bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia‐Induced Neurotransmitter Deficits in Neonatal Rats Are Partially Corrected by Exogenous GM1 Ganglioside
Authors: Hadjiconstantinou Maria , Yates Allan J. , Neff Norton H.
Published: October 2006

Abstract: Exposure of 7‐day‐old rats to 7% oxygen/balance nitrogen for 2 h results in selective changes of cholinergic, serotonergic, and dopaminergic neuronal markers in the frontal cortex, hippocampus, and striatum when evaluated 3 weeks after the insult. There is also about a 15% deficiency in brain weight. Treatment with GM1 ganglioside, 50 mg/ kg i.p., for 2 days before and for 3 weeks after the hypoxic insult partially corrects the neurodevelopmental abnormalities including the deficiency in brain weight. We conclude that GMI ganglioside might have therapeutic potential for treating suspected neonatal hypoxia.
1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐ and 1‐Methyl‐4‐(2′‐Ethylphenyl)‐1,2,3,6‐Tetrahydropyridine‐Induced Toxicity in PC 12 Cells: Role of Monoamine Oxidase A
Authors: Basma Alie N. , Heikkila Richard E. , Nicklas William J. , Giovanni Andrew , Geller Herbert M.
Published: October 2006

Abstract: The toxicity of l‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), l‐methyl‐4‐(2′‐ethylphenyl)‐1,2,3,6‐tetrahydropyridine (2'Et‐MPTP), and their corresponding pyridinium species was studied in the rat pheochromocytoma PC 12 cell line. MPTP and its analogues are known to be metabolized by monoamine oxidase (MAO) to dihydropyr‐idinium intermediates which are further transformed, either enzymatically or spontaneously, into pyridinium species. MAO activity in PC12 cells is almost exclusively of the A form, and 2'Et‐MPTP is a good substrate for both MAO‐A and MAO‐B. In contrast, MPTP is a poor substrate for MAO‐A, but a good substrate for MAO‐B. 2'Et‐MPTP caused considerably more cell death than MPTP in the PC 12 cells. However, l‐methyl‐4–(2′‐ethylphenyl)pyridinium and 1‐methyl‐4‐phenylpyridinium, the corresponding pyridinium species formed from 2'Et‐MPTP and MPTP, respectively, were equipotent as toxins. The toxic effects of the tetrahy‐dropyridines and their corresponding pyridiniums were both concentration‐ and time‐dependent. Measurements of the levels of the pyridinium species formed and the remaining tetrahydropyridine in the media indicated that 2'Et‐MPTP was converted about five to seven times more readily into its toxic pyridinium species than was MPTP. There was, moreover, an excellent correlation between amount of pyridinium formed and cell death. There was also a parallel between the capacity of clorgyline and pargyline, irreversible MAO inhibitors, to decrease the formation of the pyridinium species and their capacity to protect against the toxic actions of the tetrahydropyridines. These data are consistent with the concept that the MAO‐A‐dependent formation of the pyridinium species from the tetrahydropyridine is a prerequisite for toxicity in PC12 cells.
Use of 1,2‐Bis(2‐Amino‐5‐Fluorophenoxy)ethane‐N,N,N′,N′‐Tetraacetic Acid (5FBAPTA) in the Measurement of Free Intracellular Calcium in the Brain by 19F‐Nuclear Magnetic Resonance Spectroscopy
Authors: Badar‐Goffer Ronnitte S. , Ben‐Yoseph Oded , Dolin Simon J. , Morris Peter G. , Smith Gerry A. , Bachelard Herman S.
Published: October 2006

Abstract: We have applied the 19F‐nuclear magnetic resonance (NMR) calcium indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration ([Ca2+] i) in superfused brain slices. A mean ± SD control value of 380 ± 71 nM(n = 18) was obtained at 37°C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3 H] 5FBAPTA showed that after loading of its tetraac‐etoxymethyl ester, ∼55% was de‐esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de‐esterified 5FBAPTA, >90% was in the cytosolic fractions, with >1% in the mitochondria or microsomes. The NMR‐visible de‐esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+], was constant over a 5‐h period, although the scatter of individual results was ±20%. The [Ca2+]; was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e. the [Ca2+]i“overshot” to values lower than the original control value. The [Ca2+]i was surprisingly resistant to changes in extracellular Ca2+ concentration.
Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse
Authors: Fujii Tatsuya , Ito Masatoshi , Tsuda Hideo , Mikawa Haruki
Published: October 2006

