October 2006

Abstract: The effect of dopamine (DA) on the binding of [3H]phorbol 12,13‐dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 ± 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half‐maximal stimulation was observed with 10–6M DA. The peak response was observed at 2–3 min after addition of 10–4M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10–6M) blocked the response to NE, suggesting mediation by an α1‐adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH‐23390 (10–6M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10–6M) inhibited the response to DA by 46 ± 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF‐38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.
Pharmacological and Physicochemical Properties of Pre‐Versus Postsynaptic 5‐Hydroxytryptamine1A Receptor Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study
Authors: Radja F. , Daval G. , Hamon M. , Vergé D.
Published: October 2006

Abstract: Numerous data suggested that the pharmacological and biochemical properties of 5‐hydroxytryptaminelA (5‐HTIA) receptors exhibit some regional differences in the CNS, notably within the raphe nuclei compared with various fore‐brain areas (such as the hippocampus). This possibility has been further investigated in the dorsal raphe nucleus and two areas within the hippocampus, the dentate gyrus and the CA1 area, using the quantitative autoradiographic technique. The potencies of 5′‐guanylylimidodiphosphate to inhibit the specific binding of 125I‐Bolton‐Hunter‐8‐methoxy‐2‐(N‐propyl‐N‐propylamino)tetralin (125I‐BH‐8‐MeO‐N‐PAT) to 5‐HT1A sites and of N‐ethylmaleimide to block these sites irreversibly were identical in the dorsal raphe nucleus and the hippocampal areas in rat brain sections. In contrast, slight but significant differences were noted in the pH dependence and pharmacological properties of 5‐HT1A sites labeled by 125I‐BH‐8‐MeO‐N‐PAT in these three regions. Similarly, heat denaturation experiments and tissue exposure to either phospholipase A2 or the alkylating agent 8‐methoxy‐2‐(N‐2′‐chloropropyl,N‐propyl)aminotetraline revealed regional differences in the properties of 5‐HT1A sites. However, in most cases, the observed variations were of greater amplitude between the CA1 area and the dentate gyrus, where 5‐HT1A sites are located postsynaptically, than between any one of these areas and the dorsal raphe nucleus where they act as (presynaptic) so‐matodendritic autoreceptors. These data further support that subtypes of 5‐HT1A receptors probably exist in the rat brain, but this heterogeneity seems unrelated to the pre‐ or postsynaptic location of these receptors.
Identification of Insulin in Chick Embryo Retina During Development and Its Inhibitory Effect on DNA Synthesis
Authors: Tesoriere Giovanni , Vento Renza , Calvaruso Giuseppe , Taibi Gennaro , Giuliano Michela
Published: October 2006

Abstract: Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid‐insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time‐course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2–6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10–8M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.
Identification of Plasmolipin as a Major Constituent of White Matter Clathrin‐Coated Vesicles
Authors: Sapirstein Victor S. , Nolan Charles E. , Stern Richard , Gray‐Board Gladys , Beard Margaret E.
Published: October 2006

Abstract: We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin‐associated glycoprotein. On the other hand, these vesicles contain 2′,3′‐cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3–4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.
Drug‐Induced Changes in Blood Pressure Lead to Changes in Extracellular Concentrations of Epinephrine, Dihydroxyphenylacetic Acid, and 5‐Hydroxyindoleacetic Acid in the Rostral Ventrolateral Medulla of the Rat
Authors: Dev Bhaskar R. , Mason Patrick A. , Freed Curt R.
Published: October 2006

Abstract: Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 μm in diameter and 500 μm in length was stereotaxically implanted in the RVLM of the urethane‐anesthetized rat. Sterile physiological Ringer's solution was perfused at a rate of 1.5 μl/min. The perfusate was collected under ice‐cold conditions every 15 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5‐hydroxyindoleacetic acid (5‐HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5‐HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by nitroprusside (—20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5‐HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure. Furthermore, because changes in epinephrine concentration parallel changes in sympathetic activity as measured by heart rate, epinephrine in the extracellular fluid space of the RVLM may be a marker for sympathetic nerve activity.
Adenylyl Cyclase Activity in Postmortem Human Brain: Evidence of Altered G Protein Mediation in Alzheimer's Disease
Authors: Cowburn Richard F. , O'Neill Cora , Ravid Rivka , Alafuzoff Irina , Winblad Bengt , Fowler Christopher J.
Published: October 2006

