bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of Lead‐203 at the Blood‐Brain Barrier During Short Cerebrovascular Perfusion with Saline in the Rat
Authors: Deane R. , Bradbury M. W. B.
Published: October 2006

Abstract: Lead transport at the blood‐brain barrier has been studied by short (< 1.5 min) vascular perfusion of one cerebral hemisphere of the rat with a buffered physiological salt solution at pH 7.4 without calcium, magnesium, or bicarbonate and containing 203Pb‐labelled lead chloride. In the absence of complexing agents, 203Pb uptake was rapid, giving a space of 9.7 ml/100 g of wet frontal cortex at 1 min. Lead‐203 influx was linear with lead concentration up to 4 μM. Five percent albumin, 200 μM cysteine, or 1 μM EDTA almost abolished 203Pb uptake. Lead‐203 entry into brain was uninfluenced by varying the calcium concentration or by magnesium or the calcium blocker methoxyverapamil. Similarly, 1 μM bicarbonate or 50 μM 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid was without effect. Increasing the potassium concentration reduced 203Pb uptake. Vanadate at 2 μM, 2 μM carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (a metabolic uncoupler), or 2 μM stannic chloride all markedly enhanced lead entry into brain, as did a more alkaline pH (7.80). In conclusion, there is a mechanism allowing rapid passive transport of 203Pb at the brain endothelium, perhaps as PbOH+. Lead uptake into brain via this system is probably made less important by active transport of lead back into the capillary lumen by the calcium‐ATP‐dependent pump.
Transport of Tryptophan into Brain from the Circulating, Albumin‐Bound Pool in Rats and in Rabbits
Authors: Pardridge William M. , Fierer Gary
Published: October 2006

Abstract: Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin‐bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood‐brain barrier (BBB), the transport of tryptophan from the circulating albumin‐bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety‐Renkin‐Crone equation modified for protein‐bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 ± 0.1 and 3.9 ± 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 ± 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to value of 2.1 ± 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5‐fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia. The BBB permeability‐surface area product (PS) was estimated along with the KaD parameter, and this PS value in the rabbit brain, 0.13 ml/min/g, was identical to the quantity [(Vmax/Km) +KD] measured from estimates of the constants of saturable (Km, Vmax) and nonsaturable (KD) transport of tryptophan through the BBB. In conclusion, the present studies demonstrate that tryptophan is transported into brain from the circulating albumin‐bound pool via an enhanced dissociation mechanism and that this process varies among species and with changing anesthesia and cerebral hemodynamics.
Affinity States of Rat Brain Opioid Receptors in Different Tissue Preparations
Authors: Zajac J.‐M. , Bigeard A. , Delay‐Goyet P. , Roques B. P.
Published: October 2006

Abstract: The binding of [3H]Tyr‐D‐Ala‐Gly‐(N‐Me)PheGly‐ol ([3H]DAGO) and [3H]Tyr‐D‐Thr‐Gly‐Phe‐Leu‐Thr ([3H]DTLET), selective agonists for μ and δ‐opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris‐HCI, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs‐HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20‐μm tissue slices were used, whatever the brain area studied. The dissociation rate of (3H)DTLET was clearly dependent on the tissue preparation used, because the Koff value of this ligand in Krebs‐HEPES was 2.5‐fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs‐HEPES was 6.5‐fold greater than that measured in Tris‐HCl. In intact cells, the Koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs‐HEPES and similar to that observed in membrane preparations in Tris‐HCI supplemented with 30 mM NaCl. These data suggest (a) that the Koff constant of [3H]DTLET was regulated by the ionic environment of the δ‐opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.
Effects of Transforming Growth Factor β1 on Astroglial Cells in Culture
Authors: Toru‐Delbauffe D. , Baghdassarian‐Chalaye D. , Gavaret J. M. , Courtin F. , Pomerance M. , Pierre M.
Published: October 2006