Abstract: Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 μg of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 μg of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, nor‐adrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine‐β‐hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.
Effect of Precursors on the Synthesis of Catecholamines and on Neurotransmission in the Superior Cervical Ganglion of the Rat
Authors: Zhang C. , Bordet S. , Karoum F. , Commissiong J. W.
Published: October 2006

Abstract: Male Sprague‐Dawley rats (325–350 g) were anesthetized with urethane (1.5 g/kg i.p.) and treated with physiological saline, Aspartame (APM; 552 μmol/kg), or tyrosine (Tyr; 552 μmol/kg). Ganglionic transmission and the synthesis of dopamine (DA) and norepinephrine (NE) were measured in the superior cervical ganglion (SCG) following electrical stimulation of the cervical sympathetic trunk (CST). When the CST was stimulated with single pulses, neither APM nor Tyr affected the synthesis of NE or DA. However, in response to low‐ (5 Hz, 20 s) and high‐ (20 Hz, 20 s) frequency pulses, the metabolism of DA was increased (p > 0.05), but to the same extent after saline, APM, or Tyr. In rats stimulated with similar low‐ and high‐frequency pulses, the synthesis of NE was increased significantly (p > 0.05) after Tyr, but not after APM or saline. In saline‐treated controls, ganglionic transmission was not changed in response to single pulses, or low‐ or high‐frequency stimulation. However, after treatment with APM, ganglionic transmission was depressed significantly (p > 0.01) in response to high‐frequency stimulation (single: 0.46 ± 0.09 mV; low: 0.39 ± 0.07 mV; high: 0.27 ± 0.07 mV). After treatment with Tyr, ganglionic transmission was depressed significantly (p > 0.05) in response to both low‐ and high‐frequency stimulation (single: 0.44 ± 0.04 mV; low: 0.22 ±0.12 mV; high: 0.26 ± 0.07 mV). In the nonstimulated SCG, l‐3,4‐dihydroxyphenylalanine (25 mg/kg) caused a rapid, significant (p > 0.01) increase in the synthesis and metabolism of DA, but not of NE. Treatment with nialamide (200 mg/kg i.p.) followed by electrical stimulation (15 Hz, 15 min) of the CST caused a significant (p > 0.05) increase of both NE and DA in the stimulated SCG. It is concluded that there are both similarities and differences in the regulation of the synthesis of NE and in the modulation of ganglionic transmission after the administration of the precursors APM and Tyr. The results indicate that caution is needed in comparing the neurochemical and neurophysiological effects of different catecholamine precursors.
Leukotriene B4 Release and Polymorphonuclear Cell Infiltration in Spinal Cord Injury
Authors: Xu Jian , Hsu Chung Y. , Liu Tsung H. , Hogan Edward L. , Perot Phanor L. , Tai Hsin‐Hsiung
Published: October 2006

Abstract: Activation of arachidonic acid occurs after spinal cord injury. Leukotriene B4 is a lipoxygenase metabolite of arachidonic acid. In a rat model of experimental spinal cord injury, we found that the leukotriene B4 content was less than the sensitivity of our assay (8 pg/mg of protein) in non‐traumatized spinal cord. Leukotriene B4 was detectable in raumatized cord (mean ± SE, 25 ± 5 pg/mg of protein; n = 3). Release of leukotriene B4 from spinal cord slices into the incubation medium was also noted after trauma (9 ± 1 pg/mg of protein; n = 12) and was enhanced by exposure of traumatized spinal cord slices to the calcium ionophore A23187 (375 ± 43 pg/mg of protein; n = 12). The amount of leukotriene B4 released corresponded to the extent of post‐traumatic polymorphonuclear cell infiltration determined by a myeloperoxidase assay. Results from this study suggest that the source of leukotriene B4 in spinal cord injury is infiltrating polymorphonuclear cells.
Altered Patterns of Protein Phosphorylation and Synthesis Caused by Methyl Mercury in Cerebellar Granule Cell Culture
Authors: Sarafian Theodore , Verity M. Anthony
Published: October 2006