Abstract: The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5′‐O‐(3‐thiotriphosphate)‐, aluminum fluoride‐, and forskolin‐stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride‐stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5′‐O‐(3‐thiotriphosphate)‐, aluminum fluoride‐, and forskolin‐stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5′‐O‐(3‐thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5′‐O‐(3‐thiotriphosphate)‐, but not aluminum fluoride‐, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin‐stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein‐stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non‐disease‐related factors associated with the nature of terminal illness of individuals.
Biotin Transport in Primary Culture of Astrocytes: Effect of Biotin Deficiency
Authors: Rodriguez‐Pombo Pilar , Ugarte Magdalena
Published: October 2006

Abstract: The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of < 60 nM and linear at >60 nM. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated diffusion. Moreover, biotin uptake in astrocytes grown in biotin‐restricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10‐fold) in the Vmax of the biotin uptake without any change in the apparent Km.
Effects of Monovalent and Divalent Cations on 3‐(+)[125I]Iododizocilpine Binding to the N‐Methyl‐d‐Aspartate Receptor of Rat Brain Membranes
Authors: Rajdev Sunita , Reynolds Ian J.
Published: October 2006

Abstract: We have investigated the binding of 3‐[125I]iododizocilpine ([125I]iodo‐MK‐801) to the N‐methyl‐d‐aspartate (NMDA) receptor in well‐washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine > thienylphencyclidine > phencyclidine > ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS‐19755, 7‐chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 μM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]‐iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.
Effects of Frequency and Pattern of Medial Forebrain Bundle Stimulation on Caudate Dialysate Dopamine and Serotonin
Authors: Manley Leslie D. , Kuczenski Ronald , Segal David S. , Young Stephen J. , Groves Philip M.
Published: October 2006

Abstract: In vivo microdialysis was employed to detect changes in extracellular dopamine and serotonin in the rat caudate in response to electrical stimulation of the medial forebrain bundle. Extracellular dopamine concentrations increased linearly as a function of the frequency (4–33 Hz) of evenly spaced stimuli in both the presence and absence of cocaine added to the dialysate. Because dopamine neurons are known to fire in single‐spike and burst patterns, stimulation pulses were also delivered in a bursting pattern. The response of extracellular dopamine was augmented in both the presence and absence of cocaine when the same number of stimuli were delivered in bursts as compared to an evenly spaced pattern. Serotonin, which was only assessed in the presence of cocaine, similarly increased linearly with frequency, but, in contrast to the dopamine response, levels of serotonin were not augmented by stimuli presented in bursts. These results suggest that microdialysis can be used to detect physiological changes in synaptic transmitter concentrations.
Octanoic Acid Produces Accumulation of Monoamine Acidic Metabolites in the Brain: Interaction with Organic Anion Transport at the Choroid Plexus
Authors: Kim TC. S. , Roe C. R. , Mann J. D. , Breese G. R.
Published: October 2006

Abstract: Effects of octanoic acid on monoamines and their acidic metabolites in the rat brain were analyzed by HPLC. Octanoic acid (1,000 mg/kg i.p.) elevated homovanillic acid levels by 54% in the caudate and 338% in the hypothalamus but increased 5‐hydroxyindoleacetic acid (5‐HIAA) levels in both the caudate and the hypothalamus by ∼ 50% compared with the control. A lower dose of octanoic acid (500 mg/kg) increased 5‐HIAA levels by 29% in the caudate and 20% in the hypothalamus. However, it did not produce any changes in the concentration of homovanillic acid in either the caudate or the hypothalamus. Treatment with octanoic acid also failed to change the level of dopamine, serotonin, and 3,4‐dihydroxyphenylacetic acid in the caudate and the hypothalamus. The role of carrier‐mediated transport in the clearance of 5‐HIAA from the rabbit CSF was also evaluated in vivo by ventriculocisternal perfusion. Steady‐state clearance of 5‐HIAA from CSF exceeded that of inulin and was reduced in the presence of octanoic acid. Because this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of en‐cephalopathy in children with medium‐chain acyl‐CoA de‐hydrogenase deficiency who have elevated levels of octanoic acid systemically.
DNA–Damaging and Transcription‐Terminating Lesions Induced by AF64A In Vitro
Authors: Futscher Bernard W. , Pieper Russell O. , Barnes Diane M. , Hanin Israel , Erickson Leonard C.
Published: October 2006

