bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmitter Glutamate Release from Isolated Nerve Terminals: Evidence for Biphasic Release and Triggering by Localized Ca2+
Authors: McMahon Harvey T. , Nicholls David G.
Published: October 2006

Abstract: The kinetics of Ca2+‐dependent release of glutamate from guinea‐pig cerebrocortical synaptosomes evoked by KCl or 4‐aminopyridine are investigated using a continuous flu‐orimetric assay. Release by both agents is biphasic, with a rapid phase complete within 2 s followed by a more extensive slow phase with a half‐maximal release in 52 s for KCl‐evoked release and > 120 s for 4‐aminopyridine‐evoked release. The two phases ofglutamate release may reflect a dual localization of releasable vesicles at the active zone and in the bulk cytoplasm. Decreasing depolarization depresses the extent rather than increasing the time for half‐maximal Ca2+‐dependent release. Both the fast and the slow phases of glutamate release require external Ca2+ and cytoplasmic ATP. KCl depolarization produces a transient “spike”of cytoplasmic free Ca2+ ([Ca2+]c), which recovers to a plateau; the major component of glutamate release occurs during this plateau. Predepolarization in the absence of added external Ca2+, to inhibit transient Ca2+ channels, does not affect the subsequent glutamate release evoked by Ca2+ readdition. Thus, release involves primarily noninactivating Ca2+ channels. For a given increase in [Ca2+]c, KCl and 4‐aminopyridine cause equal release of glutamate, while ionomycin releases much less glutamate. This lowered efficiency is not due to ATP depletion. It is concluded that glutamate exocytosis is evoked by localized Ca2+ entering through noninactivating voltage‐dependent Ca2+ channels and that nonlocalized Ca2+ entry with ionomycin is inefficient.
Dopamine D2 Receptors Attenuate Phosphatidylisitol 4,5‐Bisphosphate in Synaptosomal Membranes from Rat Neostriatum
Authors: von Eule Gabriel , von Euler Anne
Published: October 2006

Abstract: The effects of dopamine on [32P]ATP‐labelled phosphatidylinositol 4‐phosphate, phosphatidylinositol 4,5‐bisphosphate, and phosphatidic acid were analyzed by TLC in synaptosomal membranes of the rat neostriatum. The incorporation of 32P into these compounds was found to be stable within 1 min and was maintained during the 30 min of incubation. Dopamine (0.1–10μM) was found to attenuate the levels of phosphatidylinositol 4,5‐bisphosphate without affecting the levels of phosphatidylinositol 4‐phosphate or phosphatidic acid. The maximal decrease (−35 ± 4%) was reached at 10 μM of dopamine after 30 min of incubation. The dopamine (0.1 μM)‐induced decrease was blocked by the D2 selective antagonist raclopride (1 μM), but not by the D1 selective antagonist SCH 23390 (1 μM). These findings indicate the existence of an intramembrane coupling of dopamine D2 receptors to phosphoinositide turnover and may underlie some of the physiological effects of D2 receptor stimulation.
Serotonin Synthesis Rate Measured in Living Dog Brain by Positron Emission Tomography
Authors: Diksic M. , Nagahiro S. , Chaly T. , Sourkes T. L. , Yamamoto Y. L. , Feindel W.
Published: October 2006

Abstract: In vivo measurements by positron emission tomography of the brain serotonin synthesis rates in the normal dog, in the dog with increased plasma tryptophan concentration, and in the dog under different arterial oxygen tensions are described. The method described here permits repeated measurements in the same brain for the first time. An increase in the plasma tryptophan concentration from 16.6 to 191.5 and then to 381 μM resulted in close to a linear increase in the brain serotonin synthesis rate. When PaO2 was raised from 76 ± 2 to 106 ± 1 mm Hg, the rate of serotonin synthesis in the dog brain increased from 39 ± 8 to 54 ± 10 pmol g−1 min−1. The estimates of the Michaelis‐Menten constants, Kappm and Vmax for the transport of tryptophan through the blood‐brain barrier are 303 ± 54 μM and 63 ± 10 nmol g−1, min−1, respectively.
The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus
Authors: Niznik Hyman B. , Fogel Evan F. , Fassos Frank F. , Seeman Philip
Published: October 2006