Abstract: The effects of transforming growth factor β1 (TGFβ1) on DNA synthesis and functional differentiation of astroglial cells cultured in serum‐free medium were investigated. TGFβ1 diminished and delayed the peak of DNA synthesis induced by serum. TGFβ1‐treated cells were larger than control cells. This factor delayed the appearance of process‐bearing cells induced by acidic fibroblast growth factor treatment and also affected the astrocyte‐specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGFβ1 inhibited the induction of GS activity by HC in a dose‐ and time‐dependent manner. Moreover, pretreatment with TGFβ1 for 4 h maintained the inhibition of GS activity for ˜16 h after removal of this factor from culture medium. These results suggest that TGFβ1 may be an important regulator of astrocyte growth and differentiation.
The Myelin‐Deficient Rat Has a Single Base Substitution in the Third Exon of the Myelin Proteolipid Protein Gene
Authors: Simons R. , Riordan J. R.
Published: October 2006

Abstract: Several genetic disorders that occur in animals and in humans result in an inability to synthesize normal myelin. Some of these disorders are inherited in an X‐linked manner. The localization of the myelin proteolipid protein (PLP) gene to the X chromosome has directed the study of X‐linked myelination disorders toward PLP. The myelin‐deficient rat is one such X‐linked dysmyelinating mutant. From a cDNA library constructed from myelin‐deficient rat brain mRNA, we have isolated and sequenced cDNAs corresponding to PLP and its alternatively spliced isoform, DM‐20. An A to C transition was detected in these cDNAs, which results in a threonine to proline change at amino acid 74 in both PLP and DM‐20. No other substitutions were seen in the cDNA sequences. Polymerase chain reaction amplification and sequencing of the corresponding genomic regions were used to confirm the single base change. This substitution occurs in a highly hydrophobic portion of the protein that is thought to be an α‐helical transmembrane segment. The presence of a helix‐breaking amino acid such as proline in this segment is likely to influence the ability of the protein to interact with the membrane.
Transmembrane 36CI− Flux Measurements and Desensitization of the γ‐Aminobutyric AcidA Receptor
Authors: Kardos Julianna , Cash Derek J.
Published: October 2006

Abstract: Some data on the concentration range of response and the concentration for half‐response (EC50) of γ‐aminobutyric acid (GABA) for the GABAA receptor are reviewed and compared. An analysis of the 36CI− flux assay demonstrates that both the EC50 and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the 36CI− concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC50 can be decreased by orders of magnitude by increasing the assay time. The EC50 measured in a finite time is less than the half‐response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC50 depends on the rate of receptor desensitization. The computer simulations showed that a GABAA receptor with a half‐response concentration of 100 μM GABA can give 36CI− flux measurements with an EC50 value 100‐fold lower.
Regional Distribution of Calcitonin Gene‐Related Peptide‐, Substance P‐, Cholecystokinin‐, Met5‐Enkephalin‐, and Dynorphin A (1–8)‐Like Materials in the Spinal Cord and Dorsal Root Ganglia of Adult Rats: Effects of Dorsal Rhizotomy and Neonatal Capsaicin
Authors: Pohl M. , Benoliel J. J. , Bourgoin S. , Lombard M. C. , Mauborgne A. , Taquet H. , Carayon A. , Besson J. M. , Cesselin F. , Hamon M.
Published: October 2006

Abstract: Biochemical mapping of five different peptide‐like materials—calcitonin gene‐related peptide (CGRP), substance P (SP), Met5‐enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1–8) (DYN)—was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3‐month‐old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4‐T2) or after neonatal administration of capsaicin (50 mg/kg s.c). In control rats, all peptide‐like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy‐induced CGRP depletion (‐85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (‐60%), a finding suggesting that this peptide is contained in both capsaicin‐sensitive (mostly unmyelinated) and ‐insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (‐50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin‐sensitive small‐diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin‐insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.
Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells
Authors: Lee Ken , Miwa Soichi , Koshimura Kunio , Hasegawa Hiroshi , Hamahata Keigo , Fujiwara Motohatsu
Published: October 2006