Abstract: Although immediate cholinergic deficits produced by AF64A can be explained adequately by inhibition of enzymes involved in acetylcholine metabolism, the structural similarity of AF64A to a number of DNA‐damaging anti‐tumor agents suggested that the observed long‐term cholin‐ergic deficits may involve damage to the cell's informational molecules. This study was initiated to determine if AF64A can damage DNA and prematurely terminate RNA transcription in vitro, and to produce cytotoxic and DNA damaging effects in cells exposed to the drug in vivo. The ability of AF64A to produce N‐7 guanine alkylations in DNA in vitro was assessed using a modified Maxam and Gilbert DNA sequencing technique, and the ability of AF64A to terminate RNA transcription was assessed by an in vitro RNA transcription system. AF64A was capable of producing extensive dose‐dependent N‐7 guanine alkylations in DNA fragments exposed to AF64A in vitro, although no sequence specificity of AF64A attack could be discerned. Furthermore, AF64A was able to produce RNA transcription‐terminating lesions in vitro, also in a dose‐dependent fashion. Transcription of AF64A‐damaged DNA resulted in RNA molecules terminated not at every alkylated guanine, but at various discrete sites along the DNA template. AF64A was also found to be cytotoxic in a dose‐dependent manner in cultured mouse leukemia L1210 cells. The induced cytotoxicity was accompanied by DNA lesions which were detected as DNA single strand breaks using the DNA alkaline elution technique. The results of these experiments support the hypothesis that AF64A may alter the structure and function of cellular DNA and may help explain the observed long‐term cholinergic deficits.
The 72‐kDa Microtubule‐Associated Protein from Porcine Brain
Authors: Takeuchi Makoto , Hisanaga Shin‐ichi , Umeyama Takashige , Hirokawa Nobutaka
Published: October 2006

Abstract: A microtubule‐associated protein (MAP) with a molecular mass of 72‐kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low‐angle rotary shadowing revealed that the 72‐kDa protein was a rodlike protein ∼55–75 nm long. The 72‐kDa protein bound to microtubules polymerized from phosphocellulose column‐purified tubulin (PC‐tubulin) with taxol and promoted the polymerization of PC‐tubulin in the absence of taxol. Microtubules polymerized by the 72‐kDa protein showed a tendency to form bundles of several microtubules. Quick‐freeze, deep‐etch electron microscopy revealed that the 72‐kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72‐kDa protein to other heat‐stable MAPs, and the results showed some resemblance of the 72‐kDa protein to MAP2. Cross‐reactivity with a monoclonal anti‐MAP2 antibody further suggested that the 72‐kDa protein and MAP2 are immunologically related. To study the relationship between the 72‐kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72‐kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72‐kDa protein from juvenile rat brain was also stained with our monoclonal anti‐MAP2 antibody also suggested that the 72‐kDa protein is an MAP2C homologue of the porcine brain.
Immunoassay and Activity of Calcium‐Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane‐Associated Fractions in Human and Mouse Brain
Authors: Takeuchi Kiyoshi H. , Saito Ken‐Ichi , Nixon Ralph A.
Published: October 2006

Abstract: The millimolar form of calcium‐activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion‐exchange chromatography on DEAE‐cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X‐100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.
Kainic Acid‐Induced Decrease in Hippocampal Corticosteroid Receptors
Authors: Lowy Martin T.
Published: October 2006

