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Notizen: An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50= 9.8 ± 2.9 nM) for displacement of [3H] flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50= 4.0 ±0.3 nM). However, at concentrations from 0.1 to 10 μM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton ±-100, a result suggesting that ∼7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1–4 h at 37°C) of neurons with 100μMγ-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H] flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154–176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.

Notizen: The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with I25I-insulin resulted in the specific labeling of insulin receptor α-subunits of ∼ 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (αβ-heterodimer) receptors affinity-labeled with I25I-insulin indicated the presence of a disulfide-linked β-subunit of ∼95 kDa. Endoglycosidase F digestion of the affinity-labeled a-subunits increased their mobility by reducing their apparent mass to ∼83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657–670 of the carboxy terminal of the human insulin receptor α-subunit, whereas this antibody did label insulin receptor a-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor β- subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose a-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Notizen: Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C] isopentenyl pyrophosphate ([1-MC] I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 μM I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (S) was stimulated by dithiothreitol. The major products were identified as C90and Q95 allyhic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product cochromatographed with authentic Poly-P-P on silica-gel-impregnated paper, (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (Q95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.

Notizen: Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmittiers (e.g., glycine and doparaine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1-, 5-, and 13-fold and by 4-, 9-, and 31-fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700-fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4-fold, but remained elevated throughout the 80-min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann-Whitney test). That glutamate and aspartate concentrations in the hippocampus co-vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury. The observation that dopamine concentrations increased independently of ischemic duration indicates a maximal release with only S min of ischemia and suggests that dopamine's role in the incremental injury seen with increasing ischemic duration is limited to prolonged high concentrations rather than increasing peak levels as seen with glutamate and aspartate. The sustained elevation of glycine concentrations after ischemia may explain the ability of excitatory amino acids to produce delayed toxicity in the reperfusion phase, as glycine has been shown to facilitate glutamate's activity at the N- methyl-D-aspartate receptor.

Notizen: Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4, 5-bisphosphate. Approximately 60–70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of ∼ 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.

Notizen: Rat sciatic nerve contains a membrane-bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 μg of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg ot protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one-half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.

Notizen: Abstract: A small acidic polypeptide, termed thymosin β10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin β10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin β10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin β10, as well as its homologue gene product, thymosin β4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin β10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time- and dose-dependent increase in the content of thymosin β10, but not that of thymosin β4. The increase in thymosin β10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin β4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on β-thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin β10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin β10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.

Notizen: Abstract: Zinc is essential for the normal development and function of the CNS, although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied 65Zn uptake by brain from blood and have measured the blood-brain barrier permeability to 65Zn in the anaesthetised rat in vivo. Adult male Wistar rats within the weight range 500–600 g were used. 65ZnCl2 and 125I-albumin, the latter serving as a vascular marker, were injected intravenously in a bolus of normal saline. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 h, after which the whole brain was removed, dissected, and analysed for radioisotope activity. Data have been analysed by graphical analysis, which suggests that after 30 min of circulation, 65Zn uptake by brain from blood is unidirectional with an influx rate constant, Kin, of ∼5 × 10−4 ml/min/g. At circulation times of 〈30 min, 65Zn fluxes between blood and brain are bidirectional, where influx has a K value of 〉5 × 10−4 ml/min/g. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for 65Zn of ∼4 ml/100 g, which is not CSF.

Notizen: Abstract: Single cell Ca2+ mobilization was studied by non-parametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 μM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 μM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 μM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1–10 μM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.

Notizen: Abstract: The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromo-cytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 μM and 7.1 μmol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36–46), for example, exhibited a Km of 108 μM and a Vmax of 6.93 μmol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.

Notizen: Abstract: In order to study the possible involvement of dopamine receptors in the pathophysiology of various neurological and psychiatric disorders, we have isolated the human D2A gene. Like the rat D2A gene, the human gene contains at least eight exons and spans at least 52 kb. Exons 2–8 are clustered within 14 kb of genome. Exon 1 is separated from exon 2 by at least 38 kb. We and others have shown that alternative utilization of exon 6 gives rise to alternative D2A transcripts. Despite the extreme size of intron 1, no alternative transcription between exons 1 and 2 can be detected in basal ganglia and pituitary using polymerase chain reaction analysis. The relative abundance and tissue distribution of the alternative D2A transcripts were examined in 18 human brain regions. The relative expression of the transcripts varied by at least 70-fold across the brain regions surveyed. As expected, high levels of transcripts were detected in caudate, putamen, and pituitary. Moderate levels were detected in regions of catecholamine-containing cell bodies and in the amygdala. In contrast to the rat brain, very low levels of transcripts were detected in cortical regions.

Notizen: Abstract: We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate ([1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [γ-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.

Notizen: Abstract: A series of genomic clones containing DNA that encodes the chicken γ-aminobutyric acidA (GABAA) receptor β4 subunit have been isolated. These have been restriction mapped and partially sequcnced to determine the structural organization and the size of the β4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor β4-subunit gene has been compared to that of the murine GABAA receptor δ-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor sub-unit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.

Notizen: Abstract: The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10−8M and 10−5M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10−6M and 10−5M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 × 10−5M propranolol.

Notizen: Abstract: Neurite-promoting activities of lipids were assessed using serum-free cultures of fetal rat septal neurons. The most potent one was phosphatidylinositol (PI), followed by PI 4-phosphate, phosphatidylserine, sphingomyelin, and phosphatidylcholine. The EC50 value for PI was 1.5 μg/ml (1.8 μM), and activity was maximal at 4 μg/ml (56% of total cells had neurites after 24 h). Cerebroside, sulfatide, and di- and triacylglycerols showed relatively low activities. Synthetic dipalmitoyl phosphatidylcholine was also active, with a maximal activity (47%) at 100 μg/ml, a finding implying that the unsaturated fatty acid moiety is not released and further used as substrate for the arachidonic acid cascade. Lysophospholipids, phosphatidylglycerol, and cardiolipin were rather cytotoxic and lacked activity, an observation suggesting that membrane perturbation is not responsible for the neuritepromoting activity. Treatment with a protein kinase C inhibitor, H-7, or an Na+,K+-ATPase inhibitor, ouabain, inhibited the PI-induced neurite outgrowth, but the cyclic AMP- and cyclic GMP-dependent protein kinase inhibitor HA1004 did not inhibit this activity, a result indicating that multiple elements (protein kinase C and Na+,K+-ATPase) are involved in the induction of neurites. Because phospholipids can be provided either as lipid vesicles or as lipoproteins produced by macrophages at regeneration sites, they may play an important role in the regeneration of certain populations of neurons.