Abstract: The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K+‐induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media pre‐equilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K+‐induced catecholamine release was inhibited by hypoxia to ∼40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura‐2) gradually increased and reached a plateau of ∼1.0 μM at 30 min, whereas the level was constant in the control medium (∼200 nM). High K+‐induced increases in [Ca2+]I were inhibited by hypoxia to ∼30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K+‐induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K+‐induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K+‐induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage‐dependent Ca2+ channels.
Is the Adenosine Receptor Modulation of Histamine‐Induced Accumulation of Inositol Phosphates in Cerebral Cortical Slices Mediated by Effects on Calcium Ion Fluxes?
Authors: Alexander Stephen P. H. , Hill Stephen J. , Kendall David A.
Published: October 2006

Abstract: In this report, we show that under conditions designed to provide an initially uniform incorporation of [3H]inositol into mouse and guinea pig cerebral cortical slices prior to agonist stimulation, the accumulation of 3H‐inositol phosphates (3H‐InsPx, x = 1–4) induced by histamine in mouse and guinea pig cerebral cortical slices increased in a quasilinear manner with increasing added calcium. Raising the ambient calcium ion concentration failed to reduce the adenosine receptor‐mediated inhibition of the histamine‐induced 3H‐InsPx response in mouse cerebral cortical slices. Similarly, the potentiation of the histamine response by adenosine receptor activation in guinea pig cerebral cortical slices was unaffected by lowering the added calcium ion concentration. The presence of the calcium ionophore A23187 (33 μM) produced 3H‐InsPx responses in both mouse and guinea pig cerebral cortical slices, which were not affected by the presence of the stable adenosine analogue 2‐chloroadenosine. A23187 also potentiated the accumulation of 3H‐InsPx induced by histamine in both species. Both the inhibitory and potentiatory modulations of the histamine response by 2‐chloroadenosine in mouse and guinea pig, respectively, were still apparent in the presence of A23187. These results indicate that the histamine‐induced 3H‐InsPx accumulations in both mouse and guinea pig cerebral cortical slices are sensitive to variations in calcium ion concentrations. However, the adenosine receptor modulations of the histamine responses are relatively insensitive to fluctuations in either extra‐or intracellular calcium ion concentrations, and thus cannot be mediated by effects on calcium ion movements.
Vasoactive Intestinal Peptide Stimulates Chick Pineal Melatonin Production and Interacts with Other Stimulatory and Inhibitory Agents but Does Not Show α1‐Adrenergic Potentiation
Authors: Zatz Martin , Kasper Gabriel , Marquez Christopher R.
Published: October 2006

Abstract: Vasoactive intestinal peptide (VIP) is known to mimic the effects of β‐adrenergic receptor stimulation in the rat pineal, including marked potentiation by α1‐adrenergic receptor stimulation, and to cause increased melatonin synthesis. In contrast, the chick pineal does not respond to β‐adrenergic stimulation, and melatonin synthesis is inhibited by norepinephrine via an α2‐adrenergic receptor. The present experiments show that chick pineal cells in primary culture do, however, respond to VIP with increased melatonin production. The effect of VIP was inhibited by addition of norepinephrine or of nitrendipine or by exposing the cells to “unexpected” white light. Stimulation by VIP was enhanced by addition of forskolin or Bay K 8644 but not by α1adrenergic receptor stimulation. Although stimulation by VIP appears similar in the chick pineal to that seen in the rat pineal and other systems, “dual‐receptor regulation,” at least with α1‐adrenergic receptors, appears to be absent.
Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro
Authors: Tischler Arthur S. , Ruzicka Laurel A. , Perlman Robert L.
Published: October 2006

Abstract: The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF‐induced neurite outgrowth from a PC12 cell subclone in a dose‐dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF‐induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF‐induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures. Staurosporine might be useful in studying both the actions of NGF and the ways in which those actions are altered in the course of normal development and tumor formation.
Cell‐Specific Expression of the Mouse Glial Fibrillary Acidic Protein Gene: Identification of the Cis‐ and Trans‐Acting Promoter Elements for Astrocyte‐Specific Expression
Authors: Miura Masayuki , Tamura Taka‐aki , Mikoshiba Katsuhiko
Published: October 2006

