bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic AMP‐Dependent Induction of Serotonin N‐Acetyltransferase Activity in Photoreceptor‐Enriched Chick Retinal Cell Cultures: Characterization and Inhibition by Dopamine
Authors: Iuvone P. Michael , Avendano Gary , Butler Bonnie Johnson , Adler Ruben
Published: October 2006

The activity of serotonin N‐acetyltransferase (NAT), a key regulatory enzyme in the melatonin biosynthetic pathway, was examined in low‐density monolayer cultures of chick embryo retinal cells prepared with three levels of photoreceptor enrichment. In cultures prepared from embryonic day 8 retinas (E8), photoreceptors represented approximately 30% of the total cell population, whereas in those prepared from embryonic day 6 retinas (E6), approximately 70% of the cells were photoreceptors. In E8 retinas treated with kainic acid to destroy neurons (E8K), the relative content of photoreceptors was increased to approximately 50%. NAT activity was detectable in the cultures under all conditions studied, and was markedly increased by drugs that increase intracellular cyclic AMP levels and cyclic AMP‐dependent protein kinase activity: 8‐bromocyclic AMP, forskolin, and 3‐isobutyl‐1‐methylxanthine (IBMX). Consistent with the hypothesis that NAT is localized in photoreceptors, the effects of the stimulatory treatments were significantly greater in E6 and E8K cultures than in E8 cultures. The stimulation of NAT activity in E6 cultures was inhibited by actinomycin D and cycloheximide, suggesting the involvement of RNA and protein synthesis. Dopamine inhibited the induction of NAT activity by forskolin and IBMX, but not that elicited by 8‐bromocyclic AMP. The dopamine‐mediated suppression of activity was significantly inhibited by pertussis toxin and by spiperone and sulpiride, both D2‐dopamine receptor antagonists, but not by SCH 23390, a D1‐dopamine receptor blocker, or antagonists of α‐adrenergic, β‐adrenergic, or serotonergic receptors. Because the inhibitory effect of dopamine on E6 and E8K cultures was at least as great as that on E8 cultures, the results suggest that dopamine acts on D2‐like receptors on photoreceptors. The receptors appear to be coupled to adenylate cyclase through an inhibitory GTP‐binding protein and to mediate inhibition of cyclic AMP synthesis and consequent induction of NAT activity.
Comparison of α1‐Adrenergic Receptor‐Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures
Authors: Wilson Karen M. , Gilchrist Siobhan , Minneman Kenneth P.
Published: October 2006

α1‐Adrenergic receptor binding sites and norepinephrine‐stimulated 3H‐inositol phosphate (3H‐InsP) accumulation were measured in primary cultures of neurons and glia from 1‐day‐old rat brains. The density of α1‐adrenergic receptor binding sites was ˜three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H‐InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine‐stimulated 3H‐InsP formation remained constant for [3H]inositol prelabelling periods of 1–14 days in neurons, whereas the response increased with time in glia and was maximal after 7–10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H‐InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pre‐treatment with phenoxybenzamine decreased norepinephrine‐stimulated 3H‐InsP formation in a dose‐dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H‐InsPs were accumulated in neurons and glial cells. These results demonstrate that α1‐adrenergic receptors exist on both neurons and glial cells and activate 3H‐InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H‐InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.
Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development
Authors: Gonzalez Bruno J. , Leroux Philippe , Bodenant Corinne , Braquet Pierre , Vaudry Hubert
Published: October 2006

Abstract: Somatostatin (SRIF) receptors (SRIF‐Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF‐Rs coincides with the expression of SRIF‐like immunoreactivity in the cerebellum. However, the cellular location of SRIF‐Rs does not overlap with the distribution of SRIF‐like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF‐Rs in the immature (13–day‐old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [125I‐Tyr0,D‐Trp8]SRIF‐14 ([125I‐Tyr0,d‐Trp8]S14) and I25I‐SMS 204–090. The pharmacological profile of cerebellar SRIF‐Rs was compared with that of adult cortical SRIF‐Rs. Saturation studies performed in 13–day‐old rat cerebellum showed that the A'D values for [125I‐Tyr0,D‐Trp8]S14 and 125I‐SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding Bmax values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high‐affinity sites (SSI). Competition studies showed that different D‐Trp‐sub‐stituted analogs displaced [125I‐Tyr0,d‐Trp8]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr0,d‐Trp8]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day‐old cerebellum and adult cerebral cortex. The higher‐affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower‐affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn2+ was the most efficient cation for promoting binding of [125I‐Tyr0,d‐Trp8]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF‐Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same KD and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF‐binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF‐Rs. The extremely high density of SRIF‐Rs found in the external granule cell layer in 13–day‐old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.
Development of Monoaminergic Neurotransmitters in Fetal and Postnatal Rat Brain: Analysis by HPLC with Electrochemical Detection
Authors: Herregodts Patrick , Velkeniers Brigitte , Ebinger Guy , Michotte Yvette , Vanhaelst Luc , Hooghe‐Peters Elisabeth
Published: October 2006