Notizen: Abstract: The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine-induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage-dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration-induced depolarization and presumably also intracellular Ca2+ mobilization.

Notizen: Abstract: Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3–72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as “functional inactivation.”Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1–3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2–6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR “functional desensitization,”which is a reversible process that occurs on shorter-term (0–5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites. Agonist-induced functional inactivation of TE671 cell nAChRs occurs under conditions previously shown to induce increases in numbers of nAChR ligand binding sites expressed on the cell surface and in total membrane pools and contrasts with coordinate down-regulation of nAChR ligand binding and function following chronic agonist treatment of nAChR-bearing cells in the periphery.

Notizen: Abstract: [3H]Kainate bound to chick cerebellar membranes with a KD of 0.6 μM and with an exceptionally high Bmax of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [3H]kainate was reduced (KD= 2.7 μM), but the Bmax was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate 〉 kainate 〉 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 〉 glutamate. Binding sites for α-[3H]amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) were much less abundant, with KD and Bmax values in membranes of 86 nM and I pmol/mg of protein, respectively. The affinity of [3H]AMPA binding was also reduced on solubilisation (KD= 465 nM), but there was an increase in the Bmax (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [3H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [3H]kainate, domoate was markedly less potent than kainate at displacing [3H]AMPA. These results suggest that [3H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [3H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non-N-methyl-D-aspartate receptors.

Notizen: 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.

Notizen: 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.

Notizen: Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and the cervical sympathetic trunk in which 〉99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed I25I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelinand non-myelin-forming Schwann cells. Endopeptidase-24. 11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.

Notizen: The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-l-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced, (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation, CCh-stimulated IP3 production and contraction are attenuated, (c) The increase in tension by a maximal effective dose of NaF (20 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation, (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively, (e) Isoproterenol (5 μM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively, (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by ∼30% of their respective controls, (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are causal by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to tie hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.

Notizen: Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 μM and a Kmax value of 3.7 nmol/mg of protein/min at 28°C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas α-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparaginc was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20–30, and this value was reduced to 5–6 by withdrawal of sodium or addition of high [KG]. A plot of log [Na+]o/{Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to 1, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [GIn]i maintained by the brain in vivo.

Notizen: Abstract: The effects of loop diuretics and ion substitution on the 2-min uptake of K (86Rb as marker) were analyzed to obtain evidence for K cotransport with Na and Cl in the choroid plexus epithelium. The isolated plexuses, which were excised from lateral ventricles of adult rats, were bathed in artificial cerebrospinal fluid (aCSF). At concentrations of 10-6 to 10-4M, the specific cotransport inhibitors, bumetanide and piretanide, suppressed uptake of K in a dose-dependent manner. Ouabain-insensitive K uptake was stimulated by preincubating the choroid plexus in aCSF very low in [Na] and [K], then incubating it in much higher concentrations of these cations; bumetanide (10-4M) blocked this stimulated uptake by 52%. Moreover, tissue preincubation in Na- or Cl-free medium, followed by incubation with normal concentrations of both ions, stimulated the ouabain-insensitive uptake of K from 15 (baseline) to 35 nmol/mg dry weight. This stimulation of K transport depended on the simultaneous presence of both Na and Cl in aCSF, and replacing either ion alone did not stimulate the ouabain-insensitive K uptake. Collectively, these findings, together with those from a previous pharmacological study of 22Na and 36Cl transport, constitute strong evidence for the cotransport of Na, K, and Cl in rat choroid plexus.

Notizen: Abstract: DNA sequences encoding two variants of a novel γ-aminobutyric acidA (GABAA) receptor β subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named β4 and β4′, respectively. The predicted mature polypeptides are 72–77% identical to the previously characterized mammalian and chicken β1,β2, and β3 subunits. Analysis of the β4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5′-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.

Notizen: Abstract: Book reviewed in this article: Nutrition and the Brain, Vol. 8 by R. J. Wurtman and J. J. Wurtman. Glossary of Biochemistry and Molecular Biology by D. M. Glick. Antiepileptic Drugs (Third Edition) edited by R. H. Levy, F. E. Dreifuss, R. H. Mattson, B. S. Meldrum, and J. K. Penry. Developmental Neurobiology (Nestlé Nutrition Workshop Series, Vol. 12) edited by P. Evrard and A. Minkowski. Allosteric Modulation of Amino Acid Receptors: Therapeutic Implications (Fidia Research Foundation Symposium Series, Vol. 1) by E. A. Barnard and E. Costa. Neurochemical Pharmacology—A Tribute to B. B. Brodie (Fidia Research Foundation Symposium Series, Vol. 2) edited by E. Costa. Neuromethods Vol. 11: Carbohydrate and Energy Metabolism edited by A. A. Boulton, G. B. Baker, and R. F. Butterworth. Adenosine and Adenosine Receptors edited by M. Williams. Laughing Death: The Untold Story of Kuru by V. Zigas. Psychoactive Drugs: Tolerance and Sensitization edited by A. J. Goudie and M. W. Emmett-Oglesby. The NMDA Receptor edited by J. C. Watkins and G. L. Collingridge. Basic and Clinical Aspects of Neuroscience (Vol. 3): The Role of Brain Dopamine edited by E. Flückiger, E. E. Müller, and M. O. Thorner. Glycine Neurotransmission edited by O. P. Otterson and J. Storm-Mathisen. The Neuropeptide Cholecystokinin (CCK): Anatomy and Biochemistry, Receptors, Pharmacology and Physiology edited by J. Hughes, G. Dockray, and G. Woodruff.