Abstract: In the preceding report we demonstrated a dose‐dependent increase in 32P‐phosphoprotein labeling after 24‐h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures. In the present study we have examined 32P‐labeled phosphoproteins by two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. At concentrations of 0.5 and 1 μM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr∼28,000 and pI ∼ 5.7–5.9 (pp 28/5.7–5.9) and a protein with Mr∼ 58,000 and pI ∼ 5.6. The pp28 cluster displayed considerable two‐dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications. Time course studies revealed that increased 32P phosphola‐beling of pp28/5.7–5.9 was detectable after 12‐h exposure to 3 μM MeHg and reached values of 300–500% of control by 24 h. These studies also showed that among the 21 proteins analyzed by two‐dimensional densitometry, 32P phosphola‐beling of four proteins increased by 20–50% and of two proteins decreased by 20–50% after 24‐h treatment. However, exposure to 10 μM MeHg produced stimulation of pp28/5.7–5.9 32P phospholabeling within 2 h. Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5–20%) enhanced. 35S and 32P double‐label analysis of cells treated with0, 0.5, and 1 μM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis. Stimulation of protein synthesis, however, was observed for proteins of Mr 32,000 and pI 6.5 and Mr 29,000 and pI 6.1. 35S incorporation into the 32,000 Mr protein following 24‐h exposure of granule neurons to 1 μM MeHg was stimulated about fivefold. The observation of enhanced synthesis or 32P phospholabeling of select proteins by MeHg is suggestive of a stress response and the electrophoretic and time‐dependent behavior of these proteins is consistent with such a role.
Metabolic Stability of 3‐O‐Methyl‐d‐Glucose in Brain and Other Tissues
Authors: Jay Thérèse M. , Dienel Gerald A. , Cruz Nancy F. , Mori Kentaro , Nelson Thomas , Sokoloff Louis
Published: October 2006

Abstract: 3‐O‐Methyl‐d‐glucose (methylglucose) is often used to study blood‐brain barrier transport and the distribution spaces of hexoses in brain. A critical requirement of this application is that it not be chemically converted in the tissues. Recent reports of phosphorylation of methylglucose by yeast and heart hexokinase have raised questions about its metabolic stability in brain. Therefore, we have re‐examined this question by studying the metabolism of methylglucose by yeast hexokinase and rat brain homogenates in vitro and rat brain, heart, and liver in vivo. Commercial preparations of yeast hexokinase did convert methylglucose to acidic products, but only when the enzyme was present in very large amounts. Methylglucose was not phosphorylated by brain homogenates under conditions that converted 97% of [U‐14C]glucose to ionic derivatives. When [14C]methylglucose, labeled in either the methyl or glucose moiety, was administered to rats by an intravenous pulse or a programmed infusion that maintained the arterial concentration constant and total 14C was extracted from the tissues 60 min later, 97–100% of the 14C in brain, >99% of the 14C in plasma, and >90% of that in heart and liver were recovered as unmetabolized [14C]methylglucose. Small amounts of 14C in brain (1–3%), heart (3–6%), and liver (4–7%) were recovered in acidic products. Plasma glucose levels ranging from hypoglycemia to hyperglycemia had little influence on the degree of this conversion. The distribution spaces for methylglucose were found to be 0.52 in brain and heart and 0.75 in liver.
Ionic Basis of Inhibitory Presynaptic Modulation in Rat Cortical Synaptosomes
Authors: Zoltay Gabor , Cooper Jack R.
Published: October 2006

Abstract: We have investigated the possibility that, regardless of the involvement of a second messenger system, the ultimate effect of presynaptic, receptor‐activated inhibitory modulation is the opening of a K channel. With the consequent hyperpolarization of the terminal, less Ca2+ would enter and this would result in the observed diminished release of a neu‐rotransmitter. This possibility was explored utilizing rat cortical synaptosomes that were prelabeled with either 86Rb or [3H]acetylcholine, depolarizing with either K+ or veratridine, and measuring either efflux of 86Rb or release of [3H]acetylcholine in the presence or absence of inhibitory presynaptic modulators. The modulating agents used were 2‐chloroadenosine, carbamylcholine, clonidine, and morphine. In all instances, these agents promoted an increased efflux of 86Rb, indicating hyperpolarization, and decreased release of acetylcholine. These results are compatible with our suggestion that an increase in K conductance may be responsible for presynaptic inhibition of the release of neurotransmitters.
Characterization and Intracellular Distribution of Microtubule‐Associated Protein 2 in Differentiating Human Neuroblastoma Cells
Authors: Kirsch Joachim , Zutra Aliza , Littauer Uriel Z.
Published: October 2006

Abstract: The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule‐as‐sociated protein 2 (MAP2) enabled us to identify two distinct high‐molecular‐mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250‐kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high‐molecular‐mass MAP2 by exposing nitrocellulose‐bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against β‐tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426–445 of the C‐terminal region of β‐tubulin. Immunological cross‐reactivity with several mAbs directed against bovine brain MAP2, taxol‐promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti‐MAP2 and anti‐β‐tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.
[3H]1‐Methyl‐4‐Phenyl‐2,3‐Dihydropyridinium Ion Binding Sites in Mouse Brain: Pharmacological and Biological Characterization
Authors: Del Zompo M. , Ruiu S. , Maggio R. , Piccardi M. P. , Corsini G. U.
Published: October 2006