Abstract: The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1‐(2‐[bis‐(4‐fluorophenyl)methoxy]ethyl)‐4‐[2‐(4‐azido‐3‐[125I]iodophenyl)ethyl]piperazine ([125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr= 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and, following complete deglycosylation, migrated as an Mr∼48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorpqration of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20–50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR‐12935 {3H‐labeled 1‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenylpropyl)piperazine}. In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR‐12935 with an estimated affinity (Ki∼ 2,000 nM) 10 times lower than in controls (Ki∼ 30 nM), while the affinity of mazindol for [3H]GBR‐12935 binding in the caudate was equal to that seen with controls (Ki∼ 50 nM). The proportion of [3H]GBR‐12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50–80%. Taken together, these data clearly suggest that (a) the dopamine transporter is not detectable in Parkinson's diseased putamen, (b) [3H]GBR‐12935 binding in the putamen of Parkinson's diseased brain is probably not to the dopamine uptake carrier, and (c) there is an interregional variation in the loss of dopamine uptake sites (lost in putamen and somewhat spared in caudate), which appears to parallel markedly the reported loss of dopamine content in idiopathic Parkinson's diseased striatum.
The Fusion of Liposomes to Rat Brain Microsomal Membranes Regulates Phosphatidylserine Synthesis
Authors: Corazzi Lanfranco , Pistolesi Roberto , Arienti Giuseppe
Published: October 2006

Abstract: Rat brain microsomal membranes were fused to liposomes prepared with several pure lipids, namely, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and mixtures of phosphatidic acid and phosphatidylcholine or phosphatidylethanolamine. The fusion between liposomes and microsomes was measured by the octadecyl rhodamine B chloride method. The extent and other properties of fusion largely depend on the lipid used to prepare liposomes; phosphatidic acid and phosphatidylinositol fuse more extensively than other lipid classes. The activity of serine base exchange is affected by the fusion between rat brain microsomes and lipids. It is strongly inhibited by phosphatidylserine, but it is activated by phosphatidic acid. The inhibition produced by phosphatidylserine on its own synthesis is proposed as a mechanism for controlling the formation of phosphatidylserine in rat brain microsomes.
Administration of 8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin in Raphe Nuclei Dorsalis and Medianus Reduces Serotonin Synthesis in the Rat Brain: Differences in Potency and Regional Sensitivity
Authors: Invernizzi Roberto , Carli Mirjana , Di Clemente Angelo , Samanin Rosario
Published: October 2006

Abstract: Following administration of 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; 0.04–5.0 μg/0.5 μl) in the raphe nucleus dorsalis (DR) or medianus (MR), the synthesis of serotonin (5‐HT), as assessed by the accumulation of 5‐hydroxytryptophan (5‐HTP) after decarboxylase inhibition, was measured in various regions of the rat CNS. At all doses, 8‐OH‐DPAT in the DR significantly reduced 5‐HTP accumulation in the striatum, nucleus accumbens, cortex, and prefrontal cortex, whereas even the highest dose had no effect in the hippocampus, hypothalamus, and spinal cord. One microgram of 8‐OH‐DPAT in the MR significantly reduced 5‐HTP accumulation in the nucleus accumbens and prefrontal cortex, and 5 μg had an effect in all the areas except the striatum and spinal cord. One and 5 μg of 8‐OH‐DPAT, administered in either the DR or MR, did not significantly modify the accumulation of dihydroxyphenylalanine in the striatum and nucleus accumbens. The results confirm that DR and MR have different sensitivities to 5‐HT1A receptor agonists, and that activation of 5‐HT1A receptors in these nuclei produces different effects on 5‐HT synthesis in different brain regions.
[3H]Citalopram Binding to Brain and Platelet Membranes of Human and Rat
Authors: Plenge Per , Mellerup Erling T.
Published: October 2006

Abstract: Citalopram, a selective serotonin (5‐HT) uptake inhibitor with antidepressant properties, was found to bind with high affinity to the 5‐HT transporter from human neuronal and platelet membranes. At 20°C, KD was about 1.5 nM in both tissues. [3H]Citalopram bound to rat neuronal membranes with higher affinity than to human neuronal and platelet membranes; at 20°C KD was about 0.7 nM. The Bmax value for the binding of [3H]citalopram to platelet membranes was close to that found using the 5‐HT uptake inhibitors [3H]imipramine and [3H]paroxetine, suggesting that all three 5‐HT uptake inhibitors bind to the 5‐HT transporter. The dissociation rate of [3H]citalopram increased twofold with each 4–5°C increase in temperature in both human and rat membranes, although at any given temperature, the dissociation rate was about four times faster in the human neuronal and platelet membranes than in rat neuronal membranes.
Calcium‐Independent γ‐Aminobutyric Acid Release from Growth Cones: Role of γ‐Aminobutyric Acid Transport
Authors: Taylor Joanne , Gordon‐Weeks Phillip R.
Published: October 2006