Abstract: The monoamines dopamine, norepinephrine, epinephrine, and serotonin and their major metabolites 3,4‐dihydroxyphenylacetic acid, homovanillic acid, 3‐methoxy‐4‐hydroxyphenylethylene glycol, and 5‐hydroxyindoleacetic acid were measured in the CNS of the rat during development from fetal day 18 to young adult. The catecholamines, serotonin, and their major metabolites remained low during fetal life. Concentrations measured in total brain started to increase around birth till the end of the fourth week of life after which steady‐state levels were measured. Our results suggest that although monoamine systems are already morphologically well developed during late gestational life, they probably become a significant functional system only around birth and early postnatal life.
Decrease of Acetylcholine Release from Cortical Slices in Aged Rats: Investigations into Its Reversal by Phosphatidylserine
Authors: Vannucchi Maria Giuliana , Casamenti Fiorella , Pepeu Giancarlo
Published: October 2006

Abstract: The release of total acetylcholine (ACh) and [3H]ACh was investigated in electrically stimulated cortical slices prepared from 4‐ and 18‐month‐old male Wistar rats. The slices were prelabeled with [3H]choline ([3H]Ch) and perfused with Krebs solution containing physostigmine. Total ACh was measured and the nature of the tritium efflux identified by HPLC. The total tritium content in the slices at the end of the incubation period was half as great in the old as in young rats. A linear relationship was found between stimulation frequencies (2, 5, and 10 Hz) and fractional [3H]ACh release in both young and old rats. In the latter the release was significantly smaller. At 10 Hz stimulation frequency the ratio between the two 2‐min stimulation periods, S2/S1, was higher in the 18‐month‐old rats than in the young rats. Specific activity of the evoked ACh release was significantly smaller in S2 than in S1 in 4‐month‐old rats only. These findings indicate that the young synthetize ACh from endogenous unlabeled Ch more than older rats. In 18‐month‐old rats both the evoked total ACh and [3H]ACh release, expressed as picograms per minute, showed an approximately 50% decrease in both S1 and S2 stimulation periods, with no significant difference in specific activity. Phosphatidylserine (PtdSer) administration (15 mg/kg, i.p. daily) for 1 week to 18‐month‐old rats prevented the reduction in total evoked ACh release but not the reduction in evoked [3H]ACh release. The specific activity of ACh release was therefore significantly smaller than that of the young and untreated old rats. This finding indicates that PtdSer is able to increase the availability of endogenous choline for de novo ACh synthesis and release. PtdSer treatment also prevented impairment of the passive avoidance conditioned response observed in old rats.
Expression of Tropomyosin Genes During the Development of the Rat Cerebellum
Authors: Faivre‐Sarrailh C. , Had L. , Ferraz C. , Widada J. Sri , Liautard J. P. , Rabié A.
Published: October 2006

Abstract: The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human‐specific α‐, β‐, γ‐, and d‐tropomyosin cDNA probes and rat‐specific α‐, β‐, and d‐tropomyosin oligonucleotide probes. The β‐ and γ‐genes do not seem to be expressed in the rat brain. The δ‐tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4‐kb mRNA. A 3‐kb mRNA is expressed by the α‐tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.
Methyl Mercury Stimulates Protein 32P Phospholabeling in Cerebellar Granule Cell Culture
Authors: Sarafian Theodore , Verity M. Anthony
Published: October 2006

Abstract: Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg). Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 μM characterized by neuron degeneration and neuritic varicosities. Dose‐response and time course profiles for cell death were established using the 5lCr release assay, which revealed that 1 μM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h. Labeling of cultures with [32P]orthophosphate following 24‐h exposure to 1 μM MeHg disclosed abnormalities in both protein and lipid phosphorylation. After 24‐h exposure to 5 μM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls. This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose‐dependent inhibition of total phosphorylation. Measurement of 32P labeling of ATP using a cyclic AMP‐dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or [32P]ATP specific activity at 1 or 4 h as a function of [MeHg]. Studies measuring 32P‐phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity. We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of [32P]ATP specific activity and protein phosphatase activity.
Hydrolysis‐Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin‐Permeabilized PC12 Cells
Authors: Carroll Alan G. , Rhoads Allen R. , Wagner Paul D.
Published: October 2006