Abstract: We have determined the whole promoter sequence and the transcriptional startpoint of the mouse glial fibrillary acidic protein (GFAP) gene and characterized the promoter function. We found that the cis elements for astrocyte specific expression are located within 256 bp from the transcription startpoint. We defined by DNase I footprinting assay three trans‐acting factor binding sites (GFI, GFII, and GFIII) using brain or C6 astrocytoma nuclear extracts. GFI, GFII, and GFIII have AP‐2, NFI, and cyclic AMP‐responsive element motifs, respectively. Mutations in GFII drastically decreased the promoter activity. Base substitution in GFI and GFIII abolished the cell‐specific expression, resulting in the GFAP promoter expression even in some non‐GFAP‐producing cells.
Small Rises in Plasma Choline Reverse the Negative Arteriovenous Difference of Brain Choline
Authors: Klein Jochen , Köppen Andrea , Löffelholz Konrad
Published: October 2006

Abstract: The concentrations of free choline in blood plasma from a peripheral artery and from the transverse sinus, in the CSF, and in total brain homogenate, have been measured in untreated rats and in rats after acute intraperitoneal administration of choline chloride. In untreated rats, the arteriovenous difference of brain choline was related to the arterial choline level. At low arterial blood levels (<10 μM) as observed under fasting conditions, the arteriovenous difference was negative (about ‐2 μM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (> 15 μM), the arteriovenous difference was positive, implying a marked net uptake of choline by the brain (3.1 nmol/g/min). The CSF choline concentration, which reflects changes in the extracellular choline concentration, also increased with increasing plasma levels and closely paralleled the gradually rising net uptake. Acute administration of 6, 20, or 60 mg of choline chloride/kg caused, in a dose‐dependent manner, a sharp rise of the arterial blood levels and the CSF choline, and reversed the arteriovenous difference of choline to markedly positive values. The total free choline in the brain rose only initially and to a quantitatively negligible extent. Thus, the amount of choline taken up by the brain within 30 min was stored almost completely in a metabolized form and was sufficient to sustain the release of choline from the brain as long as the plasma level remained low. We conclude that the extracellular choline concentration of the brain closely parallels fluctuations in the plasma level of choline. Moreover, the often described release of choline from the brain as reflected by the negative arteriovenous difference of brain choline is not a steady‐state phenomenon. Instead, the uptake of choline into and the release of choline from the brain seem to be in dynamic equilibrium that is closely related to the plasma choline level and, consequently, to nutritional choline uptake.
Soluble and Participate Forms of the Organophosphorus Neuropathy Target Esterase in Hen Sciatic Nerve
Authors: Vilanova E. , Barril J. , Carrera V. , Pellin M. C.
Published: October 2006

Abstract: Neuropathy target esterase (NTE) is the suggested “target” molecule involved in the initiation of organophosphorus‐induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P‐NTE) and soluble (S‐NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80–100 nmol/min/g tissue from which 50–60% was recovered in the soluble supernatant fraction and the remaining 40–50% in the pellet fraction. About 90% of brain tissue activity (∼ 1,800 nmol/min/g tissue) was recovered as P‐NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P‐NTE and S‐NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose‐6‐phosphatase, Na+,K+‐ATPase, 5′‐nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P‐NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S‐NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane‐bound portions of a single protein, with P‐NTE activity depending on a membrane factor from which it is separated through fractionation at high pH. The titration curve corresponding to inhibition by mipafox was studied over the 0.1–200 μM range, in the presence of 40 μM paraoxon, and data obtained were fitted to models of one or two exponential mipafox‐sensitive components plus a resistant component. Mipafox‐resistant activity was 38 and 52% of total paraoxon‐resistant activity for the particulate and soluble fractions, respectively. Particle data suggest that P‐NTE contains mainly one component with I50 of ∼5.4–7.3 μM, this representing >85% of total mipafox‐sensitive activity. However, the soluble fraction data fit better to two sensitive components: high‐ and low‐mipafox‐sensitive components with I50 of 4.9 and 43 μM, representing 35 and 65% of total paraoxon‐resistant activity, respectively.
Regional Difference in Responsiveness of Adenosine‐Sensitive Cyclic AMP‐Generating Systems in Chronic Epileptic Cerebral Cortex of the Rat
Authors: Hattori Yukio
Published: October 2006