Abstract: Because 1‐methyl‐4‐phenyl‐2,3‐dihydropyridinium ion (MPP+) appears to damage the dopaminergic neuron and cause neuronal death, we characterized [3H]MPP+ binding sites in mouse brain membranes. Among several compounds tested, debrisoquin [3,4‐dihydro‐2(1 H)‐isoquinolinecarboxamidine] and some analogues were able to antagonize [3H]MPP+ binding. Debrisoquin is able to block adrenergic transmission and inhibit the activity of monoamine oxidase A (MAO‐A). We found a certain correlation between the ability of these agents to displace [3H]MPP+ from its binding sites and their capacity to inhibit MAO‐A activity. These data and the finding of a higher number of [3H]MPP+ binding sites in human placenta compared to mouse brain suggest that these sites may correspond to MAO‐A enzymes. Recently it has been demonstrated in human brain that neurons in regions rich in catecholamines are positive for MAO‐A. Accordingly, we suggest MAO‐A as a possible accumulation site of MPP+ within the dopaminergic neuron. We also indicate the chemical structural requirement associated with the best binding of debrisoquin analogues with [3H]MPP+ sites. It would be reasonable to test the effects of debrisoquinlike drugs able to pass the blood‐brain barrier on 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine toxicity.
Localization of N‐Methyl‐d‐Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3‐(2‐Carboxypiperazin‐4‐yl)propyl‐1‐Phosphonic Acid Binding
Authors: Samuel Denise , Errami Mohammed , Nieoullon André
Published: October 2006

Abstract: The binding of [3H]3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid ([3H]CPP), a rigid analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7) and reported to be a selective N‐methyld‐aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (Kd= 272 nM) in a saturable, reversible, and protein concentration‐dependent manner, Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [[3H]CPP binding inhibitors tested were CPP, l‐glutamate, 2‐amino‐5‐phosphonovalerate, and AP7, NMDA, l‐aspartate, and α‐aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, d,l‐2‐amino‐4‐phosphonobutyrate, kainate, l‐glutamate diethylester, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6‐hydroxydopamine injections resulted, 2–3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites. These data suggest the occurrence of functional receptor‐receptor interregulations at the postsynaptic level between dopaminergic and NMDA receptors in complement with the interactions occurring at the presynaptic level between glutamatergic and dopaminergic nerve terminals.
A Specific Effect of Antipsychotic Drugs on Protein Synthesis in Human Lymphomononuclear Cells
Authors: Nimgaonkar V. L. , Whatley S. A.
Published: October 2006

Abstract: We have studied the effects of psychotropic drugs on patterns of protein synthesis in human lymphomononuclear cells by two‐dimensional gel electrophoretic analysis. Drugs effective in treatment of schizophrenia specifically increased the relative synthesis of a 30‐kDa polypeptide in cultured human lymphomononuclear cells whereas dopamine (DA) or psychoactive drugs lacking antipsychotic properties did not. The effect was stereospecific with respect to the clinically active and inactive isomers of flupenthixol. Synthesis of the 30‐kDa polypeptide appears therefore to be correlated with antipsychotic properties but not with DA receptor binding. It is possible that such effects may be associated with the clinically beneficial effect of antipsychotic drugs in the brain.
Presence of Corticotropin‐Releasing Factor‐Stimulated Adenylate Cyclase Activity in Rat Retina
Authors: Olianas Maria C. , Onali Pierluigi
Published: October 2006

Abstract: Corticotropin‐releasing factor (CRF) stimulates rat retinal adenylate cyclase activity in a concentration‐dependent manner. The half‐maximal effect is obtained at 50 nM CRF and the maximal stimulation corresponds to ∼90% increase of basal enzyme activity. The CRF effect is counteracted by the CRF antagonist α‐helical CRF 9‐41 with a Ki value of 40 nM. Other CRF‐like peptides such as sauvagine and urotensin I are as effective as CRF with a rank order of potency of urotensin I sauvagine > CRF. The sauvagine and urotensin I effects are not additive with that elicited by CRF. Moreover, the CRF stimulation is not additive with the increase of enzyme activity produced by vasoactive intestinal peptide or dopamine. The CRF effect is independent of the concentration of free Ca2+, is optimal at 5–10 mM MgCl2, and requires GTP. The results indicate that rat retinal adenylate cyclase is modulated by CRF via a receptor‐mediated mechanism.
Ion and Temperature Effects on the Binding of γ‐Aminobutyrate to Its Receptors and the High‐Affinity Transport System
Authors: Shank R. P. , Baldy W. J. , Mattucci L. C. , Villani F. J.
Published: October 2006