Abstract: The effect of the hydrolysis‐resistant GTP analogs, guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin‐permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca2+ (10 μM) was not affected by GTP7S or GMPPNP, secretion in the absence of Ca2+ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTPγS or GMPPNP was approximately 80% of that induced by 10 μM Ca2+. Half‐maximum stimulation was induced by 30 μM GTPγS or GMPPNP. Both Ca2+‐stimulated and GTPγS‐stimulated secretion were ATP dependent and inhibited by N‐ethylmaleimide. The GTPγS‐stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca2+ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12‐O‐tetradecanoyl‐phorbol 13‐acetate caused a 50% increase in both Ca2+‐stimulated and GTP7S‐stimulated secretion. Cholera and pertussis toxins did not affect Ca2+‐stimulated or GTPγS‐stim‐ulated NE secretion. Guanosine 5′‐O‐(2‐thiodiphosphate) (GDPβS) and GTP inhibited GTPγS‐stimulated secretion but not Ca2+‐stimulated secretion. The inability of GDPβS to inhibit Ca2+‐stimulated secretion indicates that the process affected by GTPγS is not an essential step in the Ca2+‐stimulated pathway.
Shark Myelin Basic Protein: Amino Acid Sequence, Secondary Structure, and Self‐Association
Authors: Milne Trudy J. , Atkins Annette R. , Warren Juanita A. , Auton Wendy P. , Smith Ross
Published: October 2006

Abstract: Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self‐association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.
Nerve Growth Factor‐Induced Transient Increase in the Phosphorylation of Ribosomal Protein S6 Mediated Through a Mechanism Independent of Cyclic AMP‐Dependent Protein Kinase and Protein Kinase C
Authors: Hashimoto Seiichi , Hagino Akihiko
Published: October 2006

Abstract: Treatment of PC12h cells with nerve growth factor (NGF) induced a transient increase in the phosphorylation of a 35,000‐dalton protein. This transient increase was observed also when extracts of NGF‐treated cells were incubated with [γ‐32P]ATP. In the intact‐cell phosphorylation system, treatment with N,2′‐dibutyryladenosine 3′,5′‐cyclic monophosphate (dBcAMP) or 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) also induced a transient increase in the phosphorylation of the 35,000‐dalton protein, but the effect was less than that of NGF. An effect comparable to that of NGF was obtained by the combination of dBcAMP and TPA. Pretreatment of PC12h cells with dBcAMP plus TPA for 3 days, which deprived the cells of their ability to respond to a re‐challenge with dBcAMP, TPA, or dBcAMP plus TPA by increasing the rate of 35,000‐dalton protein phosphorylation, caused only a slight attenuation of the NGF effect, directly indicating a minimal role of cyclic AMP (cAMP)‐dependent protein kinase and protein kinase C in the mechanism of the NGF action. Pretreatment of the cells with K‐252a, a protein kinase inhibitor, at a concentration of 300 μM almost completely blocked the action of NGF, but scarcely affected the action of dBcAMP, TPA, or dBcAMP plus TPA in intact‐cell phosphorylation experiments. This NGF‐sensitive 35,000‐dalton protein was a ribosomal protein and identified as ribosomal protein S6. The results lead us to conclude that NGF activates some NGF‐sensitive component(s), probably some specific protein kinase(s) other than cAMP‐dependent protein kinase or protein kinase C., which is suppressed by K‐252a and directly or indirectly activates a 35,000‐dalton protein kinase(s) [S6 kinase(s)] to increase the rate of phosphorylation of the 35,000‐dalton ribosomal protein (S6).
Calcium‐ Versus G Protein‐Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes
Authors: Chandler L. Judson , Crews Fulton T.
Published: October 2006

Abstract: The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0–1.0 mM), basal, potassium‐stimulated, and norepinephrine ‐stimulated formation of 3H‐inositol phosphates decreased with decreasing extracellular calcium. Potassium‐ and norepinephrine‐stimulated formation of 3H‐inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N‐methyl‐d‐glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H‐inositol phosphates. Measurement of cytosolic calcium with fura‐2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium‐free rather than sodium‐containing medium. In the absence of sodium, potassium‐stimulated formation of 3H‐inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H‐inositol phosphates from that in the presence of 1 mM carbachol or 100 μM norepinephrine revealed that the carbachol‐stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine‐stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12‐O‐tetradecanoylphorbol 13‐acetate inhibited norepinephrine‐ and, to a lesser extent, carbachol but not basal or aluminum fluoride‐stimulated formation of 3H‐inositol phosphates in sodium‐free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization‐induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis. These results also support previous evidence of two separate and distinct pathways for stimulating phosphoinositide‐linked phospholipase C (PLC) activity in brain: One pathway appears to involve a phosphoinositide‐associated guanine nucleotide binding protein‐PLC coupling process, and the other a direct activation of PLC by an increase in intracellular calcium.
Exogenous Nerve Growth Factor Increases the Activity of High‐Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats
Authors: Williams Lawrence R. , Rylett R. Jane
Published: October 2006