Abstract: Cyclic AMP accumulation in brain slices incubated with adenosine or the adenosine analogue 2‐chloroadenosine was examined in different areas of rat cerebral cortex following a unilateral injection of FeCl2 solution into the sensorimotor cortex to induce chronic epileptic activity. In the epileptic cortex, cyclic AMP accumulation in cortical slices was elicited three‐ to 11‐fold by adenosine. The elicitation by adenosine of cyclic AMP accumulation was markedly inhibited by the adenosine antagonist 8‐phenyltheophylline. In anterior cortical areas of rats in which the appearance of electrographic isolated spikes was dominant either ipsilateral or contralateral to the injection site 8 days or more after the injection, the adenosine‐elicited accumulation of cyclic AMP was greater on the side of dominant spike activity than on the other. In anterior cortical areas of rats showing nearly equal spike activity on the two sides 19 days or more after the injection, the cyclic AMP accumulation was greater on the side ipsilateral to the injection site than on the other. In anterior and posterior cortical areas of rats showing spike‐and‐wave complexes and isolated spikes 1 month or more after the injection, the cyclic AMP accumulation was greater on the ipsilateral side than on the other. Similar regional differences in the adenosine‐elicited accumulation of cyclic AMP were detected in the presence of the adenosine uptake inhibitor dipyridamole or the phosphodiesterase inhibitor dl‐4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro 20–1724). The cyclic AMP accumulation was elicited five‐ to 17‐fold by 2‐chloroadenosine, in which case the elicitation was markedly inhibited by 8‐phenyltheophylline. Regional differences in the 2‐chloroadenosine‐elicited accumulation of cyclic AMP were similar to those with adenosine and were detected in the presence of Ro 20–1724 or adenosine deaminase. The regional differences which correlated with the electrographic discharge patterns were due mainly to persistent changes in cyclic AMP accumulation on the primary epileptic side. These results suggest that alterations in adenosine‐sensitive cyclic AMP generation in the cortex are associated with the neurochemical process leading to chronic iron‐induced epilepsy.
Kynurenine Pathway Measurements in Huntington's Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid
Authors: Beal M. Flint , Matson Wayne R. , Swartz Kenton J. , Gamache Paul H. , Bird Edward D.
Published: October 2006

Abstract: Recent evidence suggests that there may be over‐activation of the N‐methyl‐D‐aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16‐sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5‐hydroxyindoleacetic acid, 5‐hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p < 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.
Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain
Authors: Coulon J. F. , Biguet N. Faucon , Cavoy A. , Delacour J. , Mallet J. , David J. C.
Published: October 2006

Abstract: Tyrosine hydroxylase, aromatic l‐amino‐acid decarboxylase, and dopamine β‐hydroxylase activities were studied in the developing fetal rat brain. A delay of 2–3 days between the detection of the tyrosine hydroxylase and the aromatic l‐amino‐acid decarboxylase and dopamine β‐hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.
Differential Phosphorylation of Myelin‐Associated Glycoprotein Isoforms in Cell Culture
Authors: Afar Daniel E. H. , Salzer James L. , Roder John , Braun Peter E. , Bell John C.
Published: October 2006