Abstract: Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K+‐stimulated release mechanisms for γ‐aminobutyric acid (GABA). Up to and including postnatal day 5, the K+‐stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2+‐independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976‐A, on K+‐stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K+‐stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K+‐stimulated GABA release from growth cones. In contrast, SKF 89976‐A profoundly depressed both basal and K+‐stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.
Developmental Changes in Enzymes Involved in Dolichyl Phosphate Metabolism in Cultured Embryonic Rat Brain Cells
Authors: Bhat Narayan R. , Frank David W. , Wolf Martha J. , Waechter C. J.
Published: October 2006

Abstract: The rates of synthesis of dolichol‐linked oligosaccharide intermediates and protein N‐glycosylation increased substantially during a developmental period corresponding to glial differentiation in primary cultures of embryonic rat brain. In this study developmental changes in three enzymes involved in dolichyl phosphate (Dol‐P) metabolism have been examined by in vitro assays and correlated with the induction pattern for lipid intermediate synthesis and protein N‐glycosylation. Dolichyl pyrophosphate (Dol‐P‐P) phosphatase activity was relatively low during the first 9 days in culture, but it increased significantly between days 9 and 25. Dol‐P‐P phosphatase did not change appreciably between days 22 and 30 in culture. A kinetic analysis of the developmental change in Dol‐P‐P phosphatase activity revealed that the Vmax increased 10‐fold between days 4 and 22, and there was also a significant change in the apparent Km for Dol‐P‐P. Dolichol kinase activity increased during the period (9–15 days) when there was a significant induction in oligosaccharide‐lipid synthesis and protein N‐glycosylation, and then declined in parallel with lipid intermediate synthesis and protein N‐glycosylation. Dol‐P phosphatase activity was present at relatively low levels for the first 9 days in culture, but it increased steadily between days 9 and 30. A kinetic comparison of the activity in membrane fractions from brain cells cultured for 9 and 25 days indicated that there was a 10‐fold increase in enzyme protein with unaltered affinity for Dol‐P. The results suggest that elevated dolichol kinase activity enhances the rate of lipid intermediate synthesis, and subsequent reciprocal changes in dolichol phosphorylation‐dephosphorylation are a regulatory factor in the deactivation of oligosaccharide‐lipid synthesis, and consequently of protein N‐glycosylation, during the period following glial differentiation in primary cultures of embryonic rat brain cells.
Arecoline‐Stimulated Brain Incorporation of Intravenously Administered Fatty Acids in Unanesthetized Rats
Authors: DeGeorge Joseph J. , Nariai Tadashi , Yamazaki Shingo , Williams Wesley M. , Rapoport Stanley I.
Published: October 2006

Abstract: Brain incorporation of [1‐14C]arachidonate ([14C]AA; 170 μCi/kg), [I‐14C]docosahexaenoate ([14C]DA; 100 μCi/kg), or [9,10‐3H]palmitate ([3H]PA; 6.4 mCi/kg) infused intravenously for 5 min was examined in the awake rat following systemic administration of the cholinomimetic arecoline (15 mg/kg i.p.). The rat was killed 15 min after infusion, and the brain was removed, frozen, and prepared for biochemical analysis and autoradiography. Brain radioactivity, normalized for plasma exposure, was increased by 41 and 45% in arecoline‐treated rats given (14C]AA and [14C]DA, respectively. Pretreatment with atropine prevented the increase in fatty acid incorporation. Arecoline treatment had no effect on brain incorporation of [3H]PA. Quantitative autoradiography indicated regionally selective increases in brain [14C]AA and [14C]DA incorporation in response to arecoline. The results suggest that intravenously administered radiolabeled fatty acids can be used to study neurotransmitter‐stimulated brain lipid metabolism in vivo.
Modification of Brain Guanine Nucleotide‐Binding Regulatory Proteins by Tryptamine‐4,5‐Dione, a Neurotoxic Derivative of Serotonin
Authors: Fishman Jordan B. , Rubins Jeffrey B. , Chen Jin‐Chung , Dickey Burton F. , Volicer Ladislav
Published: October 2006