Abstract: The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium‐sensitive high‐affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats, in 24‐month‐old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4‐month‐old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4‐month‐old animals, NGF treatment at 1.2 μg/ day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle‐treated 4‐month‐old animals; vehicle treatment had no effect on the activity of either marker. In 24‐month‐old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24‐month‐old control animals. Synaptosomal HACU in 24‐month‐old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values. Treatment of 24‐month‐old rats with NGF for 4 weeks produced further increases in ChAT activity and HACU in striatum and frontal cortex; in hippocampus, the 4‐week treatment did increase HACU, but ChAT activity was not further increased over that produced by 2 weeks of NGF administration. These data indicate an age‐related differential regulation of ChAT activity and HACU between specific brain areas. In addition, NGF stimulated phenotypic expression of these cholinergic markers in both young adult and aged rat brain; there might be an age‐associated differential sensitivity of particular brain regions to exogenous NGF.
Induction of c‐fos mRNA Expression by Afterdischarge in the Hippocampus of Naive and Kindled Rats
Authors: Shin Cheolsu , McNamara James O. , Morgan James I. , Curran Tom , Cohen Donna R.
Published: October 2006

Abstract: Periodic induction of focal electrical seizure [afterdischarge (AD)] is an absolute prerequisite for the development of kindling, an animal model of complex partial epilepsy. Once established, it is a permanent condition. The mechanism(s) that translate ADs, which last tens of seconds, into life‐long alterations in the CNS is unclear. Cellular immediate‐early genes have been implicated in the conversion of short‐term stimuli to long‐term alterations in cellular phe‐notypes by regulating target gene expression. We have investigated the contribution of one such early gene, c‐fos, to this process. The relationship between ADs and expression of c‐fos gene in the rat hippocampus, a key structure in kindling development, was studied by analysis of mRNA levels. The low constitutive expression of c‐fos mRNA in the hippocampus was not altered by kindling. There was an “all‐or‐none” relationship between induction of c‐fos and the duration of AD. The threshold for induction was approximately 30 s of AD. Above‐threshold ADs induced c‐fos in both naive and kindled animals to the same extent and with identical temporal profiles. Although the expression of c‐fos is unchanged with kindling, c‐fos may nonetheless contribute to many long‐term changes of kindling, both adaptive and epileptogenic.
The Large Isoform of Myelin‐Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain
Authors: Fujita Nobuya , Sato Shuzo , Ishiguro Hideaki , Inuzuka Takashi , Baba Hiroko , Kurihara Tadashi , Takahashi Yasuo , Miyatake Tadashi
Published: October 2006

Abstract: Two polypeptide isoforms of myelin‐associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L‐MAG) is scarcely expressed, whereas that of small MAG isoform (S‐MAG) is overexpressed. In the present study, we prepared antisera specific to the S‐MAG and L‐MAG amino acid residues, respectively. Immunoblots showed that the L‐MAG band was scarcely detectable in the quaking mouse brain, whereas the S‐MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L‐MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L‐MAG mRNA and abnormal glycosylation in the quaking mouse brain.
Preloading In Vivo: A Rapid and Reliable Method for Measuring γ‐Aminobutyric Acid and Glutamate Fluxes by Microdialysis
Authors: Young Andrew M. J. , Foley Paul M. , Bradford H. F.
Published: October 2006

Abstract: In vivo microdialysis was used in conjunction with a novel dual‐label preloading method, to monitor changes in extracellular levels of γ‐aminobutyric acid (GABA) and glutamate in the striatum of conscious, unrestrained rats. [3H]GABA and [14C]glutamate were applied in the dialysis stream fora prelpading period of 30 min, after which dialysis perfusion was continued for up to 6 h, and dialysate samples were collected for scintillation counting. Veratridine (Vtd; 100 μM in the dialysate) caused significant rises in both 3H and 14C content measured in the dialysates, the majority of which remained associated with the preloaded GABA and glutamate, respectively. The Vtd‐stimulated release of GABA and glutamate measured in this way was blocked by tetrodotoxin and was Ca2+ dependent. Thus, by reproducing results obtained using other techniques, we have shown that the preloading method provides a quick and reliable method for measuring the effects of drugs on the release of neurotransmitter GABA and glutamate in vivo by dialysis. It should enable sample times as low as 1 min to be used, thus allowing resolution of transient stimulated responses taking place over a time course of minutes.
Blockade of the Noradrenaline Carrier Increases Extracellular Dopamine Concentrations in the Prefrontal Cortex: Evidence that Dopamine Is Taken up In Vivo by Noradrenergic Terminals
Authors: Carboni E. , Tanda G. L. , Frau R. , Chiara G. Di
Published: October 2006