Abstract: The alternative splicing of myelin‐associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L‐MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and ammonium vanadate, but not by dibutyryl‐cyclic AMP. The shorter isoform, S‐MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl‐cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S‐MAG. 32P labeling of v‐sretransformed NIH 3T3 cells that express L‐MAG also show that L‐MAG is likely to be an in vivo substrate for pp60v‐src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.
Differential Sensitivity to Cations of the Melatonin Receptors in the Rat Area Postrema and Suprachiasmatic Nuclei
Authors: Laitinen Jarmo T. , Saavedra Juan M.
Published: October 2006

Abstract: We studied the effects of cations on the binding of the melatonin (MT) agonist, 125I‐MT, to MT receptors in the rat area postrema (AP) and suprachiasmatic nuclei (SCN), by using quantitative autoradiography in vitro. Ca2+ promoted agonist binding in the SCN but was without effect in the AP. Na+ induced a dose‐dependent loss of agonist binding in both areas. This effect was more drastic in the SCN and also in the absence of divalent cations. The presence of 0.1–4.0 mM Ca2+ or Mg2+ partially and nonselectively reversed this Na+‐elicited inhibition. The data agree with known cationic effects on agonist binding to other G protein‐coupled receptors and deepen our understanding of the mammalian MT receptor.
Evidence of a Monoaminergic‐Cholinergic Imbalance Related to Visual Hallucinations in Lewy Body Dementia
Authors: Perry Elaine K. , Marshall Elizabeth , Kerwin Janet , Smith Carthage J. , Jabeen Sabiha , Cheng Anthony V. , Perry Robert H.
Published: October 2006

Abstract: Senile dementia of Lewy body type is characterized clinically by a relatively acute onset of fluctuating memory loss and confusion, frequently accompanied by visual hallucinations. Neurochemical analyses of temporal cortex has revealed a distinction between hallucinating and nonhallucinating patients in both cholinergic and monaminergic transmitter activities. In contrast with the cholinergic enzyme choline acetyltransferase, which was more extensively reduced in hallucinating individuals, serotonergic S2 receptor binding and both dopamine and serotonin metabolites were significantly decreased in nonhallucinating cases. These results suggest that an imbalance between monaminergic and cholinergic transmitters is involved in hallucinogenesis in the human brain.
Involvement of Dihydropyridine‐Sensitive Ca2+ Channels in Adenosine‐Evoked Inhibition of Acetylcholine Release from Guinea Pig Ileal Preparation
Authors: Katsuragi Takeshi , Shirakabe Katsuya , Ogawa Shusuke , Soejima Osamu , Furukawa Tatsuo
Published: October 2006

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 μM), was enhanced by Bay K. 8644 (0.5 μM; a Ca2+ antagonist) or 4‐aminopyridine (30 μM; a K+ channel blocker) but not by theophylline (100 μM; a P1 purinoceptor antagonist) or atropine (0.3 μM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two‐ to fourfold in the presence of theophylline, atropine, Bay K 8644, 4‐aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10–30 μM), nifedipine (0.1–0.3 μM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1–3 μM) in a concentration‐related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K. 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4‐aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine‐sensitive Ca2+ channel activities in the cholinergic neurons, and thus L‐type Ca2+ channels may exist on the nerve terminals. It was also demonstrated that the HPLC‐ECD method used for detecting endogenous ACh was suitable for evaluating mechanisms of the neuromodulation.
Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics
Authors: García‐Martín Elena , González‐Cabanillas Soledad , Gutiérrez‐Merino Carlos
Published: October 2006

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP‐dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the KC.5 of inhibition of calcium uptake are the following: 23 μM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2+‐dependent Mg2+‐ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP‐dependent Ca2+ uptake, e.g., 80–700 μM dibucaine, 2–10 mM lidocaine, and 1–3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.
Inhibition of Brain Type A Monoamine Oxidase and 5‐Hydroxytryptamine Uptake by Two Amphetamine Metabolites, p‐Hydroxyamphetamine and p‐Hydroxynorephedrine
Authors: Arai Yuichiro , Kim Se Kyung , Kinemuchi Hiroyasu , Tadano Takeshi , Satoh Shin‐Etsu , Satoh Nobunori , Kisara Kensuke
Published: October 2006