Abstract: We have recently characterized a novel oxidation product of serotonin (5‐hydroxytryptamine, 5‐HT), tryptamine‐4,5‐dione, which increases 5‐HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide‐binding regulatory proteins Gi and Go to [3H]tryptamine‐4,5‐dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the α subunits of Gi and Go (∼40,000). The binding of [35S]guanosine‐5′‐O‐(3‐thiotriphosphate) ([35S]GTP‐γ‐S) to Gi and Go and pertussis toxin‐catalyzed [32P]ADP‐ribosylation of the G protein α subunits were both inhibited in a dose‐dependent manner by tryptamine‐4,5‐dione. Thus, neurotoxins such as tryptamine‐4,5‐dione may exert their effects through specific interactions with G proteins.
Neural 22‐Carbon Fatty Acids in the Weanling Rat Respond Rapidly and Specifically to a Range of Dietary Linoleic to of‐Linolenic Fatty Acid Ratios
Authors: Dyer John R. , Greenwood Carol E.
Published: October 2006

Abstract: Changing the dietary ratio of the essential fatty acids (EFA), 18:2n6 and 18:3n3, while keeping the amounts of other fatty acids in the diet constant can rapidly and specifically alter the proportions of n6 and n3 22‐carbon fatty acids in the brain of the weanling rat. A dietary 18:2n6/18:3n3 ratio of 165 versus 1.8 caused higher n6 and lower n3 22‐carbon fatty acid levels, without changing total 22‐carbon fatty acid levels, in phosphatidylethanolamine and phospha‐tidylcholine from several neural membrane fractions. This was apparent after only 2 weeks and showed no sign of plateauing after 12 weeks. Other neural fatty acids were essentially unaffected. The three most abundant 22‐carbon fatty acids responded somewhat differently to increments in the dietary 18:2n6/18:3n3 ratio (1.8, 9, 36, and 165). Levels of 22:4n6 increased by similar absolute amounts for each fourfold increase in dietary 18:2n6/18:3n3 ratio; in contrast, the largest absolute changes in 22:5n6 and 22:6n3 levels occurred as the 18:2n6/18:3n3 ratio increased from 36 to 165. This study shows that the 18:2n6/18:3n3 ratio of diets high in fat (40% of energy) and adequate in EFA, both typical of diets in developed countries, can substantially and relatively quickly affect the 22‐carbon fatty acids in the brain, even after the rapid accumulation of these fatty acids during neural growth has ceased.
Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion
Authors: Bhat Ratan V. , Turner Saralinda L. , Selvaag Sandra R. , Marks Michael J. , Collins Allan C.
Published: October 2006

Abstract: Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 μmol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo‐α‐bungarotoxin (α‐[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in α‐[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.
Phosphodiesterase of Cone Photoreceptors from the Lizard, Anolis carolinensis
Authors: Booth Doris P. , Hurwitz Richard L. , Lolley Richard N.
Published: October 2006

Abstract: Cone and rod photoreceptors utilize cyclic guanosine monophosphate (cGMP) in the light regulation of membrane polarization. The prototype for visual transduction is established for rod photoreceptors, which utilize a cascade of reactions to regulate a cyclic nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) and thereby control the intracellular concentration of cGMP. Although cones appear to utilize a comparable cGMP cascade for their phototransduction, evidence exists that the PDE from cone photoreceptors may be different from that of rods. Dissociated cone photoreceptors, isolated retinas, and cone outer segments from the lizard, Anolis carolinensis, have been used to identify and characterize a PDE enzyme complex that shares several features in common with the rod outer segment (ROS) PDE complex. Immunoadsorption and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis have identified a subunit of lizard cone PDE that has an apparent electrophoretic mobility of 84 kDa and a subunit of lizard rod PDE that migrates at ∼90 kDa. The lizard cone PDE complex is similar in size, extraction, activation, and immunological characteristics to the PDE complex of rod photoreceptors from lizard, bovine, and human retinas. The lizard cone PDE complex, and perhaps that from cone photoreceptors in general, differs from that of ROS in its chromatographic properties on anion‐exchange resins. The sharing of physical and activation properties of the rod and cone PDE complex is compatible with the phototransduction process occurring by a similar mechanism in both cell types. The differences in light sensitivity and speed of response may be attributable to features of the individual proteins that form the PDE complexes of rods and cones or to other undisclosed features of the respective cascades.
Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue
Authors: Regnouf F. , Rendon A. , Pradel L. A.
Published: October 2006

Abstract: Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the participate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca2+‐dependent phospholipid‐ and F‐actin‐binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F‐actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 × 10−6M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca2+‐independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca2+‐dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross‐react with these antibodies. Anti‐p37 and antilipocortin 1 cross‐react on the same 37‐ and 34‐kDa polypeptides; anti‐p36 and antilipocortin 2 cross‐react only on the 36‐kDa polypeptides.
Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism
Authors: Fukui Shinsuke , Schwarcz Robert , Rapoport Stanley I. , Takada Yoshiaki , Smith Quentin R.
Published: October 2006