Abstract: A set of procedures was developed to study the binding of γ‐[3]‐aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na+‐dependent transport carrier, at 25 and 37°C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze–thawing or treatment with Triton X‐100. Isoguvacine, (–)‐baclofen, and (–)‐nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high‐ (KD < 30 nM), middle‐ (KD 100–500 nM), and low‐affinity (KD > 5 μM) binding sites. However, the binding data in the middle‐affinity region (100–1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl− at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl−. Apparent binding to the transport carrier required both Na+ and Cl−. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high‐affinity site of the GABAA receptor and the transport binding site.
Drug Effects on the Release of Endogenous Acetylcholine In Vivo: Measurement by Intracerebral Dialysis and Gas Chromatography–Mass Spectrometry
Authors: Marien Marc R. , Richard Jean W.
Published: October 2006

Abstract: Intracerebral microdialysis was combined with a sensitive and specific gas chromatographic–mass spectrometric assay to measure the release of endogenous acetylcholine in the rat striatum in vivo. In rats anesthetized with urethane (1.2 g/kg i.p.), the levels of striatal acetylcholine dialyzed into a Ringer's perfusate were: (a) reliably measurable only in the presence of physostigmine; (b) stable at between 3 and 8 h of perfusion (30–75 pmol/20 min in the presence of 75 μM physostigmine); (c) reduced by calcium‐free Ringer's solution, tetrodotoxin (0.1 μM), and vesamicol (1.0 μM); and (d) increased by elevated potassium (100 mM), atropine (3–300 μM), and haloperidol (0.75 mg/kg i.p.). In conscious unrestrained rats, the spontaneous release of striatal acetylcholine was not altered significantly following the administration of urethane. The changes in acetylcholine release observed in this study are consistent with the known actions of some drugs or ionic conditions on striatal cholinergic neurotransmission and are evident under the condition of urethane anesthesia. The present results demonstrate the sensitivity and suitability of this method for monitoring endogenous striatal acetylcholine release in vivo.
Target Regulation of Axotomy‐Sensitive Proteins
Authors: Savage Mary J. , Buriani Alessandro , Goldberg Daniel J.
Published: October 2006

Abstract: Large changes in the production of certain proteins often follow axotomy. How the cell body is signaled to make these changes, or terminate them after regeneration is finished, is unclear. This issue was addressed by studying an axotomized giant identified neuron, the giant cerebral neuron of the sea slug Aplysia, both in vivo and in culture. One week after axon crush in vivo, there were increases of 1.5–18‐fold in the 5‐h incorporation of [35S]methionine into seven proteins identified by two‐dimensional gel electrophoresis. There were decreases of five‐to 28‐fold in the labeling of four other proteins. An axotomized giant cerebral neuron grows vigorously when placed in culture and forms chemical synapses with appropriate target cells while continuing unabated growth. The labeling of two of the proteins that up‐regulate after axotomy in vivo was suppressed by the presence of target cells in culture. For one of the proteins, this effect was also produced by membranes of target cells, but not by medium conditioned by exposure to target cells. These results are consistent with the idea that loss of membrane–membrane contact with target cells (or its restoration) is involved in the initiation (or termination) of the up‐regulation of certain proteins after axotomy.
Identification and Characterization of Cysteinyl‐Leukotriene Formation in Tissue Slices from Human Intracranial Tumors: Evidence for Their Biosynthesis Under In Vivo Conditions
Authors: Simmet Thomas , Luck Werner , Winking Michael , Delank Wolfgang K. , Peskar Bernhard A.
Published: October 2006