Two amphetamine metabolites, p‐hydroxyam‐phetamine (p‐OHA) and p‐hydroxynorephedrine (p‐OHN), selectively inhibited the A form of monoamine oxidase (MAO) in rat and mouse forebrain homogenates. Of these two metabolites, p‐OHA inhibited MAO‐A more strongly than p‐OHN. This MAO‐A‐selective inhibition by p‐OHA or p‐OHN was found to be competitive with respect to deamination of its substrate, 5‐hydroxytryptamine (5‐HT). The degree of MAO‐A inhibition was not changed by 90 min of preincubation of the enzyme preparations with either metabolite, and the activity inhibited by p‐OHA after the preincubation recovered completely to the control level after repeated washing. Uptake of 5‐HT or dopamine into mouse forebrain synaptosomes was highly reduced by both p‐OHA and p‐OHN. Both metabolites were more potent in reducing dopamine uptake than in reducing 5‐HT uptake. In reduction of 5‐HT and of dopamine uptake, p‐OHA was more potent than p‐OHN. These results indicate that p‐OHA is a more selective inhibitor of brain MAO‐A activity and 5‐HT uptake than its subsequent metabolite, p‐OHN. These two actions of p‐OHA might, together with possible 5‐HT efflux into the synaptic cleft, greatly contribute to head twitch, a brain 5‐HT‐mediated animal behavior induced by p‐OHA.
Increased γ‐Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors
Authors: Lummis Sarah C. R. , Gundlach Andrew L. , Johnston Graham A. R. , Harper Peter A. W. , Dodd Peter R.
Published: October 2006

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine‐mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral γ‐aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high‐ and low‐affinity binding sites for the GABA agonist [3H]muscimol were found (KD= 9.3 ± 1.5 and 227 ± 41 nM, respectively), whereas only the high‐affinity site was detectable in controls (KD= 14.0 ± 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA‐stimulated benzodiazepine binding. The affinity for t‐[3H]butylbicyclo‐o‐benzoate, a ligand that binds to the GABA‐activated chloride channel, was significantly increased in ICM brain membranes (KD= 148 ± 14 nM) compared with controls (KD= 245 ± 33 nM). Muscimol‐stimulated 36Cl‐ uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low‐affinity, physiologically relevant GABA receptors. Study of the GABA receptor complex in ICM calves may provide a greater insight into the interactions of the various binding sites on the GABA‐benzodiazepine receptor protein molecule.
Gangliosides Stimulate Calcium Flux in Neuro‐2A Cells and Require Exogenous Calcium for Neuritogenesis
Authors: Wu Gusheng , Vaswani Kuldeep K. , Lu Zi‐Hua , Ledeen Robert W.
Published: October 2006

The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal and neuroblastoma systems, but the mechanism is not understood. Involvement of Ca2+ is suggested by this study which demonstrates that treatment of Neuro‐2A cells with bovine brain gangliosides (BBG) in Ca2+‐depleted medium failed to produce neurite outgrowth. This was in contrast to treatment with retinoic acid or dibutyryl cyclic AMP which induced differentiation under the same conditions. Addition of BBG to Neuro‐2A cells caused small, but significant, increases in both influx and efflux of Ca2+. It thus appears that although neuritogenesis can proceed by more than one mechanism, that induced by BBG requires exogenous Ca2+ and involves stimulation of Ca2+ flux.
Exogenous Gangliosides Stimulate Breakdown of Neuro‐2A Phosphoinositides in a Manner Unrelated to Neurite Outgrowth
Authors: Vaswani Kuldeep K. , Wu Gusheng , Ledeen Robert W.
Published: October 2006

Gangliosides administered exogenously are wellknown effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro‐2A cells with bovine brain ganglioside mixture (BBG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BBG concentration of 25 μM. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on gangliosidestimulated neurite outgrowth. A similar result was obtained with ψ‐tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro‐2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded.
Neurotoxins Distinguish Between Different Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations
Authors: Luetje Charles W. , Wada Keiji , Rogers Scott , Abramson Stewart N. , Tsuji Kuniro , Heinemann Steve , Patrick Jim
Published: October 2006