Abstract: To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood–brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L‐kynurenine (L‐KYN), 3‐hydroxykynurenine (3‐HKYN), 3‐hydroxyanthranilic acid (3‐HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital‐anesthetized rats using an in situ brain perfusion technique. L‐KYN was found to be taken up into brain at a significant rate [permeability—surface area product (PA) = 2–3 × 10−3 ml/s/g] by the large neutral amino acid carrier (L‐system) of the blood–brain barrier. Best‐fit estimates of the Vmax and Km of saturable L‐KYN transfer equalled 4.5 × 10−4μmol/s/g and 0.16 μmol/ml, respectively. The same carrier may also mediate the brain uptake of 3‐HKYN as D,L‐3‐HKYN competitively inhibited the brain transfer of the large neutral amino acid L‐leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7–1.6 × 10−3 ml/s/g), and at far lower rates (PA, 2–7 × 10−5 ml/s/g) for 3‐HANA, KYNA, and QUIN. Transfer for KYNA, 3‐HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L‐KYN across the blood–brain barrier and suggest that circulating L‐KYN, 3‐HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3‐HANA cross the blood–brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.
Expression of Heat Shock Protein 70 and Heat Shock Cognate 70 Messenger RNAs in Rat Cortex and Cerebellum After Heat Shock or Amphetamine Treatment
Authors: Miller E. Katherine , Raese Joachim D. , Morrison‐Bogorad Marcelle
Published: October 2006

Abstract: The expression of strictly inducible hsp70 mRNAs and constitutively expressed hsc70 mRNAs was compared in cerebellum and cerebral cortex of control rats, heat‐shocked rats, and rats made hyperthermic with amphetamine. An hsc70‐specific oligonucleotide probe identified a 2.55‐kb mRNA in cerebellum and cerebral cortex of all rats. An hsp70‐specific oligonucleotide probe identified a 3.05‐kb mRNA and a 3.53‐kb mRNA in cerebellum and cerebral cortex of heat‐shocked and amphetamine‐treated rats, but not in control rats. Quantitation demonstrated that both hsp70 and hsc70 mRNA levels, relative to 18S rRNA levels, were increased following each treatment. The relative levels of both mRNAs were higher in cerebellum than in cerebral cortex. In amphetamine‐treated rats, hsc70 mRNA relative levels increased at body temperatures greater than 39°C, whereas hsp70 mRNA synthesis was induced at temperatures greater than 40°C. Total thermal response values and relative levels of both mRNAs were compared. The results suggested that both the transcription and turnover of hsp70 mRNAs differed between cerebellum and cerebral cortex. At equivalent total thermal response values, amphetamine‐treated rats had higher relative levels of hsp70 mRNAs than heat‐shocked rats, suggesting that amphetamine enhanced the induction of hsp70 mRNAs.
Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development
Authors: Southam E. , East S. J. , Garthwaite J.
Published: October 2006

Abstract: The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N‐methyl‐D‐aspartate (NMDA) and glutamate were highly efficacious agonists, whereas α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK‐801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist for ionotropic non‐NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK‐801. Cyclic GMP accumulations induced by NMDA and non‐NMDA agonists alike were Ca2+‐dependent and could be antagonized by competitive NO synthase inhibitors in an arginine‐sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L‐NG‐nitroarginine, a highly potent component (IC50= 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under “basal” conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.
Gangliosides Inhibit Glucosylceramide Synthase: A Possible Role in Ganglioside Therapy
Authors: Shukla Girja S. , Shukla Arti , Radin Norman S.
Published: October 2006

Abstract: Gangliosides stimulate the hydrolysis of glucosylceramide (GlcCer), their precursor, and therefore may lower the level of cellular GlcCer and exert a feedback control effect to slow the formation of gangliosides. Tests were made to see if a similar effect on GlcCer levels can be exerted by the action of gangliosides on GlcCer synthesis. Using a new assay procedure, we showed that gangliosides do inhibit the synthase in brain membranes quite effectively, the most active being those lipids with more sugar and sialic acid moieties. Mice injected with a mixture of brain gangliosides for 5 days were found to have a lower level of ceramide:UDP‐Glc glucosyl‐transferase activity in brain, liver, and kidney. The inhibition seems to be exerted by competition for the active site and binding to effector site(s) on the enzyme. It is possible that the reported therapeutic actions of gangliosides on the nervous system are, in part, the result of lowered levels of GlcCer. Malignant tumors shed gangliosides into the extracellular fluid, which are believed to block the generation of antibodies by the host's immunodefense system; this effect also may be due, in part, to reduction in the GlcCer level of immunogenic cells. A new finding is that a ceramide containing phytosphingosine is a markedly better substrate for GlcCer synthase than one containing the more common base.
Four Forms of Tyrosine Hydroxylase Are Present in Human Adrenal Medulla
Authors: Haycock John W.
Published: October 2006

