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Notes: Abstract: The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1–2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.

Notes: Abstract: Synthetic n-butyl β-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]β-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD= 2.7 ± 0.1 nM) with a Bmax of 1.16 ± 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax= 2.36 ± 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10−11-10−5M butyl β-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KDi= 0.6–1.5 nM and KD2= 12–18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl β-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1. γ-Aminobutyric acid (GABA) stimulated the binding of [3H]flunitrazepam, whereas the effect on the binding of [3H]butyl β-carboline was negative. This finding, together with some pharmacological experiments in mice, suggests that n-butyl β-carboline-3-carboxylate acts as an inverse agonist on the central benzodiazepine receptor, inhibiting GABAergic neurotransmission.

Notes: Abstract: The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.

Notes: Abstract: The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-d-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.

Notes: Abstract: Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysinecoated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) not detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.

Notes: Abstract: pros-Methylimidazoleacetic acid (p-MIAA; 1-methylimidazole-5-acetic acid), an isomer of the histamine metabolite, tele-methylimidazoleacetic acid (t-MIAA), is present in brain and CSF. Its relationship to histamine synthesis and catabolism was assessed in brains of rats. p-MIAA distribution in brain regions was heterogeneous although the concentrations in regions with the highest (hypothalamus) and the lowest (medulla-pons) levels differed less than fourfold. There was no significant correlation between the regional distributions of p-MIAA with those of histamine or its metabolites. pros-Methylhistidine (1 g/kg, i.p.) produced a 20-fold increase in mean levels of p-MIAA and up to a 50-fold increase in levels of pros-methylhistamine (p-MH), a putative intermediate; levels of histamine and its metabolites were unaltered. l-Histidine (1 g/kg, i.p.) or α-fluoromethylhistidine (100 mg/kg, i.p.), the irreversible inhibitor of histamine synthesis, did not alter the levels of p-MIAA in brain. Like the levels of t-MIAA, the levels of p-MIAA were unaltered after probenecid administration. Contrary to its effects in lowering t-MIAA levels, pargyline (75 mg/kg, i.p.) produced a slight rise in levels of p-MIAA in all regions. These findings suggest that, in brain, the metabolic pathways of histamine are independent of pathways that generate p-MIAA. Further, since brain is capable of p-MH formation, its use as an internal standard in analytical methods merits caution.

Notes: Abstract: The main objective of these studies was to determine whether the acute administration of choline to rats provides supplemental precursor that can be used to support acetylcholine synthesis when the demand for choline is increased by increasing neurotransmitter release. For these experiments, hippocampal and striatal slices were prepared from rats that had received saline or an acute injection of choline. Slices were incubated in a choline-free buffer containing 4.74–35 mM KC, and acetylcholine synthesis and release and choline production were measured. The initial tissue contents of acetylcholine and choline did not differ between experimental groups for either brain region. When hippocampal slices from the controls were incubated for 10 min with depolarizing concentrations of KCl, acetylcholine release increased and the tissue content decreased in a concentration-dependent fashion; no net synthesis of acetylcholine occurred. In contrast, hippocampal slices from the choline-injected animals maintained their tissue content in the presence of high concentrations of KCl, despite an increase in acetylcholine release that was similar in magnitude to that of the controls; positive net synthesis of acetylcholine resulted. Although the molar concentration of choline achieved in the incubation media at the end of the 10-min period did not differ between groups, the mobilization of free choline from bound stores was significantly greater in hippocampal slices from the choline-injected group than the controls. In addition, the synthesis of acetylcholine by hippocampal slices from the cholineinjected group was prevented by the presence of hemicholinium-3 (1 μM) in the media. Acetylcholine synthesis and choline mobilization by striatal slices did not differ between groups. Results demonstrate that acute supplementation with choline provides precursor to support acetylcholine synthesis when the release of neurotransmitter is increased.

Notes: Abstract: The voltage-dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage-sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of 〈500 μM, the initial (5-s) calcium uptake declined by ∼ 20–50% in 12- and 24-month-old rats relative to 3-month-old adults. Depolarization of synaptosomes from 3-month-old rats in a calcium-free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage- or voltage-and-calcium-dependent inactivation) to ∼ 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 μM nifedipine resulted in a 15–25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24-month-old rats, inactivation of 45Ca2+ uptake in calcium-free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3-month-old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 μM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24-month-old rats. These results indicate that the decrease in calcium channel-mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine-resistant and -sensitive calcium channels. It is interesting that the large decrease in calcium uptake via calcium channels that occurs in 24-month-old rats is correlated with a slower dephosphorylation of the synaptosomal phosphoprotein P96, a process that is intimately related to the opening and ensuing calcium flow through voltage-gated calcium channels.

Notes: Abstract: Vitamin A (retinol) and some of its analogs exhibited varying degrees of inhibition on induced iron and ascorbic acid lipid peroxidation of rat brain mitochondria. Malonyldialdehyde production was used as an index of the extent of in vitro lipid peroxidation. The fat-soluble vitamins retinol, retinol acetate, retinoic acid, retinol palmitate, and retinal at concentrations between 0.1 and 10.0 mmol/L inhibited brain lipid peroxidation. Retinol and retinol acetate were the most effective inhibitors. It is concluded from this study that retinol and its analogs can be considered as potential antioxidant factors, more potent than some of the well-known antioxidants such as α-tocopherol and butylated hydroxytoluene.