Abstract: The effect of systemic administration of desmethylimipramine (DMI) and oxaproptiline (OXA), two inhibitors of the noradrenaline (NA) reuptake carrier, on the in vivo extracellular concentrations of dopamine (DA) was studied by transcerebral dialysis in the prefrontal cortex and in the dorsal caudate of freely moving rats. In the NA‐rich prefrontal cortex, either drug increased extracellular DA concentrations whereas in the dorsal caudate neither was effective. Haloperidol increased extracellular DA concentrations more effectively in the dorsal caudate than in the prefrontal cortex. Pre‐treatment with DMI or OXA. which failed to modify the effect of haloperidol in the dorsal caudate, potentiated its action in the prefrontal cortex. 6‐Hydroxydopamine lesioning of the dorsal NA bundle prevented the ability of OXA to increase DA concentrations. The results suggest that reuptake into NA terminals is an important mechanism by which DA is cleared from the extracellular space in a NA‐rich area such as the prefrontal cortex. The elevated extracellular concentrations of DA resulting from blockade of such mechanism by tricyclic antidepressants may play a role in the therapeutic effects of these drugs.
Characterization of Recombinant Human Aromatic l‐Amino Acid Decarboxylase Expressed in COS Cells
Authors: Sumi Chiho , Ichinose Hiroshi , Nagatsu Toshiharu
Published: October 2006

Abstract: The expression vector containing the full‐length cDNA of human aromatic l‐Amino acid decarboxylase (EC 4.1.1.28) was transfected in COS cells by a modified calcium phosphate coprecip‐itation method. The cells transfected with plasmids that had a true direction of the cDNA gave a major immunoreactive band at 50 kDa. This expressed enzyme catalyzed the decarboxylation of l‐3,4‐dihydroxyphenylalanine (l‐DOPA). l‐5‐hydroxytryptophan (l‐5‐HTP) and l‐threo‐3,4‐dihydroxyphenylserine. The optimal pH of the enzyme activity with l‐DOPA as a substrate was 6.5, whereas the enzyme had a broad pH optimum when l‐5‐HTP was used as a substrate. Addition of pyridoxal phosphate to the incubation mixture greatly enhanced the activity for both l‐DOPA and l‐5‐HTP.
1,2‐Diacylglycerol Content and Its Arachidonyl‐Containing Molecular Species Are Reduced in Sciatic Nerve from Streptozotocin‐induced Diabetic Rats
Authors: Zhu Xi , Eichberg Joseph
Published: October 2006

Abstract: The content of 1,2‐diacylglycerol (DAG) was determined in sciatic nerves from normal and streptozotocin‐induced diabetic rats. In nerves frozen in situ, DAG content was reduced 22% in the proximal region and 77% in the distal region of diabetic nerve, principally because of the loss of associated fat. DAG levels in freshly dissected and desheathed diabetic nerve were decreased from 23 to 30% as compared with normal nerve. Determination of DAG molecular species distribution in desheathed normal nerve indicated that 18:0/20:4 accounted for 34%, 16:0/18:1 for 17%, and several other polyunsaturated fatty acid‐containing species for 17% of the total. In diabetic nerve, the quantity of the 18:0/20:4 DAG species was reduced by 37%, and this drop was 62% of the reduction in all molecular species. The content of the minor species, 16:0/20:4 DAG, was decreased by 48%. Our results suggest that nerve DAG arises in large part from phosphoinositide degradation. Moreover, these results provide support for the hypothesis that reduced Na+,K+‐ATPase activity in diabetic nerve is a consequence of decreased phosphoinositide turnover, which thereby generates insufficient DAG to maintain a protein kinase C‐mediated step necessary for activation of Na+,K+‐ATPase.
In Vivo Mechanisms Underlying Dopamine Release from Rat Nigrostriatal Terminals: I. Studies Using Veratrine and Ouabain
Authors: Fairbrother Iain S. , Arbuthnott Gordon W. , Kelly John S. , Butcher Steven P.
Published: October 2006