Notizen: Abstract: The effect of the anxiogenic β-carboline methyl-β-carboline-3-carboxyamide (FG 7142) on dopamine release in prefrontal cortex and striatum in the awake freely moving rat was determined using the technique of microdialysis. FG 7142 (25 mg/kg, i.p.) caused a time-dependent increase in dopamine release in prefrontal cortex which was statistically significantly greater than the response to vehicle administration. Dopamine release in striatum was unaltered by FG 7142. Pretreatment of animals with the benzodiazepine antagonist Ro 15-1788 (30 mg/kg, i.p., 15 min prior to FG 7142 administration) completely abolished the increase in dopamine release caused by FG 7142 in prefrontal cortex. These data indicate that the anxiogenic benzodiazepine inverse agonist FG 7142 can selectively increase dopamine release in prefrontal cortex, and that this effect appears to be mediated via the γ-aminobutyric acid/benzodiazepine receptor complex.

Notizen: Abstract: The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 μl/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)-containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, caused an approximately twofold increase in the HA output 30–60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by ∼70% 4–5 h after treatment with α-fluoromethylhistidine (α-FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in α-FMH-treated rats. These results suggest that both HA-N-methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.

Notizen: Abstract: We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequence identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.

Notizen: Abstract: The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5′-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.

Notizen: Abstract: We describe a simple, rapid, and efficient method, based on separation on a Percoll centrifugation gradient, to purify glial progenitor cells from newborn rat brains. Cytofluorimetry analysis of the isolated cell population showed that 75 ± 8 and 86 ± 7% of the cells were A2B5- and R24-positive, respectively. Transmission electron microscopy examination of the purified cell population confirmed their homogeneity and illustrated their typical morphology, as previously described in situ. Assay of UDP-galactose-ceramide galactosyltransferase, 3′-phosphoadenosine 5′-phosphosulfate galactosylceramide sulfotransferase, and 2′,3′-cyclic nucleotide 3′-phosphohydrolase activities showed that the levels of these enzymes were 446, 76, and 11 times lower, respectively, than the levels measured in mature oligodendrocytes. Low levels of mRNA coding for 2′,3′-cyclic nucleotide 3′-phosphohydrolase and myelin proteolipid protein, but not for myelin basic protein, were present in the glial progenitor cells. At the time of isolation, 40% of the cells in the population were dividing, and the cells could easily be expanded in culture. After 3 weeks of culture in the presence of 1% fetal calf serum, 75% of the cells had differentiated into galactosylceramide-positive oligodendrocytes. When the culture took place in the presence of 10% fetal calf serum, only 2% of the cells expressed galactosylceramide, and 60% were glial fibrillary acidic protein-positive astrocytes; half of them were also A2B5 positive.

Notizen: Abstract: Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.

Notizen: Abstract: γ-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (α, β, γ, and δ). The γ2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine γ2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids, LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the γ2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.

Notizen: Abstract: Cell surface binding, internalization, and biological effects of insulin-like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF-I receptor α-subunit (Mr 125,000) binds IGF-I with a KD of 1 nM and IGF-II with 10 times lower affinity. The mannose-6-phosphate (Man-6-P)/IGF-II receptor (Mr 250,000) binds IGF-II with a KD of 0.5 nM and IGF-I with 100 times lower affinity. Surface-bound IGF-I and IGF-II are internalized by their respective receptors and degraded to amino acids. Man-6-P increases the receptor binding and internalization of IGF-II but not those of IGF-I. Neuronal synthesis of RNA and DNA is increased twofold by IGF-I with 10 times higher potency than IGF-II. Antibody 3637, which blocks receptor binding of IGF-II, has no effect on the DNA response to IGF-I or IGF-II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that 〉80% of the bromodeoxyuridine-positive cells become neurofilament positive. It is concluded that IGF-I and IGF-II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF-I and IGF-II. Proliferation of neuronal precursor cells is stimulated by IGF-I and IGF-II via activation of the IGF-I receptor.

Notizen: Abstract: In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor sub-type, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.

Notizen: Abstract: The release of putative neurotransmitters [aspartate, glutamate, and γ-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 μm thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatog-raphy with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice ± SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 ± 2.62 and 15.41 ± 3.56), or GABA (17.28 ± 2.90 and 12.73 ± 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 ± 0.69) than in the B6 mice (3.31 ± 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.

Notizen: Abstract: One of the major clinical findings in Alzheimer's disease (AD) is the formation of deposits of β-amyloid protein in amyloid plaques, derived from the β-amyloid precursor protein (β-APP). To determine the possible use of β-APP as a diagnostic marker for AD in CSF, a monoclonal antibody-based immunoassay specific for this protein was developed. The assay does not differentiate between β-APP695 and β-APP751 forms but does preferentially recognize β-APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of β-APP in AD and Parkinson's disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.

Notizen: Abstract: Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine-enkephalin (Met-Enk) metabolism in the striatum. We administered a dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met-Enk-like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP-induced dopaminergic deficit, partially corrected the Met-Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met-Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met-Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met-Enk.

Notizen: Book Reviewed in this article: Neuromethods, Volume 16: Molecular Neurobiological Techniques edited by A. A. Boulton, G. B. Baker, and A. T. Campagnoni.

Notizen: Abstract: In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane-bound form. A major part (82%) of the membrane-bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane-containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of ∼50% of membrane-bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.

Notizen: Abstract: A cDNA probe specific to microtubule-associated protein 2 (MAP2) was used to study the expression of the mRNAs encoding the high- and low-molecular-weight MAP2 variants in cultured neurons and astrocytes. The timing and relative abundance of these MAP2 transcripts and of their encoded proteins were also studied in the developing cerebral hemispheres and cerebellum of the mouse. A 9-kb mRNA, known to encode high-molecular-weight MAP2, was expressed in cultured astrocytes, albeit at a lower level than in neurons. The 6-kb transcript, recently shown to encode low-molecular-weight MAP2 (MAP2c), was expressed in neurons and was the predominant MAP2 transcript of the astrocytes. The level of the 9- and 6-kb transcripts decreased at late stages of astroglial and neuronal cell culture. The 9-kb mRNA was detected in the cerebellum and cerebral hemispheres at every developmental stage. Although the levels of this mRNA varied slightly in the cerebral hemispheres, its expression was biphasic in the cerebellum. This might be explained by the differences in timing of development of the various neuronal cell types formed in these two brain areas. The 6-kb transcript was detected only at early developmental stages in the two brain areas. Correlating the temporal expression of the 9-kb mRNA to that of high-molecular-weight MAP2 indicates that the accumulation of this protein is in part regulated at a cytoplasmic level.