Abstract: In previous studies, it had been shown that human gray and white matter tissue slices have the capacity to synthesize large amounts of cysteinyl‐leukotrienes (cysteinyl‐LT) in vitro. This study was initiated to investigate cysteinyl‐LT formation by tissue slices from intracranial tumors in comparison with cyclooxygenase products such as prostaglandin (PG) F2α, and thromboxane (TX) B2. Tissue slices from meningiomas and astrocytomas were found to release large amounts of cysteinyl‐LT spontaneously and even higher amounts after ionophore A 23187 stimulation, which could not be accounted for by blood possibly remaining in the tissue slices. Cysteinyl‐LT were identified by their immunoreactive characteristics, their biological activity in the guinea pig ileum bioassay, and their retention time on reversed‐phase HPLC. With increasing malignancy, astrocytomas were shown to have an increasing biosynthetic capacity for cysteinyl‐LT and TXB2 in vitro. In comparison with meningioma patients, malignant astrocytoma patients had an enhanced urinary excretion of LTE4, the major urinary metabolite of cysteinyl‐LT in humans, which dropped in level within 7 days after operation by 79%. A correlation exists between the in vivo cysteinyl‐LT production in patients with malignant astrocytomas and that observed under basal conditions in the tissue slices in vitro. The results provide evidence that in malignant astrocytoma patients, the tumor tissue produces large amounts of cysteinyl‐LT, which may be detected in the patients' urine.
Potentiation by γ‐Aminobutyric Acid of α1‐Agonist‐Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex
Authors: Crawford Melissa L. A. , Young J. Michael
Published: October 2006

Abstract: Noradrenaline‐induced accumulation of 3H‐labeled inositol mono‐, bis‐, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium‐treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by γ‐aminobutyric acid (GABA). However, the effect on [3H]IP3 accumulation was much greater than that on [3H]IP3 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration–response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of >1 mM. Prazosin (1 μM) blocked the noradrenaline‐induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mMGABA. 3H‐IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 μM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3‐aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (–)‐Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline‐induced 3H‐IP formation was still apparent in the absence of Li+, but the increase of [3H]IP content was less than that of [3H]IP1 content.
Alteration of α‐Tocopherol Content in the Developing and Aging Peripheral Nervous System: Persistence of High Correlations with Total and Specific (n‐6) Polyunsaturated Fatty Acids
Authors: Clément M. , Bourre J. M.
Published: October 2006

Abstract: In contrast to brain, the scialic nerve concentration of vitamin E in rats increased rapidly during the postnatal period (approximately fivefold between days 1 and 8), then decreased dramatically (about twofold between days 8 and 30), and further decreased slowly between days 30 and 60 and remained constant up to 2 years. Although the sciatic nerve concentration of vitamin E decreased by 58% between days 8 and 30, the concentration of vitamin E in serum presented a marked decrease (∼75%). The vitamin E concentrations varied in a similar pattern in whole sciatic nerve and in endoneurium and showed a very close correlation (r= 0.94). The age‐related changes in fatty acid concentration of the endoneurial fraction of the sciatic nerve were characterized by a large increase in content of saturated and monounsaturated fatty acids up to 6 months (twofold for saturated and fourfold for monounsaturated fatty acids). Then, up to 24 months, the amount of these fatty acids decreased very slowly. The content of (n‐6) polyunsaturated fatty acids (PUFAs) decreased rapidly up to 1 year and slowly afterward. In contrast, during development the amount of (n‐3) PUFA was relatively stable and decreased during aging. A highly significant correlation between vitamin E and (n‐6) PUFA [18:2(n‐6), 20:4(n‐6), and total (n‐6)) was observed but not between (n‐3) PUFA and vitamin E. It is suggested that there may be a relationship between vitamin E and (n‐6) PUFA in the PNS membranes during development and aging.
Neurotransmitter and Depolarization‐Stimulated Accumulation of Inositol 1,3,4,5‐Tetrakisphosphate Mass in Rat Cerebral Cortex Slices
Authors: Challiss R. A. John , Nahorski Stefan R.
Published: October 2006

Abstract: [32P]Inositol 1,3,4,5‐tetrakisphosphate ([32P]Ins(1,3,4,5)P4) binds to a rat cerebellar membrane site with high affinity (KD=2.8 ± 0.6 nM) and low capacity (Bmax= 176 ± 34 fmol/mg of protein). Evidence for a low‐affinity site (KD=164 ± 48 nM) was also apparent. The high‐affinity site displayed marked specificity for the lns (1,3,4,5)P4 isomer, compared with several other inositol poly phosphates, and has been used as the basis of a radioreceptor assay for Ins(1,3,4,5)P4 in extracts of rat cerebral cortex slices. The resting Ins(1,3,4,5)P4 concentration (1.89 ± 0.11 pmol/mg of protein) in the slices was rapidly and dramatically increased by carbachol and quisqualate. K+ depolarization of cerebral cortex slices also stimulated Ins(1,3,4,5)PP4 accumulation, with at least 50% of the response being sensitive to atropine, a result indicating that muscarinic receptor stimulation by released acetylcholine contributes significantly to the K+ effect
Astrocytes Are Target Cells for Endothelins and Sarafotoxin
Authors: Marsault Robert , Vigne Paul , Breittmayer Jean‐Philippe , Frelin Christian
Published: October 2006