Abstract: Alternative splicing can result in up to four forms of human tyrosine hydroxylase (HTH) mRNA (mRNAHTH). In the adrenal gland, a major site of catecholamine biosynthesis, the presence of all four mRNAHTH forms is controversial. In the present study, postmortem human adrenal medullary tissue was analyzed for the presence of multiple forms of HTH protein by blot immunolabeling. Electrophoretic transfers were developed with affinity‐purified rabbit anti‐HTH antibodies raised against peptides that reproduced the unique amino acid sequences predicted by the four mRNAHTH forms. All four of the predicted HTH protein forms were present in the adrenal glands from a diverse sample population.
Modulation of Protein Kinase C Translocation by Excitatory and Inhibitory Amino Acids in Primary Cultures of Neurons
Authors: Vaccarino Flora M. , Liljequist Sture , Tallman John F.
Published: October 2006

In primary cultures of neurons from rat cerebral cortex and neostriatum, excitatory amino acids stimulate the translocation of protein kinase C (PKC) from the cytoplasm to the membrane. In the presence of a physiological concentration of Mg2+ in the extracellular medium, glutamate induces PKC translocation by binding to both TV‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionic acid (AMPA) excitatory amino acid receptors. Quisqualate translocates the enzyme by stimulating primarily AMPA receptors and possibly metabotropic receptors. NMDA receptor‐induced PKC translocation is sodium independent, whereas quisqualate receptor‐induced PKC translocation is sodium dependent; none of the agonists is active in the absence of calcium from the extracellular medium. Muscimol does not modify excitatory amino acid stimulation; however, blockade of γ‐aminobutyric acidA receptors by bicuculline greatly enhances glutamate‐induced PKC translocation. This enhancement is blocked by the NMDA receptor antagonist (+)‐5‐methyl‐10, 11‐dihydro‐5H‐ dibenzo[a,d]cyclohepten‐5, 10‐imine hydrogen maleate (MK‐801) and by tetrodotoxin.
Purification and Properties of p 103, a Novel 103‐kDa Component of Postsynaptic Densities
Authors: Hayes Nandini V. L. , Rayner Denise A. , Baines Anthony J.
Published: October 2006

A 103‐kDa protein present in membrane cytoskeletal preparations from bovine brain has been identified. We have purified this protein to >95% homogeneity using gel filtration and ion‐exchange chromatography. This protein, p 103, is an asymmetric dimer in dilute solution and has two major variants that can be distinguished by isoelectric focussing, pI 5.60 and 5.75. Using subcellular fractionation, it is most enriched in postsynaptic densities. Immunolocalization with anti‐p 103‐specific antibodies reveals that it is confined to the dendrites and perikarya; it is apparently absent from spinal cord axons. It coextracts from brain membrane‐skeletal preparations with brain spectrin and actin, but in vitro, it does not interact with them.
Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin
Authors: Whitaker J. N. , Herman P. K. , Sparacio S. M. , Zhou S. R. , Benveniste E. N.
Published: October 2006

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were >95% astrocytes and <3% microglia. Under serum‐free conditions, leupeptin induced a 1.4‐ to 2.0‐fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon‐γ, human recombinant tumor necrosis factor‐α, human recombinant interleukin‐1β, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.
Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve
Authors: Padilla Stephanie , Pope Carey N.
Published: October 2006

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo‐[3H]inositol into the rat sciatic nerve (50–55 mm distal to L4 and L5 dorsal root ganglia), a time‐dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10–12‐fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water‐soluble label in the dorsal root ganglia still remained ∼ 1.0, whereas the corresponding ratio in the chloroform/methanol‐soluble fraction was ∼20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injec tion site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.
Purification and Characterization of a Nonvesicular Vesamicol‐Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain
Authors: Hicks Barry W. , Rogers Gary A. , Parsons Stanley M.
Published: October 2006

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that docs not contain synaptic vesicles. The purified vesamicol‐binding protein (VBP) has a molecular mass of 470 kDa composed of 30‐ and 24‐kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30‐kDa subunit is also sensitive to endo‐β‐galactosidase. The dissociation constant of the VBP vesamicol complex is 0.9 μM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30‐kDa subunit cross‐reacts with the 24‐kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP‐like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.
Effect of Soman and Sarin on Phosphatidylcholine Asymmetry in the Electroplax from the Electric Eel
Authors: Supernovich Carrie , Grain Richard , Rosenberg Philip
Published: October 2006