Notizen: Abstract: The changes in the levels of protein kinase C [PKC(α, βII, γ)] were studied in cytosolic and particulate fractions of striatal homogenates from rats subjected to 15 min of cerebral ischemia induced by bilateral occlusion of the common carotid arteries and following 1 h, 6 h, and 48 h of reperfusion. During ischemia the levels of PKC(βII) and -(γ) increased in the particulate fraction to 390% and 590% of control levels, respectively, concomitant with a decrease in the cytosolic fraction to 36% and 20% of control, respectively, suggesting that PKC is redistributed from the cytosol to cell membranes. During reperfusion the PKC(βII) levels in the particulate fraction remained elevated at 1 h postischemia and decreased to below control levels after 48 h reperfusion, whereas PKC(γ) rapidly decreased to subnormal levels. In the cytosol PKC(βII) and -(γ) decreased to 25% and 15% of control levels at 48 h, respectively. The distribution of PKC(α) did not change significantly during ischemia and early reperfusion. The PKC activity in the particulate fraction measured in vitro by histone IIIS phosphorylation in the presence of calcium, 4β-phorbol 13-myristate 12-acetate, and phosphatidylserine (PS) significantly decreased by 52% during ischemia, and remained depressed over the 48-h reperfusion period. In the cytosolic fraction PKC activity was unchanged at the end of ischemia, and decreased by 47% after 6 h of reperfusion. The appearance of a stable cytosolic 50-kDa PKC-immunoreactive peptide or an increase in the calcium-and PS-independent histone IIIS phosphorylation was not observed. Consequently, during ischemia PKC, preferentially PKC(γ) and PKC(βII), is translocated from the cytosol and inserted into cell membranes, concomitant with a decrease in PKC activity. In the reperfusion phase the depression of PKC activity persists and the enzyme is degraded. The observed translocation and downregulation of PKC during ischemia and reperfusion may be of significance for the development of ischemic neuronal damage.

Notizen: Abstract: Enhanced cocaine concentrations in brain and blood observed after an intraperitoneal challenge dose in rats exposed to cocaine for 10 days by subcutaneous administration are traced to a change in the absorption process from the site of an intraperitoneal injection to general circulation. This conclusion is reached by three sets of corroborating results: (a) Adipose tissue of rats treated for 10 days with repeat subcutaneous injections of cocaine did not reveal a buildup of cocaine in sufficient concentrations to account for the twofold increase in brain and blood concentrations seen during intraperitoneal administration; (b) administration of the drug by an intravenous route after 10-day cocaine treatment did not show a significant difference between treatment and control groups; (c) nonlinear regression on the intravenous and intraperitoneal data sets using a two-compartment open model indicated a difference in the absorption process but not in the metabolic and blood-brain transfer processes.

Notizen: Abstract: The annexins are a group of highly related Ca2+-dependent membrane-binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with ∼20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane-containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67-kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X-114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.

Notizen: Abstract: HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas-phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin β10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin β10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20-fold reduction) by postpartum day 14. The elevated levels of thymosin β10 in fetal and neonatal brain correlate with high levels of thymosin β10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15-fold reduction in specific mRNA. In comparison, the levels of thymosin β4 polypeptide, a homologue of thymosin β10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin β4 is elevated in fetal brain, and its levels decrease approximately four-fold to a stable value around the time of birth. The reason for this discrepancy between thymosin β4 protein and mRNA levels is unknown. Thymosin β10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin β10 also declines following birth. It is concluded that β-thymosin expression (as measured by steady-state mRNA and polypeptide levels) is both up-and down-regulated during different phases of maturation of the mammalian nervous system.

Notizen: Abstract: [3H]Harman (1-[3H]methyl-β-carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO-A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO-A in the CNS of different mammalian species as well as in extraneuronal tissues.

Notizen: Abstract: In PC12 pheochromocytoma cells whose phospholipids had been prelabeled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1–2 min, before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells; bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.

Notizen: Abstract: The stereoenantiomers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 μM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 ± 14 and 75 ± 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 ± 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthioinosine, both at a concentration of 100 μM, reduced the levels and rates of L-[3H]adenosine accumulation by 〉44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.

Notizen: Abstract: The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 μM Pb2+ to intact synaptosomes in Ca2+-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2+-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1–50 nM (EC50± 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2–5 μM (EC50± 0.5 μM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.

Notizen: Abstract: Intact human neuroepithelioma SK-N-MC cells bound the β-adrenergic antagonist (–)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to β-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol norepinephrine 〉 epinephrine, which is indicative of a β1-subtype receptor. Membranes prepared from the cells bound (–)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the β1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the β2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only β1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the β1- and β2-adrenergic receptors revealed the presence of a very strong β1-adrenergic receptor mRNA signal, while under the same conditions no β2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of β1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.

Notizen: Abstract: In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LANS is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Notizen: Abstract: Significant differences were demonstrated between the long-sleep (LS) and short-sleep (SS) selected mouse lines in the abilities of barbiturates and γ-aminobutyric acid (GABA) to inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to well-washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor-chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.

Notizen: Abstract: An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is substantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7: 1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.

Notizen: Abstract: We have studied the metabolic and functional effects of two new platelet-activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14-min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high-energy phosphates, carbohydrate content, and thiobarbituric acid-reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14-min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726-treated ischemic brains than in DMSO-treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO-treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger. The PAF antagonists act at a different level, possibly restoring normal mitochondrial function or preventing some of the adverse effects of increased excitatory amino acid and/or intracellular Ca2+ levels. This is the only drug combination currently known to promote such complete short-term metabolic and functional recovery during postischemic reoxygenation.

