Journal of Neurochemistry
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Role of Serotonin<sub>2A</sub> and Serotonin<sub>2B/2C</sub> Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation Abstract : This study investigates, using in vivo microdialysis, the role of serotonin2A (5‐HT2A ) and 5‐HT2B/2C receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and 5‐hydroxyindoleacetic acid (5‐HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane‐anesthetized rats. Following DRN stimulation (300 μA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5‐HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5‐HT2A/2B/2C antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5‐HT2B/2C antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5‐HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5‐HT2A but not 5‐HT2B/2C receptors participate in the facilitatory control exerted by endogenous 5‐HT on accumbal DA release. Conversely, 5‐HT2B/2C receptors tonically inhibit basal DA release in both brain regions.
Journal of Neurochemistry - Tập 73 Số 3 - Trang 1033-1042 - 1999
Phospholipase A<sub>2</sub> and Its Role in Brain Tissue Abstract: Phospholipase A2 (PLA2 ) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn ‐2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2 ‐catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet‐activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA2+ ‐dependent and Ca2+ ‐independent, based on their catalytic dependence on Ca2+ . Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.
Journal of Neurochemistry - Tập 69 Số 3 - Trang 889-901 - 1997
Identification and Purification of Calcium‐Independent Phospholipase A<sub>2</sub> from Bovine Brain Cytosol Abstract: Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KC1. However, when buffer with 1 M KC1 was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero‐3‐phosphoethanolamine, whereas the latter was active with both diacyl‐glycero‐3‐phosphoethanolamine and 1‐alk‐1′‐enyl‐2‐acylglycero‐3‐phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel nitration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high‐resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40‐kDa molecular mass and two minor bands with 14‐ and 25‐kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high‐resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The optimal pH values of phospholipase A2 obtained from peaks I and II were 7.4 and 8.0, respectively. Neither phospholipase required Ca2+ for activity. The optimal concentrations of Triton X‐100 for peak I and peak II enzymes were 0.05 and 0.1%, respectively.
Journal of Neurochemistry - Tập 59 Số 2 - Trang 708-714 - 1992
Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood–brain barrier Abstract The brain vascular basement membrane is important for both blood–brain barrier (BBB ) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCEC s), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCEC s (mBCEC s). mBCEC s, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL /6 mice. The mBCEC s were grown as monoculture, in co‐culture with pericytes or mixed glial cells, or as a triple‐culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER ) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT ‐qPCR , mass spectrometry and immunocytochemistry. Co‐culturing mBCEC s with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER . The mBCEC s expressed all major basement membrane proteins such as laminin‐411, laminin‐511, collagen [α1(IV )]2 α2(IV ), agrin, perlecan, and nidogen 1 and 2 in vitro . Increased expression of the laminin α5 subunit correlated with the addition of BBB ‐inducing factors (hydrocortisone, Ro 20‐1724, and pCPT ‐cAMP ), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP . In conclusion, BCEC s cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation.
image Cover Image for this issue: doi: 10.1111/jnc.13789 .
Journal of Neurochemistry - Tập 140 Số 5 - Trang 741-754 - 2017
Expression of the mRNA for τ Proteins During Brain Development and in Cultured Neurons and Astroglial Cells Abstract: Two τ cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the τ mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6‐kb mRNA was detected with the two probes. In the astrocytes a 6‐kb mRNA hybridized clearly only with the 1.6‐kb probe. (2) During brain development the abundance of τ mRNA increases from a late fetal stage (— 4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of τ mRNA parallels that of α‐tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) τ mRNAs of the same size encode several τ proteins differing in molecular weight: several τ proteins are expressed either during early stages of development (juvenile τ proteins of 48 kilodaltons) or in adulthood (mature τ proteins of 50–70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and τ proteins, seems to be developmentally coordinated.