Abstract: The potential role of excitatory amino acids in the regulation of brain corticosteroid receptors was examined using systemic administration of kainic acid. Administration of kainic acid (5, 10, and 15 mg/kg) to 24‐h adrenalectomized rats that were killed 3 h later produced large, dose‐related decreases in glucocorticoid receptors (GR) in hippocampus (23–63%), frontal cortex (22–76%), and striatum (41–49%). Kainic acid did not decrease hypothalamic GR. Hippocampal mineralocorticoid receptors (MR) were also markedly decreased (50–71%) by kainic acid. Significant decreases in corticosteroid receptors could be detected as soon as 1 h after kainic acid (10 mg/kg) administration. Decreases in hippocampal, cortical, and hypothalamic GR as well as hippocampal MR were observed 24 h after administration of kainic acid (10 mg/kg) to adrenalectomized rats. Kainic acid (10 mg/kg) also significantly decreased hippocampal GR and MR as well as GR in the other three brain regions when administered to adrenal‐intact rats that were subsequently adrenalectomized and killed 48 h after drug administration. The kainic acid–induced decreases in hippocampal GR and MR binding were due to decreases in the maximum number of binding sites (Bmax) with no change in the apparent affinity (KD). Kainic acid when added in vitro did not displace the GR and MR radioligands from their respective receptors. These studies demonstrate that excitatory amino acids play a prominent role in the regulation of hippocampal corticosteroid receptors. In addition, the data indicate that noncorticosterone factors are involved in corticosteroid receptor plasticity.
Real‐Time Monitoring of Electrically Stimulated Norepinephrine Release in Rat Thalamus: I. Resolution of Transmitter and Metabolite Signal Components
Authors: Ghasemzadeh M. Behnam , Capella Peter , Mitchell Kim , Adams Ralph N.
Published: October 2006

Abstract: Electrical stimulation of an ascending path of the locus ceruleus‐norepinephrine system was used to elicit release of norepinephrine at noradrenergic terminal fields of the rat thalamus. Overflow into the extracellular fluid space was measured by fast in vivo chronoamperometry. At pretreated carbon fibers, the electrochemical signal consists of a sharp peak of ∼20–30‐s duration followed by a slower, plateau‐like decay to baseline. The peak, characterized by a variety of pharmacological manipulations and dialysis perfusion, is primarily due to norepinephrine. The plateau was shown to correspond to metabolite efflux of 3,4‐dihydroxyphenylacetic acid. By varying the degree of electrochemical pretreatment, the response time and sensitivity of the fibers can be tuned to follow the entire signal or to select the separate components for detailed evaluation. This approach can be used to provide new information on the spatial and temporal characteristics of stimulated neurotransmitter release.
Real‐Time Monitoring of Electrically Stimulated Norepinephrine Release in Rat Thalamus: II. Modeling of Release and Reuptake Characteristics of Stimulated Norepinephrine Overflow
Authors: Capella Peter , Ghasemzadeh M. Behnam , Adams Ralph N. , Wiedemann Donna J. , Wightman R. Mark
Published: October 2006

Abstract: As in the preceding study, electrical stimulation was used to effect release overflow of norepinephrine in the rat thalamus. Using a weak electrochemical pretreatment of a carbon fiber electrode, it was possible to “tune in” the electrochemical response signal for norepinephrine without metabolite interference. This reasonably selective signal was then used to study the degradation of norepinephrine release ability caused by prolonged stimulation. Further, the signals were modeled by the method used successfully for stimulated dopamine overflow, providing hitherto unavailable information on the temporal and spatial characteristics of norepinephrine release overflow. Pertinent comparisons between the release characteristics of the dopamine and norepinephrine systems show that the half‐life for norepinephrine in the extracellular fluid space is ∼1 s in thalamus compared with 33 ms for dopamine in caudate.
Neuronotypic Differentiation Results in Reduced Levels and Altered Distribution of Synaptophysin in PC 12 Cells
Authors: Lah James J. , Burry Richard W.
Published: October 2006