Notizen: Abstract: Heterogeneity of rat brain angiotensin II receptors was revealed by quantitative autoradiography after incubation with 125I-Sar1-angiotensin II and displacement with the angiotensin II antagonists CGP 42112 A and DuP-753 and by receptor sensitivity to dithiothreitol. Receptors in areas involved in cardiovascular and fluid control—the subfornical organ, nucleus of the solitary tract, paraventricular nucleus, and area postrema—are displaced by DuP-753 with an IC50 of 1 × 10−7M, are sensitive to 5 mM dithiothreitol, and thus are angiotensin II type-1. Receptors in the inferior olive are displaced by CGP 42112 A (IC50, 1 × 10−9M) but not by DuP-753 in concentrations up to 10−4M, are insensitive to 5 mM dithiothreitol, and thus are angiotensin II type-2.

Notizen: The release of γ-aminobutyric acid (GABA) in rat dorsolateral striatum was studied using in vivo microdialysis. Dialysis was conducted 2 days after probe implantation in awake, freely moving rats using a modified Ringer solution. Calcium induced a reversible increase in GABA release that was abolished by tetrodotoxin but was only slightly attenuated by a maximally effective dose of pergolide, a D2 receptor agonist. It was thus concluded that pergolide inhibits calcium-stimulated release of GABA presynaptically by a mechanism distinct from that of tetrodotoxin.

Notizen: In l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-treated mouse brain, there was no significant increase or decrease in the content of an endogenous amine, 1,2,3,4-tctrahydroisoquinoline (TIQ), which is well noted for inducing parkinsonism, whereas another endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), was markedly reduced. This result agrees with the finding in human idiopathic parkinsonianism, confirmed by our previous research. In addition, pretreatment with 1-MeTIQ completely prevented MPTP- or TIQ-inducing bradykinesia, a symptom of parldnsonism. This study confirmed that 1-MeTIQ plays an important role in preventing the pathogenesis of parldnsonism and is a possible leading compound of anti-parkinsonism agents.

Notizen: An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P-like immunoreactivity, measured with an antiserum directed against the COOH-terminal region of mammalian substance p and neurokinin b-like immurtoreactivity, measured with an antiserum directed against the NH2-terminus of neurokinin B. The primary structure of the substance p-related peptide (ranakinin) was established as: Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin b-related peptide was established as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence is the same as that of mammalian neurokinin B.Ranakinin was equipotent with substance p and [Sar9,-Met(O2)11]substance p in inhibiting the binding of 125I-Bolton-Hunter-[Sar9, Met(O2)11]substance p, a selective radio-ligand for the NK1 receptor, to binding sites in rat subman-dibular gland membranes (IC50 1.6 ± 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.

Notizen: Elevated iron concentrations in the substantia nigra (SN) pars compacta have been implicated in the development of idiopathic Parkinson's disease. Because, as a transitional metal, iron promotes free radical formation, the role of iron in the degeneration of the nigrostriatal dopamine neurons in Parkinson's disease has received much attention. This study further investigates the cytotoxic effects of iron in the SN. Various concentrations of FeCl3 (1, 5, and 50 μg of Fe3+ in 5 μl) were unilaterally injected into the SN of adult rats. The two lower doses of iron had no effect on striatal dopamine levels or on the behavioral responses of the rats. However, injection of 50 μg of Fe3+ resulted in a substantial selective decrease of striaial dopamine (95%), 3,4-dihydroxyphenylacetic acid (82%), and homo-vanillic acid (45%), without any change in norepinephrine concentration. Dopamine-related behavioral responses, such as spontaneous movements in a novel space and rearing, were significantly impaired, whereas amphetamine administration induced ipsilatcral rotation in the iron-treated rats. The present study indicates that the nigrostriatal dopamine neurons are susceptible to the presence of ionic iron and thus supports the assumption that iron initiates dopaminergic neu-rodegeneration in Parkinson's disease.

Notizen: The study of the regulation of low-abundance blood-brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single-step method for isolation of polyfA)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-de-oxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was—15-19 #g/g of brain or choroid plexus, 14-17 μg per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 μg/107 ECL cells. Northern blot analysis showed characteristic and undegraded 2.1-and 1.7-kb actin transcripts in brain capillaries and a 2.1-kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up-regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two-step method.

Notizen: : The effect of hypothermia on the ischemia-induced changes in the subcellular distribution of protein kinase C (PKC)(γ), -(βII), and -(α) and the activity of PKC was studied in striatal homogenates of rats subjected to 20 min of cerebral ischemia. The effect of post-ischemic cooling was also studied. During normothermic ischemia, PKC(γ) and -(βII) increased 3.9-and 2.9-fold, respectively, in the particulate fraction, signifying a translocation of PKC to cell membranes. The levels of PKC(α) did not change significantly. PKC activity decreased during ischemia by 52% and 47% (p 〈 0.05) in the paniculate and cytosolic fractions, respectively, and remained inhibited for the 1 h recovery period. In hypothermic animals, there was no evidence of translocation, and the inhibition of PKC activity was completely abolished. Hypothermia induced in the recovery phase, however, did not affect PKC distribution or activity. The protective effect of intraischemic hypothermia may in part be due to the prevention of the ischemia-induced translocation and subsequent downregulation of PKC, possibly through a temperature-dependent modification of the cell membranes.

Notizen: Two rabbit arylamine N-acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, ρ-aminosalicylic acid, and ρ-aminobenzoic acid. In the pineal gland, serotonin is metabolized to N-acetylserotonin by an unknown N-acetyltransferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N-acetyltransferase (AA-NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as ρ-phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N-acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross-hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA-NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N-acetylation of serotonin.