Journal of Neurochemistry - Tập 50 Số 6 - Trang 1894-1899 - 1988
Activation of NADPH oxidase and extracellular superoxide production in seizure‐induced hippocampal damage Abstract We sought to determine whether the extracellular compartment contributed to seizure‐induced superoxide (O2 .− ) production and to determine the role of the NADPH oxidase complex as a source of this O2 .− production. The translocation of NADPH oxidase subunits (p47phox , p67phox and rac1) was assessed by immunoblot analysis and NADPH‐driven O2 .− production was measured using 2‐(4‐hydroxybenzyl)‐6‐(4‐hydroxyphenyl)‐8‐benzyl‐3,7‐dihydroimidazo [1,2‐α] pyrazin‐3‐one‐enhanced chemiluminescence. Kainate‐induced status epilepticus resulted in a time‐dependent translocation of NADPH oxidase subunits (p47phox , p67phox and rac‐1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate‐injected rats showed increased NADPH‐driven and diphenylene iodonium‐sensitive O2 .− production in comparison to vehicle‐treated rats. The time‐course of kainate‐induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate‐induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2 .− .
Journal of Neurochemistry - Tập 92 Số 1 - Trang 123-131 - 2005
<i>t</i>‐[<sup>35</sup>S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues Abstract Specific high affinity binding of the cage convulsant t ‐[35 S] butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35 S] TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM ) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22°C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4°C temperature and 150‐min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (K D = 50 nM , Bmax = 250 fmol/mg protein in housefly; K D = 25 nM , B max = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k +1 =8 × 106 M ‐1 min‐1 ), but dissociation was described better by two rate constants (k ‐1 , = 0.28 min‐1 and 0.042 min‐1 ; calculated K d values of 80 nM and 12 nM ). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35 S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM ), aldrin, and lindane (200 nM ), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35 S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1‐2 μM ). Stereospecific inhibition by cypermethrin isomers agreed partially with biological activity, suggesting the possible role of this site in some toxicological actions of pyrethroids, although these compounds have more potent effects on voltage‐regulated sodium channels. [35 S]TBPS binding sites in crayfish muscle appear identical to γ‐aminobutyric acid (GABA) receptor‐chloride channels labeled by radiolabeled picrotoxin in this tissue. The sites in both invertebrate tissues examined are similar to those in mammalian brain and appear to be the target of important drugs.
Journal of Neurochemistry - Tập 52 Số 4 - Trang 1311-1318 - 1989
Lead Enters Bovine Adrenal Medullary Cells Through Calcium Channels Abstract: Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 μM Pb2+ , depolarization by 77 mM K increases the rate of Pb uptake from 12 ± 1 to 47 ± 5 μmol/(L cells × min). K‐induced Pb uptake has an apparent K m for Pb2+ of 2.6 μM , and is antagonized by Ca2+ with a K 0.5 of 1.4 mM . The Ca channel blocker D‐600 inhibits Pb entry with a K 0.5 of 0.4 μM . Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.
Journal of Neurochemistry - Tập 48 Số 2 - Trang 383-389 - 1987
Transport of Lead‐203 at the Blood‐Brain Barrier During Short Cerebrovascular Perfusion with Saline in the Rat Abstract: Lead transport at the blood‐brain barrier has been studied by short (< 1.5 min) vascular perfusion of one cerebral hemisphere of the rat with a buffered physiological salt solution at pH 7.4 without calcium, magnesium, or bicarbonate and containing 203 Pb‐labelled lead chloride. In the absence of complexing agents, 203 Pb uptake was rapid, giving a space of 9.7 ml/100 g of wet frontal cortex at 1 min. Lead‐203 influx was linear with lead concentration up to 4 μM . Five percent albumin, 200 μM cysteine, or 1 μM EDTA almost abolished 203 Pb uptake. Lead‐203 entry into brain was uninfluenced by varying the calcium concentration or by magnesium or the calcium blocker methoxyverapamil. Similarly, 1 μM bicarbonate or 50 μM 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid was without effect. Increasing the potassium concentration reduced 203 Pb uptake. Vanadate at 2 μM , 2 μM carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (a metabolic uncoupler), or 2 μM stannic chloride all markedly enhanced lead entry into brain, as did a more alkaline pH (7.80). In conclusion, there is a mechanism allowing rapid passive transport of 203 Pb at the brain endothelium, perhaps as PbOH+ . Lead uptake into brain via this system is probably made less important by active transport of lead back into the capillary lumen by the calcium‐ATP‐dependent pump.
Journal of Neurochemistry - Tập 54 Số 3 - Trang 905-914 - 1990
MICRODETERMINATION OF MONOAMINE OXIDASE AND 5‐HYDROXYTRYPTOPHAN DECARBOXYLASE ACTIVITIES IN NERVOUS TISSUES
Journal of Neurochemistry - Tập 12 Số 1 - Trang 15-23 - 1965
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