Abstract: Endothelin‐I, endothelin‐3, and the snake venom toxin sarafotoxin 56b stimulate the hydrolysis of phosphatidylinositol by phospholipase C with similar potencies in primary cultures of astrocytes prepared from rat brain cortex. In indo I‐loaded cells, endoth elin‐I, endothelin‐2, endothelin‐3, and sarafotoxin induce the rapid mobilization of intracellular Ca2+ stores and promote a more slowly developing influx of Ca2*+. These responses were insensitive to pertussis toxin and to inhibitors of cyclooxygenase and lipoxygenase. Similar actions of endothelins and sarafotoxin were observed using astrocytes from the cerebellum and glioma cells from the C6 and NN cell lines. The endthelin receptor of astrocytes differs from the receptor previously characterized in endothelial cells from brain microvessels in that it has a high affnity for endothelin‐3. Thus, brain endothelin‐l and endothelin‐3 have different target cells in the brain and may have different functions.
Differential Expression of mRNA and Protein Encoding Retinal and Pineal S‐Antigen During the Light/Dark Cycle
Authors: Craft Cheryl M. , Whitmore Donald H. , Donoso Larry A.
Published: October 2006

Abstract: S‐Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well‐characterized molecule that participates in light‐induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S‐antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S‐antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7‐kb mRNA for S‐antigen was observed in both the pineal gland and the retina. Retinal S‐antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S‐antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S‐antigen, identified with two S‐antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb CIOC10, was analyzed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS‐PAGE revealed a single 46‐kDa protein in retina. In contrast, two bands of ∼43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S‐antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland‐specific forms of S‐antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue‐specific structural components for S‐antigen in the retina and pineal gland that are not regulated in the same manner.
Evidence that Phosphoinositide Metabolism in Rat Cerebral Cortex Stimulated by Pilocarpine, Physostigmine, and Pargyline In Vivo Is Not Changed by Chronic Lithium Treatment
Authors: Honchar Michael P. , Vogler George P. , Gish Beverly G. , Sherman William R.
Published: October 2006

Abstract: The effect of chronic versus acute administration of lithium on receptor‐linked phosphoinositide metabolism was assessed by comparing the change in the cerebral cortex levels of myo‐inositol 1‐phosphate in response to pilocarpine, physostigmine, or pargyline in rats. Rats were exposed to either 29 consecutive days of LiCl injections or 27 and 39 days of dietary Li2CO3, followed by injected LiCl at the end of the diet to insure a constant level of exposure to the drug. In each experiment, an acute group received a single injection of LiCl 20–24 h before they were killed. One hour before being killed, some of the animals acutely exposed to lithium and some of the animals chronically exposed to lithium each received pilocarpine, physostigmine, or pargyline. At the conclusion of the experiment, the rats were killed and brain levels of myo‐inositol 1‐phosphate and lithium were determined. A differential production of myo‐inositol 1‐phosphate in groups receiving acute versus chronic lithium would provide evidence of a change in receptor‐linked phosphoinositide metabolism due to the chronic administration of lithium. Brain levels of mvo‐inositol 1‐phosphate are dependent on tissue lithium concentrations; consequently, significant differences observed in brain lithium levels between the groups receiving acute versus chronic lithium prevented a meaningful assessment of the effect of the mode of lithium administration on the production of myo‐inositol 1‐phosphate in those groups. Stepwise multiple regression analysis and the measured brain lithium levels were used to assess the response of myo‐inositol 1‐phosphate levels to stimulation in animals receiving acute or chronic lithium treatment. The results of the analysis indicate that there would have been no differences in the response to drugs when comparing the two routes of lithium administration if the lithium levels had been identical in both the acute and chronic lithium groups. These results also suggest that multiple regression analysis can be used with an established data base to aid in the interpretation of the results of experiments in which changes in brain myo‐inositol 1‐phosphate levels are used as an index of phosphoinositide metabolism. The possibility is discussed that the effects of lithium on phosphoinositide signalling are already present in the group receiving a single acute dose of LiCl and that this effect persists over the course of chronic administration.
Manipulation of Plasma Membrane Fatty Acid Composition of Fetal Rat Brain Cells Grown in a Serum‐Free Denned Medium
Authors: Park C. C. , Hennessey T. , Ahmed Z.
Published: October 2006