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the α3β2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The α2β2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the α1β1γδ muscle subunit combination. The α2β2 and α3β2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog‐1, whereas complete blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog‐1. The α1β1γδ muscle subunit combination was blocked by the α‐conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both α‐conotoxins. Thus, neosurugatoxin and the α‐conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog‐1 distinguish between different neuronal subunit combinations on the basis of differing α subunits.
Liver, Brain, and Heart Metallothionein Induction by Stress
Authors: Hidalgo Juan , Borrás Marta , Garvey Justine S. , Armario Antonio
Published: October 2006

To date, stress has been reported to induce metallothionein (MT) synthesis in the liver only. In the present experiment, the effects of food and water deprivation alone or of immobilization stress plus food and water deprivation on liver, brain, and heart MT have been studied in adult male rats. Liver and brain MT levels were increased by immobilization stress as soon as 6 h after the onset of stress. Eighteen hours of immobilization, which is accompanied by food and water deprivation, further increased liver and brain MT levels and significantly increased heart MT content. A specific effect of immobilization was evident in all three tissues, because the effect of food and water deprivation alone was significantly lower than that of immobilization plus starvation. Changes in MT apparently were not related to changes in cytosolic Zn.
In Vivo Autoxidation of Dopamine in Guinea Pig Striatum Increases with Age
Authors: Fornstedt Bodil , Pileblad Erik , Carlsson Arvid
Published: October 2006

Catechols are known to react readily with molecular oxygen to form the corresponding quinones together with reduced oxygen species. These products have been shown to be toxic in in vivo and in vitro systems. 5‐S‐Cysteinyl adducts of catechols are believed to be formed through the spontaneous reaction between quinones and thiol‐containing compounds, like cysteine and glutathione (GSH). Thus, the brain levels of these adducts probably indicate the autoxidation rate of catechols in vivo. In the present study, the striatal concentrations of 5‐S‐cysteinyldopamine (5‐S‐cysteinyl‐DA), 5‐S‐cysteinyl‐3,4‐dihydroxyphenylalanine (5‐S‐cysteinyl‐DOPA), and 5‐S‐cysteinyl‐3,4‐dihydroxyphenylacetic acid (5‐S‐cysteinyl‐DOPAC) were determined in 2‐week‐, 2‐month‐and 3‐year‐old guinea pigs. In addition, brain levels of DA, the DA metabolite DOPAC, and GSH were assessed. The concentration of 5‐S‐cysteinyl‐DA increased markedly with age. The 3‐year‐old guinea pigs had the highest level, i.e., 248% of the concentration in the 2‐week‐old animals and 219% of the concentration in the 2‐month‐old animals. Furthermore, the striatal 5‐S‐cysteinyl‐DOPA level in the 3‐year‐old group was 68% higher than in the 2‐week‐old group and 46% higher than in the 2‐month‐old group. No age difference in the striatal concentration of DA was found. In contrast, the concentration of DOPAC increased with age: The DOPAC level in the 3‐year‐old animals was 153% of the level in the 2‐week‐old animals and 116% of the level in the 2‐month‐old animals. A slight decrease in brain GSH content was observed between 2 weeks and 2 months, whereas the level in the 3‐year‐old animals was not significantly different from the concentration in the 2‐month‐old animals. Thus, the present study has shown an increase in 5‐S‐cysteinyl‐DA and 5‐S‐cysteinyl‐DOPA levels with age in guinea pig striatum. The rise in 5‐S‐cysteinyl‐DA content was larger than the increase in content of the DA metabolite DOPAC, a finding indicating that DA to a greater extent undergoes autoxidation with increasing age. This may be part of the mechanism underlying the loss of dopaminergic neurons in senescence and in Parkinson's disease.