Some effects of organophosphorus anticholinesterase compounds that are unrelated to cholinesterase inhibition and that are sometimes long lasting may be due to alterations at the cellular membrane level. Phosphatidylcholine exchange protein was used to assess the effects of sarin and soman on Phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the electroplax. Exposure of electroplax (30 min in vitro) to soman (10−4, 10−6M) or sarin (10−4, 10−6, 5 × 10−9M) increased the percentage of Phosphatidylcholine in the outer monolayer of the innervated plasma membrane bilayer and decreased the percentage in the inner monolayer. These changes by sarin were observed at concentrations that produced 100% cholinesterase inhibition (10−4, 10−6 M) and at a concentration (5 × 10−9 M) where no inhibition occurred, suggesting that these effects are not directly due to cholinesterase inhibition. A 1‐week exposure of live eels to soman (10−8M) in vivo caused an increase in Phosphatidylcholine labeling in the outer monolayer of the innervated and noninnervated surfaces of the electroplax. Two weeks after stopping exposure to soman, increased labeling was still observed, suggesting that this may be a long‐term effect. Because the organophosphates did not increase the permeability of the electroplax, all of these changes in labeling appear to be due to a redistribution of Phosphatidylcholine from the inner to the outer monolayer of the plasma membrane bilayer.
One‐Line Monitoring of Extracellular Brain Glucose Using Microdialysis and a NADPH‐Linked Enzymatic Assay
Authors: van der Kuil Jan H. F. , Korf Jakob
Published: October 2006

A method to monitor extracellular glucose in freely moving rats, based on intracerebral microdialysis coupled to an enzyme reactor is described. The dialysate is continuously mixed with a solution containing the enzymes hexokinase and glucose‐6‐phosphate dehydrogenase, and the fluorescence of NADPH formed enables the on‐line registration of extracellular glucose. The method is applied to monitor changes in extracellular brian glucose during the infusion of glucose, electrically induced seizure, immobilization stress, and repetitive hypoxia. After glucose loading or after seizure, hippocampus dialysate glucose concentration was increased transiently. During immobilization, there was a short‐lasting decrease and, therafter, an increase in the extracellular hippocampus glucose. During repetitive hypoxia in rats with a unilaterally occluded carotid artery, the content of glucose of striatal dialysates followed closely changfes in blood pressure. These results illustrate the usefulness of the method in studying changes in brain glucose concentrations underpathological and physiological conditions.
Increase in β‐1,4‐Galactosyltransferase Activity During PC12 Cell Differentiation Induced by Forskolin and 2‐Chloroadenosine
Authors: Roth Jerome A. , Marcucci Kenda , Lin Wei‐hsung , Napoli Joseph L. , Wagner John A. , Rabin Richard
Published: October 2006

Galactosyltransferase (GAL Tase) activity was measured in differentiating PC12 cells induced by either forskolin or 2‐chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2‐chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2‐chtoroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2‐chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)‐ and protein kinase A‐dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine F9 carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.
The Slow Cyclic GMP Increase Caused by Serotonin in NG108‐15 Cells Is Not Inhibited by Antagonists of Known Serotonin Receptors: Possible Existence of a New Receptor Subtype Coupled with Membrane‐Bound Guanylate Cyclase
Authors: Tohda Michihisa , Sakuma Ichiro , Nomura Yasuyuki
Published: October 2006

Characterization of the serotonin (5‐HT>induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin‐ and ANP‐induced elevations in NG108‐15 cells. At 20 s, l,2‐bis(2‐aminophenoxy)ethaae‐N,N,N′,N′‐tetraaoetic acid tetraacetoxymethyl ester (BAPTA‐AM, 100 μM), a membrane‐permeabilized Ca2+ chelator, or N‐monomethyl‐L‐arginine (NMMA, 300 μM), an inhibitor of L‐arginine‐derived nitric oxide (NO) synthesis, inhibited 5‐HT‐induced elevation by approximately 40%, and completely inhibited bradykinin‐induced response. Neither 5‐HT‐ nor ANP‐induced cGMP elevation at 10 rain was affected by BAPTAAM or NMMA. The cGMP elevated by 5‐HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5‐HT stimulates two subtypes of 5‐HT receptors in NG108‐15: first, 5‐HT3 subtype stimulating Ca2+‐sensitive cytosolic guanylate cyclaje through NO derived from L‐arginine and second, a probably novel 5‐HT receptor subtype involved in activation of membrane‐bound guanylate cyclase.
Involvement of Nerve Growth Factor and Its Receptor in the Regulation of the Cholinergic Function in Aged Rats
Authors: Alberch Jordi , Pérez‐Navarro Esther , Arenas Ernest , Marsa Jordi
Published: October 2006