Abstract: The in vivo mechanisms underlying the dopamine (DA)‐releasing actions of veratrine and ouabain in the striatum of halothane‐anaesthetised rats have been investigated using brain microdialysis. Relevant catecholamines and indoleamines were separated and quantified using HPLC combined with an electrochemical detection system. Veratrine (10 μg/ml‐1 mg/ml) and ouabain (10 μM‐1 mM) were added to the medium perfusing the dialysis probes. Both compounds increased dialysate DA content in a dose‐related manner. Dialysate levels of the DA metabolites 3,4‐dihydroxyphenylacetic acid and homovanillic acid and the serotonin metabolite 5‐hydroxyindoleacetic acid were reduced by both veratrine and ouabain. Veratrine‐induced DA efflux was maximal in the first 20‐min sample collected after drug infusion began, whereas the maximal effect of ouabain was not observed until 20–40 min after administration began. Veratrine‐induced DA efflux was unaffected by systemic injection of the DA uptake inhibitor nomifensine but was inhibited by either coperfusion of tetrodotoxin (TTX) or removal of calcium from the perfusing buffer. These data suggest that veratrine induces release of DA via a carrier‐independent mechanism, perhaps involving an exocytotic release process. In contrast, ouabain‐induced DA release was reduced by nomifensine but was inhibited to a lesser degree by calcium depletion and TTX. Detailed analyses of these data suggest that although ouabain initially induces release of DA via a carrier‐dependent mechanism, an exocytotic process may also be involved. The finding that ouabain‐induced DA efflux exhibits a degree of TTX and calcium sensitivity suggests that membrane depolarisation caused by Na+,K+‐ATPase blockade opens voltage‐gated sodium channels and initiates an exocytotic release of DA. The intracellular pools of DA involved in the release of DA induced by veratrine and ouabain were also examined. Depletion of vesicular pools of DA by pretreatment with reserpine reduced the amount of DA release induced by both agents, although this effect was only significant in the case of veratrine. However, in reserpinised animals the residual amount of DA release induced by veratrine was inhibited by nomifensine, a result suggesting that DA may be released via a carrier‐dependent process in the absence of vesicular DA. Newly synthesised pools of DA were also depleted by pretreatment with the DA synthesis inhibitor α‐methyl‐p‐tyrosine. Under these conditions, both veratrine‐ and ouabain‐induced DA efflux was reduced. In the case of veratrine, newly synthesised DA is presumably used to replenish the vesicular store released by depolarisation. A similar process may also operate with ouabain, although the newly synthesised pool of DA may also be released directly via the carrier‐dependent release process.
Effect of Chronic Typical and Atypical Neuroleptic Treatment on Proenkephalin mRNA Levels in the Striatum and Nucleus Accumbens of the Rat
Authors: Angulo Jesus A. , Cadet Jean Lud , Woolley Catherine S. , Suber Freeman , McEwen Bruce S.
Published: October 2006

Abstract: We measured proenkephalin (PEK) mRNA levels in the anterior and medial aspects of the caudate‐putamen (CPU) and in the nucleus accumbens (NAc) of the rat by in situ hybridization histochemistry after chronic treatment for 21 days with typical (haloperidol and prolixin) and atypical (molindone, thioridazine, and clozapine) neuroleptics. Chronic administration with these drugs resulted in PEK mRNA levels that were 60–80% higher than controls in the anterior and medial aspects of the CPU but only 25–30% over controls in the NAc. All three atypical neuroleptics studied increased PEK mRNA in the following order: anterior‐CPU, thioridazine > clozapine and molindone; medial‐CPU, thioridazine and molindone > clozapine; and NAc, thioridazine > > molindone and clozapine. Chronic treatment with the specific dopamine D2 antagonist sulpiride also caused elevation in PEK mRNA levels in all three brain regions studied whereas the specific serotonin S2 receptor blocker, cinanserin, had no significant effects on PEK mRNA levels. These results are consistent with the hypothesis that elevated levels of the enkephalins in the mesolimbic system may be necessary for antipsychotic activity. They also support the idea that the undesirable motoric signs and symptoms observed after chronic treatment with typical neuroleptics may not be the result of increased levels of enkephalins in the basal ganglia because atypical neuroleptics which are almost totally devoid of these side effects caused similar increases in PEK mRNA in the CPU.
Evidence That Secondary Rat Schwann Cells in Culture Maintain Their Differentiated Phenotype
Authors: Rutkowski Lynn , Needham Leila , Frayer Karen , Carson Daniel , McKhann Guy , Tennekoon Gihan I.
Published: October 2006