Abstract: Several synaptic vesicle proteins including synap‐tophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultra‐structural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor‐stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.
Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes
Authors: Kamisaki Yoshinori , Hamada Toshihiro , Maeda Kazuhisa , Ishimura Masahiko , Itoh Tadao
Published: October 2006

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ‐amino‐butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) evoked 2.7‐, 1.5‐, and 2.9‐fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K+‐evoked overflow of Glu to 56% of the control overflow with a potency (IC50) of 17 nM, but it did not affect K+‐evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K+‐evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α2‐adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α2 adrenoceptors localized on the primary afferent terminals.
Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals
Authors: Doyu Manabu , Sobue Gen , Ken Eishuku , Kimata Koji , Shinomura Tamayuki , Yamada Yoshihiko , Mitsuma Terunori , Takahashi Akira
Published: October 2006

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady‐state levels were high, but when the nerves were crushed, the steady‐state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five‐ to eightfold over that of control levels as the total RNA and β‐actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady‐state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β‐actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up‐regulated by axonal or neuronal contact, but they do not express A chain mRNA.
Support for Radiolabeling of a Glycine Recognition Domain on the N‐Methyl‐d‐Aspartate Receptor Ionophore Complex by 5,7‐[3H]Dichlorokynurenate in Rat Brain
Authors: Yoneda Yukio , Suzuki Takeo , Ogita Kiyokazu , Han Daiken
Published: October 2006

Abstract: Pretreatment with Triton X‐100 more than doubled the binding of radiolabeled 5,7‐dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N‐methyl‐d‐aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [3H]DCKA and [3H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration‐dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [3H]kainic acid and α‐amino‐3‐hydroxy‐5‐[3H]methylisoxazole‐4‐propionic acid (AMPA) to the non‐NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7‐dinitroquinoxaline‐2,3‐dione, was active in displacing the binding of [3H]glutamic ([3H]Glu) and D,L‐(E)‐2‐amino‐4‐[3H]propyl‐5‐phosphono‐3‐pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non‐NMDA receptors. In addition, the proposed antagonist at the AMPA‐sensitive receptor, 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline, not only displaced weakly the binding of both [3H]‐ Gly and [3H]DCKA, but also inhibited the binding of (+)‐5‐[3H]methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ([3H]MK‐801) to an ion channel associated with the NMDA‐sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [3H]DCKA binding and [3H]Gly binding, in addition to between the potencies to displace [3H]‐DCKA or [3H]Gly binding and to potentiate or inhibit [3H]MK‐801 binding. All quinoxalines tested were invariably more potent displacers of [3H]DCKA binding than [3H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [3H]Gly and [3H]‐DCKA. These results undoubtedly give support to the proposal that [3H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [3H]Gly and [3H]DCKA.
In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L‐690,330
Authors: Atack John R. , Cook Susan M. , Watt Alan P. , Fletcher Stephen R. , Ragan C. Ian
Published: October 2006

Abstract: We have previously described the synthesis of bis‐phosphonate‐containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L‐690,330, is described. L‐690,330 is a competitive inhibitor of inositol monophosphatase with a K1, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000‐fold more potent in vitro than lithium, in muscarinic m1 receptor‐transfected Chinese hamster ovary cells prelabelled with [3H]inositol, L‐690,330 only produced 40% of the accumulation of [3H]inositol monophosphates achieved by lithium at the same concentration (10 mM), suggesting that the ability of L‐690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L‐690,330 were able to increase brain inositol(1)phosphate levels in vivo to three‐ to fourfold control levels. This effect was dose dependent (ED50= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L‐690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L‐690,330 were much less than seen with lithium, consistent with the blood‐brain barrier restricting access of the polar L‐690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.
Changes in Heat Shock Protein 70 and Ubiquitin mRNA Levels in C1300 N2A Mouse Neuroblastoma Cells Following Treatment with Iron
Authors: Uney James B. , Anderton Brian H. , Thomas Siân M.
Published: October 2006