Notizen: The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 μM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KC1. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 μM choline did not lead to a further increase, but preincubation with 25 or 50 μM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 μM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 μM KC1, ACh release from slices preincubated with 50 μM choline was greater than from slices preincubated with 0, 10, or 25 μM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KC1, the ACh content of slices preincubated with 0 or 10 μM choline decreased significantly, whereas that of slices preincubated with 25 or 50 μM choline did not. Although ACh synthesis by slices incubated with KRB did not differ significantly among groups, synthesis by slices incubated with 25 mM KC1 was significantly greater for tissues preincubated with 50 μM choline than for those preincubated with 0, 10, or 25 μM choline. Preincubation in the absence of choline decreased choline levels by 43%, whereas preincubation with 50 μM choline increased levels by 55%; choline levels were unaltered by preincubation with 10 μM choline. Following incubation with 25 mM KCl, the choline content of slices preincubated with 50 μM choline decreased by 0.59 nmol, whereas the content of slices preincubated with 10 μM choline decreased by 0.33 nmol, suggesting that the former used more of its tissue choline to support ACh synthesis; choline levels did not change in slices preincubated in the absence of choline. These results suggest that prior exposure of hippocampal slices to choline provides precursor that is used during a subsequent incubation with 25 mM KCl to support the synthesis of ACh.

Notizen: The effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extrasynaptosomal concentration of glutamate ([Glu]cxt) under aerobic conditions was 2.3 μM and increased to 4.9 μM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]cxt The accumulation of glutamate in the external medium during anoxia was Ca2+ in dependent and insensitive to a significant reduction of the Ca2+-dependent release of the amino acid. These results indicate that a Ca2+-independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.

Notizen: The mouse monoclonal antibody SMP has previously been demonstrated to react immunohistochemically with neurofibrillary tangles, argyrophilic plaques, and lep-tomeningeal vascular amyloid deposits in the brain tissue of individuals dying from pathologically diagnosed Alzheimer's disease. In preliminary studies the antibody was shown, by size exclusion chromatography, to bind to a protein with an apparent molecular mass of 260 kDa present in the CSF and serum of demented individuals. Chromatographic separation of a 40% ammonium sulphate precipitate of CSF and serum yielded immunoreactive fractions that were subjected to 9% sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by western blotting. Probing the nitrocellulose blot with the antibody revealed that the antibody selectively binds to a protein chain with an apparent molecular mass of 100 kDa. By using a combination of affinity chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, coupled with western blotting, the serum component with which the antibody reacts has been identified as complement factor 4. In addition, the antibody has been shown to react specifically with an epitope on the α-chain of this protein.

Notizen: The effects of 6R-5, 6, 7, 8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KC1. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6-methyl-5, 6, 7, 8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isotner of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R- BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KC1-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization-induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.

Notizen: Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.

Notizen: In synaptosomal membranes from rat brain cortex, in the presence of 150 mM NaC1, the opioid antagonist [3H] naltrexone bound to two populations of receptor sites with affinities of 0.27 and 4.3 nM, respectively. Guanosine-5′-(3-thiotriphosphate) had little modulating effect and did not alter the biphasic nature of ligand binding. On the other hand, receptor-selective opioids differentially inhibited the two binding components of [3H] naltrexone. As shown by nonlinear least-squares analysis, the μ opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol or sufentanil abolished high-affinity [3H] naltrexone binding, whereas the δ-selective ligands [D- Pen2, D-Pen5] enkephalin, ICI 174, 864, and oxymorphindole inhibited and eventually eliminated the low-affinity component in a concentration-dependent manner. These results indicate that, in contrast to the guanine nucleotide-sensitive biphasic binding of opioid-alkaloid agonists, the heterogeneity of naltrexone binding in brain membranes reflects ligand interaction with different opioid-receptor types.

Notizen: Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin-like immunoreactivity (SRIF-LI) was measured by means of a radioimmunoassay procedure. High KC1 (9-50 mM) concentration dependently evoked SRIF-LI release; the evoked overflow reached a plateau at 25 mM KC1 and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KC1 used. The 15 mM K+-evoked release of SRIF- LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K+-evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 μM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF-LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1-50 μM) induced release of SRIF-LI in a concentration-dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF-LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium-deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that 〉90% of the SRIF-LI released either during the spontaneous outflow or by 15 mM KC1 was represented by SRIF-14 (SRIF-2814-28). These values reflected the ratio SRIF-14/SRIF-28 found in synaptosomes at the end of the experiments.

Notizen: Regulation of prostaglandin (PG) E2 receptors was investigated in a 3-[(3-cholamidopropyl) dimethylammonio]- 1-propanesulfonate-solubilized fraction from the synaptic membrane of porcine temporal cortex. The fraction was preincubated with exogenous protein kinases, and then the binding of PGE2 was measured. PGE2 binding was increased approximately twofold by pretreatment with the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase) or calmodulin-dependent protein kinase II but not by that with protein kinase C. The increase was dependent on the ATP concentration, with ED50 values being close to the ATmvalues of these protein kinases. Protein kinase inhibitors specific for A kinase and for calmodulin-dependent protein kinase II abolished the effect in a dose-dependent manner, with IC50 values being similar to those reported. Further study using the catalytic subunit of A kinase revealed that the maximal binding capacity apparently increased without affecting the affinity and the rate constants for association and dissociation. On the other hand, acid phosphatase treatment reduced the binding activity to the level of nonspecific binding. In addition, treatment by A kinase did not affect the binding of guanosine 5′-(3-thiotriphosphate) by the GTP-binding proteins and the activation of adenylate cyclase mediated by stimulatory guanine nucleotide-binding regulatory protein, and therefore the phosphorylation is believed to occur on the receptor protein. The results suggest that the PGE2 receptor can take active phosphorylated and inactive dephosphorylated forms, of which only the phosphorylated one can bind PGE2.

Notizen: Mice were anesthetized with 5a-[3H]pregnan-3a-o1-20-one. Brain levels for 5α-pregnan-3α-o1–20-one and its five major metabolites (5α-pregnanedione, ko, k1, k2, k3) were compared at behavioral endpoints that are characteristic of the anesthetized state. The results support the hypothesis that 5α-pregnan-3α-o1–20-one mediates the anesthetic response, and they weigh against the hypothesis that any of its metabolites is solely responsible for the onset or the maintenance of the anesthetized state. For an administered dose of 3 mg/kg, brain levels (means ± SEM) for 5α-pregnan-3α-o1–20-one at the time of the loss of the righting response (n = 10) and at the time of the return of the righting response (n = 6) were 7.24 ± 0.61 pmol/mg of brain tissue and 3.63 ± 0.26 pmol/mg of brain tissue, respectively. No metabolite level was lower at the return of the righting response than at the loss of the righting response. 5α-Pregnan-3′-o1–20-one brain levels increased consistently with the percentage of anesthetized mice. This was not the case for any of the metabolites. Fifty percent of the mice were anesthetized when the 5α-pregnan-3α-o1–20-one level was 4.5 pmol/mg of brain tissue.