Abstract: Schwann cells, on receiving the correct signal, will encircle an axon and wrap it with a myelin sheath. To begin examining some of the mechanisms underlying the process of myelination in vitro, we isolated Schwann cells from the sciatic nerves of neonatal rats and generated large cell populations with cholera toxin. The immunological and biochemical properties of these secondary Schwann cells were characterized after five to seven passages in the absence of axonal contact. These cells continued to express antigens found in both myelinating (P0 and 2′,3′‐cyclic nucleotide phosphohydrolase) and nonmyelinating cells in vivo (A5E3 and glial fibrillary acidic protein) in addition to the markers common to both types of cells (Ran‐1, 217c, S‐100, and laminin). Biochemical analyses showed that these cells synthesize the very‐long‐chain fatty acids (22–26 carbon atoms) found in myelin membranes. Moreover, the enzymes required for the synthesis of myelin glycolipids (including sphingosine acyltransferase, UDP‐galactose:ceramide galactosyltransferase, and cerebroside sulfotransferase) were still active, and metabolic labeling studies showed that galactocerebroside and sulfatide were synthesized even though the galactocerebroside pool was insufficient to be detected by immunostaining. Secondary Schwann cells also synthesized four species of myelin basic protein and the major structural glycoprotein in myelin, P0. The pathway necessary for glycosylation of P0 protein remained active, and an analysis of the oligosaccharide chain revealed that 70% was processed to a complex form. In summary, we found that secondary Schwann cells still express most of the immunological markers of differentiated cells and continue to synthesize low levels of myelin components. Therefore, Schwann cells do not dedifferentiate in culture, as previously believed.
Ischemia‐Induced Alterations in Lipid Metabolism of the Gerbil Cerebral Cortex: I. Changes in Free Fatty Acid Liberation
Authors: Nakano Shinichi , Kogure Kyuya , Abe Koji , Yae Tetsuji
Published: October 2006

Abstract: Does the impaired lipid metabolism during nonlethal transient ischemia truly recover within a few hours after recirculation? In an attempt to answer this question, we first investigated the time course of the changes in the amount and composition of free fatty acids (FFAs) accumulated during 5‐min ischemia and after various postischemic recirculation durations (3 min, 1 h, 24 h, 3 days, and 6 days) in the gerbil cerebral cortex. Then those of FFAs liberated in response to the second 5‐min ischemia at various recirculation intervals (3 min, 1 h, 3 days, and 6 days) following the initial one were also measured to evaluate the changes in the cellular response. The former study disclosed that the FFA levels transiently returned to the control levels at 1‐h recirculation, increased again a few days after the onset of recirculation, followed by the final return to the control levels after 6‐day recirculation. The latter study disclosed that the cellular response to the second ischemia was quite different from that to the initial one even after 6‐day recirculation, suggesting that membrane lipid metabolism had not yet been recovered even at such a late period. We discuss the significance of the alterations in lipid metabolism.
Dissociation of Muscimol, SR 95531, and Strychnine from GABAA and Glycine Receptors, Respectively, Suggests Similar Cooperative Interactions
Authors: Maksay Gábor
Published: October 2006

Abstract: Binding to γ‐aminobutyric acid‐A (GABAA) receptors was studied in synaptosomal membranes of rat brain. Dissociation of [3H]muscimol and the GABAA antagonist [3H]2(3‐carboxypropyl)‐3‐amino‐6‐p‐methoxyphenylpyridazinium bromide ([3H]SR 95531) binding elicited by 100‐fold dilution was accelerated by excess of GABA or SR 95531. Control dissociation might be retarded by rebinding. The contribution of a rapid first phase of dissociation of the agonist [3H]muscimol was preferentially enhanced by SR 95531. In contrast, the dissociation of [3H]SR 95531 binding was preferentially accelerated by GABA. These opposite preferential accelerations can be explained by negative heterotropic cooperativity and a reversed affinity relationship of agonists and antagonists to GABAA binding sites with different affinities. Modification of the membranes by p‐diazobenzenesulfonic acid (DSA) selectively decreased the accelerating effect of GABA on the dissociation of [3H]SR 95531 binding. [3H]Strychnine binding was studied in a membrane preparation of rat spinal cord. The dissociation of the antagonist [3H]strychnine elicited by dilution was preferentially accelerated by glycine. Again, pretreatment with DSA decreased selectively this negative heterotropic (i.e., allosteric) interaction. Chemical modification by DSA might be attributed to tyrosine residues responsible for similar allosteric interactions for the GABA‐ and glycine‐gated chloride channels.
Mechanism of Calcium Transport Across the Plasma Membrane of Bovine Chromaffin Cells
Authors: Kao Lung‐Sen , Cheung Nam Sang
Published: October 2006

Abstract: The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30‐fold, based on the α‐bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 nM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 μM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.
Guanine Nucleotide Effects on 8‐Cyclopentyl‐1,3‐[3H]Dipropylxanthine Binding to Membrane‐Bound and Solubilized A1 Adenosine Receptors of Rat Brain
Authors: Klotz Karl‐Norbert , Keil Roger , Zimmer Franz J. , Schwabe Ulrich
Published: October 2006