Abstract: We have shown that following heat shock (42.5°C for 30 min), mouse‐derived C1300 N2A neuroblastoma cells contain increased levels of mRNA coding for the inducible form of heat shock protein 70 and for ubiquitin. Incubation of C1300 cells with iron also induces an elevation in content of mRNAs coding for the same two proteins that can be blocked by α‐tocopherol and desferrioxamine. Iron was shown to increase mitochondrial and lysosomal activities in differentiated C1300 N2A cultures, as shown by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and neutral red cytotoxicity assays. These responses were not initially associated with any loss of viability, as assessed by the lactate dehydrogenase release assay. These results suggest that there is production of cytoprotective heat shock proteins in response to iron‐mediated cell damage, probably involving free radical generation, in neural cells. The apparent stress response of vulnerable neurones in human neurodegenerative diseases, particularly Parkinson's disease, may be induced by iron‐mediated free radical production in degenerating neurones, making investigation of the mechanism of free radical‐induced responses in neuronal cells of special interest.
Effects of Chronic Nicotine Treatment on the Accumulation of [3H]Tetraphenylphosphonium by Cerebral Cortical Synaptosomes
Authors: Hillard Cecilia J. , Pounds Jody J.
Published: October 2006

Abstract: Chronic exposure of rats to nicotine increases the number of [3H]nicotine binding sites in the brain; however, it is not clear whether nicotinic cholinergic receptor function is altered as well. In this study, we have used [3H]tetraphenylphosphonium as a probe of synaptosomal membrane potential to investigate whether exposure to nicotine in vivo alters the ability of cerebral cortical synaptosomes to maintain a potential difference and to depolarize in response to in vitro nicotine. Treatment of rats for 14 days with 0.475 mg of nicotine base/day via subcutaneously implanted minipumps resulted in a decrease in the synaptosomal accumulation of [3H]tetraphenylphosphonium in physiological buffer, corresponding to a decrease in estimated membrane potential from –55 mV to –50 mV. The onset of the decrease in membrane potential occurred after 7 days of in vivo nicotine treatment and was significantly correlated with an increase in [3H]nicotine binding to cerebral cortical synaptosomal (P2) membranes. Nicotine, at in vitro concentrations of 3–1,000 μM, decreased [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes from control animals. When compared to accumulation in buffer alone, in vitro nicotine and other nicotinic agonists did not significantly decrease [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes prepared from rats treated with nicotine in vivo. These studies provide evidence that chronic treatment with nicotine results in an average lower membrane potential in cerebral cortical synaptosomes and in functional down‐regulation of the depolarization response to nicotinic cholinergic receptor stimulation.
GT1b Ganglioside Prevents Tetanus Toxin‐Induced Protein Kinase C Activation and Down‐Regulation in the Neonatal Brain In Vivo
Authors: Aguilera José , Padrós‐Giralt Carme , Habig William H. , Yavin Ephraim
Published: October 2006

Abstract: A single dose of 0.25 ng of tetanus toxin (TeTx), equivalent to ∼5 minimal lethal doses, injected intracerebrally to 1‐day‐old rats, caused translocation, i.e., activation, of Ca2+‐phosphatidylserine‐dependent protein kinase C (PKC) from the cytosolic to the membrane compartment within 1 h. Six hours after treatment with the toxin, a 40–50% reduction in the total brain PKC (cytosolic plus membrane) activity was noticed. GT1b (2 μg per brain) ganglioside, a putative receptor for TeTx, completely prevented enzyme translocation when injected intracerebrally 30 min before toxin administration and abolished down‐regulation after 6 h from the time of toxin injection. GM1 (2 μg per brain), a ganglioside of lesser affinity for TeTx, produced by itself a 20–30% reduction of the total PKC activity and did not reverse TeTx‐induced PKC down‐regulation after 6 h. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) phorbol ester, administered at a concentration of 5 × 10−5M, caused activation and down‐regulation of the enzyme, although with several orders of magnitude lesser potency. GT1b prevented the TPA‐induced down‐regulation.