Abstract: Modifications of plasma membrane acyl‐linked phospholipid fatty acid composition were produced by supplementing the culture medium with essential fatty acids. The plasma membrane fraction was purified by Percoll gradient centrifugation from dissociated fetal rat brain cells grown in a serum‐free culture medium. Both the concentration dependence and the time course of the modifications were examined. Supplementation of the medium with essential polyunsaturated fatty acid, linolenic acid (18:3ω3) or lin‐oleic acid (18:2ω6), produced incorporation of the elongated and desaturated products of ω3 or ω6 class, respectively, i.e., the incorporation was class specific. Within each class, the most unsaturated and elongated members, i.e., terminal members, were preferentially incorporated until they reached a maximum concentration within 6–7 days. At higher concentrations of supplemented fatty acids, additional class specific incorporation in plasma membrane was produced by an increase in the concentration of intermediate members. At the same time, the concentration of monounsaturated fatty acids declined and that of saturated fatty acids remained unchanged. The modifications in fatty acid composition were reversible, with the time course similar to that of incorporation. The total plasma membrane phospholipid and sterol contents did not change with alterations of fatty acid composition, but did change with time in culture. This preparation should prove useful for investigating the role of polyunsaturated fatty acids in brain cell functions, including neuronal excitability.
Solubilization of the Membrane‐Bound Sialidase from Pig Brain by Treatment with Bacterial Phosphatidylinositol Phospholipase C
Authors: Chiarini A. , Fiorilli A. , Siniscalco C. , Tettamanti G. , Venerando B.
Published: October 2006

Abstract: The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5′‐nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37°C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5′‐nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase, whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 ± 10−‐4 and 0.98 ± 10−‐4M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4°C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase. Both enzyme activities could be separated by Sepharose 6B column filtration in a unique fraction that was eluted between catalase and bovine serum albumin. These results suggest that (a) the plasma membrane fraction from pig forebrain contains two sialidases (pH 4.2 and 6.6) that are presumably linked via a glycosylphosphatidylinositol anchor, sensitive to PIPLC action, and (b) pH 6.6 sialidase may be in the membrane under the inhibition of a factor that is liberated by PIPLC treatment and removed during ammonium sulfate fractionation and subsequent dialysis.
Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy
Authors: Toda Ken‐Ichi , Kobayashi Takuro , Goto Ikuo , Ohno Kousaku , Eto Yoshikatsu , Inui Koji , Okada Shintaro
Published: October 2006

Abstract: We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep‐Pak column, derivatization with o‐phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9–35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (∼2 pmol/ mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223–1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3–45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.
Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte‐Enriched and Neuronal Primary Cultures from Rat Brain
Authors: Agulió Luis , Picatoste Fernando , García Agustina
Published: October 2006

Abstract: Histamine stimulates cyclic AMP accumulation in astrocyte‐enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax= 304 ± 44% over basal, EC50= 43 ± 5 μM) was much higher than in neuronal cultures (Emax= 24 ±2%, EC50= 14 ± 7 μM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki=1.1 ± 0.2 μM) and ranitidine (Ki‐ 46 ± 10 nM) but was insensitive to the H1 antagonist mepyramine (1 μM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2‐thiazolylethylamine (0.01–1 mM) did not potentiate the response to impromidine (10 μM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine‐induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.
Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors
Authors: de Carvalho Roberto Paes , Braas Karen M. , Snyder Solomon H. , Adler Ruben
Published: October 2006

Abstract: We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in ∼20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while >50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine. and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K+‐stimulated conditions. Potassium‐enhanced release was blocked by 10 mM CoCl2 or in Ca2+‐free, Mg2+‐rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.
5‐Hydroxytryptamine Amplifies β‐Adrenergic Stimulation of N‐Acetyltransferase Activity in Rat Pinealocytes
Authors: Sugden David
Published: October 2006

Abstract: In cultured rat pinealocytes β‐adrenergic induction of N‐acetyltransferase, a key enzyme in the synthesis of melatonin, is amplified by addition of 5‐hydroxytryptamine (5‐HT) to the culture medium. However, 5‐HT when added alone has no effect on enzyme activity. Pharmacological experiments with a range of agonists and antagonists suggest that this action is not mediated by 5‐HT1, 5‐HT2, 5‐HT3, or 5‐HT4 receptor subtypes but may involve a site similar to the 5‐HTip receptor described in the enteric nervous system. The potential role of 5‐HT in modulating adrenergic stimulation of N‐acetyltransferase activity is discussed.