Abstract: The effects of guanine nucleotides on binding of 8‐cyclopentyl‐1,3‐[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration‐dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide‐induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N‐ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP‐ or NEM‐induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane‐bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high‐affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.
Pharmacological Evidence for the Modulation of Monoamine Release by Adenosine in the Invertebrate Nervous System
Authors: Barraco Robin A. , Stefano George B.
Published: October 2006

Abstract: An in vitro preparation from the pedal ganglia of the marine bivalve, Mytilus edulis, was used to examine the modulation of transmitter release by adenosine and its analogs from invertebrate nervous tissue. The ganglia of this organism contain the monoamines dopamine (DA), serotonin (5‐HT), and norepinephrine (NE), and the presynaptic release of these substances is known to be calcium‐dependent. This organism also contains a DA‐sensitive adenylate cyclase system which resembles that seen in mammals. Neural tissue from the pedal ganglia was incubated with labeled monoamines, and release studies were then conducted in superfusion chambers; release of monoamines was evoked by the addition of 50 mM KCl. Addition to the superfusion medium of the adenosine analog, 5′‐N‐ethylcarboxamidoadenosine (NECA; 10 nM), inhibited the release of 5‐HT and DA, and to a lesser extent NE, whereas 100‐fold higher concentrations of adenosine itself and the adenosine analog, R‐N6‐phenylisopropyladenosine, were required to achieve comparable levels of inhibition. The inhibitory effects of NECA on neurotransmitter release were blocked by the adenosine receptor antagonist, theophylline (IC50= 10–14 μM). The results from this study indicate for the first time the possible role of adenosine as a modulator of neurotransmitter release in the invertebrate nervous system.
Alzheimer's β‐Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid
Authors: Klunk William E. , Pettegrew Jay W.
Published: October 2006

Abstract: β‐Amyloid protein is a major protein component of neuritic plaques in the brain of Alzheimer's disease patients. A major advance in understanding the molecular biology of Alzheimer's disease came with the purification and sequencing of this protein. Because β‐amyloid protein is very insoluble, extreme conditions such as 88% formic acid were commonly used to dissolve its fibrils. We now report that 88% formic acid covalently modifies β‐amyloid protein fragments, probably by the formation of a formate ester to a serine in the protein. The t1/2 of the formylation is ∼3.5 h, and the t1/2 for hydrolysis of the formylated peptide is much longer, being 9.9 h in water and 66 h in HPLC eluant. This suggests that if formic acid is used in the purification of β‐amyloid protein or peptide fragments of this protein, it is likely that some formylated peptide will be present in subsequent studies. Although unrecognized modification of a protein is inherently undesirable, it is uncertain what effects this formylation will have on ensuing studies. Certainly, investigations into the immunologic, physical, and physiologic properties of β‐amyloid protein could be influenced.
D1 and D2 Dopaminergic Regulation of Acetylcholine Release from Striata of Freely Moving Rats
Authors: Bertorelli Rosalia , Consolo Silvana
Published: October 2006

Abstract: The effects of selective D1 and D2 dopaminergic agents on the extracellular acetylcholine (ACh) content in striata of freely moving rats were determined by the microdialysis technique. LY 171555, a selective D2 agonist, reduced ACh output by ∼30% within 20 min at the dose of 0.2 mg/kg, i.p., whereas the D2 antagonists (‐)‐remoxipride (10 mg/kg, s.c.) and L‐sulpiride (50 mg/kg, i.p.) induced maximal increases of ∼‐50% within 10 and 20 min, respectively. In contrast, the D1 antagonist SCH 23390 (0.25 mg/kg, s.c.) decreased the extracellular ACh content by ∼30% in 20 min, but lower doses‐0.025 and 0.05 mg/kg‐had no such effect. The stimulation of ACh release by LY 171555 was prevented by (‐)‐remoxipride but not by SCH 23390(0.25 mg/kg, s.c.) In addition, the D1 agonist SKF 38393 failed to modify the ACh increasing effect of (‐)‐remoxipride. Thums, the D1 and D2 receptors subserve opposing functions on ACh release. The D1/D2 dopaminergic agonist R‐apomorphine, at the dose of I mg/kg, i.p., reduced ACh output by ∼‐35% only when D1 receptors were blocked by SCH 23390(0.025 mg/kg, s.c.). The results provide clear in vivo evidence of the tonic inhibition exerted by dopaminergic nigrostriatal input on the cholinergic system of the basal ganglia through D1 and D2 receptors.