Notizen: Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.

Notizen: Insights into the etiology and pathophysiology of Parkinson's disease may derive from elucidation of the neurotoxic mechanisms of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and its active metabolite, I-methyl-4-phenylpyridinium (MPP+). In previous studies, MPP+ provoked oxidation of cytochrome b and K+ leakage into the extracellular space of rat striatal slices. Magnitudes of these time-dependent responses were far greater than expected had the MPP+ effects been limited to dopaminergic terminals. To determine whether cytochromes become oxidized from K+-induced increases in ion transport activity or from electron transport inhibition at complex I, oxygen consumption was measured because this should be increased by the former and decreased by the latter mechanism. Low MPP+ concentrations (1 μM) decreased O2 consumption (∼40% in 3 h) in striatal slices. This decrease was diminished by mazindol and did not occur in hippocampal slices. High toxin concentrations (100 μM) inhibited oxygen consumption to a greater extent (∼60%) in striatal slices; this inhibition was still greater in hippocampal slices. These results support the hypothesis that acute effects of low (“selective”) MPP+ concentrations require the presence of dopaminergic terminals to trigger a sequence of destructive metabolic events but that the metabolic consequences of MPP+ spread to neighboring cells. In contrast, high MPP+ concentrations nonselectively inhibit metabolic and ion transport activity without requiring the presence of dopaminergic terminals. These results also suggest that physiological effects of “selective” MPP+ concentrations extend to nondopaminergic cells.

Notizen: Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24–34 and 176–185 from the NH2 terminus, respectively, were conjugated to keyhole limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-AP-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H] YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner, a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H] YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25–30% decrease in Bmax (203.5 ± 11.0 and 164.6 ±3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD. These results suggest that polyclonal antisera raised against peptide I exhibited specific antibodies for the dopamine D2 receptor protein. The primary epitope for this antibody is at or near the ligand binding site which can be recognized in both denatured and native receptor protein in striatal membranes.

Notizen: In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 μM to 1 mM evoked a saturable release of -γ-[3H] aminobutyric acid ([3H] GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50), 100–150 nM) in releasing [3H] GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H] tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H] GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-B-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(±)-2-carboxypiperazin-4-yl] propyl-1-phosphonic acid (IC50range, 〉50 μM) and the non-NMDA-receptor antagonist, 6, 7-dinitroquinoxalinedione (IC50 range, 5–50 nM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H] GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 pMexcitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 fiMSKF-89976-A, a non- transportable inhibitor of theGABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are dearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.

Notizen: Pretreatment of striatal membranes with N-ethylmaleimide in the presence of a D1specific agonist inactivated endogenous guanine nucleotide binding proteins (G proteins), but not D, dopamine receptors, resulting in a loss of high-affinity agonist binding sites. Such D1 receptors were solubilized, mixed with exogenous G proteins from cells not containing D1receptors, and reconstituted into phospholipid vesicles. These reconstituted receptors were able to couple to the exogenous G proteins, and the proportion of agonist high-affinity sites of the receptor (40–57%) was similar to levels obtained with naive receptors coupling to endogenous G proteins (40%) upon solubilization and reconstitution. These hybrid high-affinity sites were fully modulated by guanine nucleotides. Pretreatment of cells with pertussis toxin prior to extraction of G proteins resulted in a 50% decrease in the proportion of high-affinity sites; these sites remained sensitive to guanine nucleotides. When D, receptors were reconstituted with extracts of cyc− cells, which lack stimulatory G proteins, the proportion of high-affinity sites was reduced to 31% of the total. Pertussis toxin treatment of the cyc− cells completely abolished the formation of high-affinity sites. These results demonstrate that D1-dopaminergic receptors are able to couple to not only stimulatory G proteins (Gs), but also to inhibitory G proteins (Gi).

Notizen: Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15–30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).

Notizen: Chronic ethanol treatment is known to alter the function of the γ-aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the α1α3, α6, γ2s, γ2t, and γ3 receptor subunits in withdrawal seizure-prone and -resistant (WSP and WSR, respectively) mice fed an ethanol-containing liquid diet or a control diet Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit-specific probes. Chronic ethanol treatment decreased the content of α1, mRNA in WSP but not WSR mice and decreased the content of α6 mRNA in WSR but not WSP mice. The content of γ3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of α3 and α6 mRNA than the WSR line. Thus, a decrease in the content of α1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of α6 and α3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for γ2S and γ2L subunits do not appear to be altered in ethanol dependence.

Notizen: The present study was designed to examine the steady-state density and the turnover rates of D1 dopamine (DA) receptors in the striatum, nucleus accumbens, substantia nigra, and retina of the rat. The turnover rates were measured by monitoring the repopulation kinetics of D1 DA receptors labelled with [3H]SCH 23390 after the irreversible inactivation induced by a single dose of N-ethoxycarbonyl-2-ethoxy- 1, 2-dihydroquinoline (10 mg/kg s.c.). The repopulation of D1 DA receptors could be described adequately in all the neural tissues investigated by a theoretical model that assumes a constant rate of receptor production (i.e., zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e., first order). The quantitative analysis of the experimental data using this theoretical model revealed significant regional differences in the rates of receptor production and degradation. Thus, the receptor production rates determined in the nucleus accumbens and striatum (8.03 and 9.96 fmol/mg of protein/h, respectively) were four- to sixfold larger than those measured in the substantia nigra (1.80 fmol/mg of protein/h) and retina (1.50 fmol/mg of protein/h). On the other hand, the receptor degradation rates in the striatum, nucleus accumbens, and retina (0.0093 h−1, 0.0110 h−1, and 0.0123 h−1, respectively) were 2.6–3.5-fold larger than the receptor degradation rate in the substantia nigra (0.0035 h−1).