: The role of nerve growth factor (NGF) and its receptor (NGFR) in the regulation of cholinergic activity has been studied during the aging process. NGFRs were quantified in cortical membranes using a radioactive binding assay. NGF levels and choline acetyltransferase (ChAT) activity were determined in cortex, hippocampus, neostriatum, and septum. These assays were performed in both adult (6‐month‐old) and aged (36‐month‐old) rats. High‐and low‐affinity 125I‐NGF binding sites were present in cortex of adult and aged rats. Furthermore, we observed a decrease in number and affinity of both NGFRs in aged rats. ChAT activity in these rats was lower (⋍30%) than in adult rats in all the brainregions examined. NGF levels were not modified in cortex and hippocampus and were decreased in neostriatum (55%) and septum (35%). In conclusion, our results suggest that, during the aging process, the cholinergic impairment is related to a decrease in NGF levels in neostriatum but not in cortex and hippocampus. The reduction in level of NGF protein in septum could be due to a decrease in number of high‐affinity 125I‐NGF binding sites.
Tryptophan Hydroxylase Synthesis Is Induced by 3′, 5′‐Cyclic Adenosine Monophosphate During Circadian Rhythm in the Rat Pineal Gland
Authors: Ehret Mireille , Pevet Paul , Maitre Michel
Published: October 2006

: Tryptophan hydroxylase in the rat pineal gland undergoes diurnal rhythmic activity. Rat pineal glands exhibit increased tryptophan hydroxylase activity when incubated with a cyclic AMP analogue in vitro. Cyclic AMP‐dependent protein kinase phosphorylates tryptophan hydroxylase, purified from rat brain, without any modification of its enzyme activity under our experimental conditions. Actinomycin O or cydoheximide decreases the stimulating effect of the cyclic AMP analogue on pineal tryptophan hydroxylase activity. Incubation of pineal glands in the presence of [35S]mcthionine showed a cyclic AMP‐induced increase in tryptophan hydroxylase synthesis. These results explain the circadian rhythm of tryptophan hydroxylase activity in the rat pineal gland and suggest that the regulation of tryptophan hydroxylase expression by cyclic AMP occurs probably either at the translational level or via transient expression of a transcriptional regulatory element
Differential Involvement of the Arachidonic Acid Cascade on the α1‐Adrenergic Potentiation of Vasoactive Intestinal Peptide‐Versus β‐Adrenergic‐Stimulated Cyclic AMP and Cyclic GMP Accumulation in Rat Pinealocytes
Authors: Chik Constance L. , Young Indranie , Ho Anthony K.
Published: October 2006

: In the rat pineal gland, α1‐adrenergic agonists, which stimulate arachidonic add release, also potentiate vasoactive intestinal peptide.(VIP)‐or β‐adrenergic‐stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation. In this study, the possible involvement of the arachidonic acid pathway in the potentiation mechanism was examined in dispersed rat pinealocytes using two inhibitors of the arachidonic acid cascade, indomethacin and nordihydroguaiaretic acid. These two inhibitors appeared to have differential effects on the α1‐adrenergic potentiation of VIP‐or β‐adrenergic‐stimulated cAMP and cGMP responses. Whereas nordihydroguaiaretic acid was effective in suppressing both the α1‐adrenergic potentiation of VIP‐or β‐adrenergic‐stimulated cAMP and cGMP responses, indomethacin inhibited selectively the VIP‐mediated cAMP and cGMP responses. The role of arachidonic acid metabolites was further determined using several prostaglandins—A2, I2, E2, and F2α—and leukotrienes—B4, C4, and D4. Of the seven compounds tested, prostaglandins E2 and F2α stimulated basal cAMP but not cGMP accumulation. The prostaglandin E2‐and F2α‐stimulated cAMP responses were additive to those stimulated by VIP or β‐adrenergic receptors. The other five compounds had no effects on basal or VIP‐or β‐adrenergic‐stimulated cAMPor cGMP accumulation. Taken together, these findings indicate that the arachidonic acid cascade is likely involved in the α1‐adrenergic potentiation of VIP‐or β‐adrenergic‐stimulated cAMP and cGMP accumulation. However, the specific arachidonic acid metabolite involved in the potentiation mechanisms of VIP‐versus β‐adrenergic‐stimulated cyclic nucleotide responses may be different.