Notizen: Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte-type 2 astrocyte (O-2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia-inducing molecule (AIM). Using cultures enriched in O-2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240-fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin-agarose, followed by gel nitration chromatog- raphy. In crude preparations, AIM activity migrated at 50 kDa by gel nitration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of 〉9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O-2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects, AIM may be a novel differentiation factor.

Notizen: The wide-ranging neuronal actions of glutamate arc thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca2+-depeodent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important rote in transmitter interactions in die striatum.

Notizen: The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radio-labeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.

Notizen: The biosynthesis of the hormone melatonin (MEL) by the mammalian pineal gland has been thought to be regulated strictly by stimulatory factors, most predominantly norepinephrine (NE), released from the sympathetic nerve fibers which heavily innervate the gland. Evidence from many investigators suggests that sympathetic fibers may colocalize other neuroactive factors in addition to NE. One of these factors is neuropeptide Y (NPY), which has been found in the nerve fibers of the pineal gland. The present study sought to explore potential interactions between NE and NPY in the regulation of pineal MEL secretion. Specific, saturable, and reversible binding of 125I-NPY to intact cultured pine- alocytes was measured with an affinity constant of 1 nM and an NPY binding site density of 0.04 pmol/mg of protein. In addition, cell culture studies revealed that NPY represents a potent (IC50 of 0.4 nM) endogenous inhibitor of NE-stimulated MEL secretion. However, this inhibition is accompanied by only a modest reduction (35%) of cyclic AMP accumulation. These findings reinforce the view that the mammalian pineal gland, which appears to integrate both inhibitory as well as stimulatory signals, is an important model of autonomic function, particularly in the context of biological rhythmicity.

Notizen: A nervous system-specific mRNA that is rapidly induced in PC 12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.

Notizen: Delta-sleep-inducing peptide (DSIP) stimulates the release of Met-enkephalin (Met-ENK) from superfused slices of the rodent lower brainstem in vitro. In our present study, DSIP (10–10–10–9M) induced a significant release of Met-ENK from medullary synaptosomes of rats. This DSIP-evoked release of Met-ENK was Ca2+ dependent and tetro-dotoxin (TTX) insensitive. Furthermore, DSIP (10–11–10–9M) significantly increased 45Ca2+ uptake in medullary synaptosomes. These results demonstrate that DSIP acts directly on the nerve endings of Met-ENK-containing neurons to release this pentapeptide by generating a Ca2+ influx into these neurons. Effects of DSIP on Met-ENK release in other discrete brain regions were also studied. Significant DSIP-evoked Met-ENK release from synaptosomes was observed in the cortex, hypothalamus, and midbrain (at concentrations of 10–10 and 10–9M) and in the hippocampus and thalamus (only at 10–9M), but not in the striatum. In the hypothalamus, the release of Leu-enkephalin from its synaptosomes was slightly, but not significantly, enhanced by DSIP (10–10–10–8M). Our findings demonstrate that DSIP triggered a Ca2+ influx in nerve endings to induce a subsequent release of Met-ENK from neurons in only certain brain regions.

Notizen: The release of γ-[3H]aminobutyric acid ([3H]GABA) newly synthesized from [3H]glutamine was estimated in the superior colliculus of ketamine-anesthetized rats superfused via a push-pull cannula. A significant amount of [3H]GABA was spontaneously released in the superior colliculus (582 ± 49 pCi/10 min). A major part of the large K+-evoked increase of the [3H]GABA release was Ca2+ dependent. When neuronal activity of the substantia nigra was enhanced by nigral application of K+ (30 mM) or bicuculline (10–4M), a persistent increase of the collicular [3H]GABA release was observed (60 and 80%, respectively). Conversely, when nigral activity was reduced by nigral application of GABA (10–4M) or superfusion with a Ca2+ free medium, a sustained decrease of the collicular [3H]GABA release was observed (-30 and -40%. respectively). Following the nigral application of a selective D2-receptor agonist, RU 24926 (10–5M), for 30 min in 6-hydroxydopamine-lesioned rats, a phasic increase (60%) of the collicular [3H]GABA release was detected. This effect could result from an activation of nigrocollicular GABAergic neurons by D2-receptor stimulation, because nigral activity and collicular release of [3H]GABA changed in a parallel direction.

Notizen: In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (〉96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.

Notizen: The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 γg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a-spectrin and a 62-kDa calmodulin-binding protein (p62) in the nuclei of neurons and glial cells by irnmunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by irnmunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.

Notizen: Four inhibitors of Oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the Oligosaccharide chains of the Po and 19-kDa glycoproteins. Several different inhibitors of Oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21–24 days of age) and the uptake of 14C-amino acid and [3H] fucose or [3H] mannose was measured in Po and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H] Mannose was not found as suitable as [3H] fucose as an Oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H] mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated Oligosaccharide chains. Endoglycosidase H cleaved ∼50% of the Po labeled with [3H] fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H] fucose label on the protein, whereas one-half remained on the Oligosaccharide chain of the undegraded Po, indicating that at least one-half the Pohas a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H] fucose into the Oligosaccharide chains of Po and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. Po synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed Po and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.

Notizen: Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3-methoxytyramine (3-MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3-MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3-MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3-MT. Increases in DA and 3-MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3-MT in the dialysate. γ-Butyrolactone also greatly decreased DA and 3- MT. Finally, d-amphetamine produced a large increase in DA and 3-MT in animals that had been treated previously with γ-butyrolactone. The Pearson correlation coefficients for DA and 3-MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3-MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3-MT may not reliably reflect DA release following certain pharmacological manipulations.

Notizen: The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca2+-dependent release of glutamate evoked by 4-aminopyridine (4-AP) was inhibited by arachidonic acid (0.5–10 μM), but the KC1-evoked release was not modified. The Ca2+-independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 μM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca2+-dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4-AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K+-channel Mocker tetraethyl-ammonium.

Notizen: Abstract: In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic-and ischaemic-type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic-type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic-type conditions. Evidence is presented to suggest that the effect of l-lactate was due to the lactate moiety itself rather than the associated acidosis.