Notes: Abstract: 1-Methyl-4-phenylpyridinium (MPP+) was taken up into human and rat striatal synaptosomes by a saturable system, similar to that for dopamine, with Km values of 0.24 and 0.17 μM, respectively, and similar Vmax values. Uptake of MPP+ and dopamine into both rat and human synaptosomes was inhibited by cocaine and amfonelic acid, with the latter being five to 10 times more potent than the former. MPP+ uptake was potently inhibited by dopamine in preparations from both species. In general, the characteristics of human and rat synaptosomal MPP+ uptake were very similar. It seems unlikely that species differences in toxicity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or reaction to dopamine uptake blockers stem from this system.

Notes: Abstract: Activities of choline acetyltransferase (ChAT) were microassayed in individual cell bodies of motor neurons, isolated from freeze-dried sections after autopsy of lumbar spinal cords from four patients with sporadic amyotrophic lateral sclerosis (ALS) and four control patients with nonneurological diseases. Numerous large neurons were found in the anterior horn at the early degeneration stage of ALS, but the cell bodies atrophied and decreased in number at the late advanced stage. The small, atrophied neurons were very fragile and were easily destroyed during the isolation procedure with a microknife. The average activity, expressed on a dry weight basis, of 58 ALS neurons was lower than that of 67 control neurons. The large, well-preserved neurons at the early nonadvanced stage had markedly lower ChAT activities than control neurons. The specific activity gradually increased with the progress of atrophy but did not return to the control level.

Notes: Abstract: Lumbar punctures were performed on four occasions over a 5-day period (8:30 a.m. on days 1, 3, and 5; 2: 30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day-to-day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total γ-aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5-HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.

Notes: Abstract: Aromatic l-amino acid decarboxylase (AAAD) activity of the rat retina increases when animals are placed in a lighted environment from the dark. The rise of activity can be inhibited by administering α2 adrenoceptor agonists. In the dark, the enzyme activity can be made to increase by administering α2 adrenoceptor antagonist drugs. Kinetic analysis indicates that the maximum velocity of the enzyme increases with little change of the Km for the substrate l-3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5′-phosphate. The rise of activity in the light and in the dark after α2 antagonists can be blocked by administering cycloheximide, suggesting that protein synthesis is needed for the response. We speculate that epinephrine released in the dark from a subpopulation of retinal amacrine cells onto α2 receptors suppresses AAAD activity that is associated with dopaminergic amacrines.

Notes: Abstract: 5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50= 200 ± 40 nM) and human (IC50= 190 ± 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50= 1,200 ± 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 ± 0.04 in rat, 0.69 ± 0.08 in human, and 0.96 ± 0.02 in bovine cortex. Scatchard analysis of (±)-(3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 ± 0.04 nM and a Bmax of 1.5 ± 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.

Notes: Abstract: The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5′N-[3H]ethylcarboxyamidoadenosine ([3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.

Notes: Abstract: N-Methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N-methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic-L-amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of ∼7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 ± 25 μM and 0.29 ± 0.06 pmol/min/mg protein, and 463 ± 43 μM and 0.16 ± 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.

Notes: Books review in this article: The Biology of Taurine: Methods and Mechanisms edited by R. J. Huxtable, F. Franconi, and A. Giotti Synaptic Transmitters and Receptors edited by Stanislav Tuček

Notes: Abstract: The activation of kainic acid and quisqualic acid receptors in cultured cerebellar granule cells stimulated the release of preaccumulated d-[3H]aspartate. The effect of kainate could be distinguished from that of quisqualate by its sensitivity to the antagonists kynurenic acid and 2,3-cis-piperidine dicarboxylic acid. At a concentration of kainic acid (50 μM) close to its half-maximal releasing effect, simultaneous addition of quisqualic acid (10–50 μM) resulted in a significant dose-dependent inhibition of the kainate-induced component of d-[3H]aspartate release, which was monitored by the progressive decrease in sensitivity of the evoked release to kynurenic acid. In contrast, when kainic acid was used at a subeffective concentration (10 μM), addition of low doses of quisqualate (2–5 μM) resulted in a synergistic effect on d-[3H]aspartate release. Under these conditions, the effect of the two agonists was sensitive to kynurenic acid. Kainic acid (50–100 μM) also caused a dose-dependent, kynurenic acid-sensitive accumulation of cyclic GMP (cGMP) in granule cell cultures. Quisqualic acid was, by itself, ineffective and prevented, in a dose-dependent manner, the kainate-induced cGMP formation (IC50= 5 μM). Finally, the guanylate cyclase activator sodium nitroprusside greatly enhanced cGMP formation but had no effect on d-[3H]aspartate release. Together, these results demonstrate the existence of complex interactions between quisqualic and kainic acids and indicate that the effects of the two glutamate agonists on d-[3H]aspartate release and on cGMP accumulation are independent.

Notes: Abstract: We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N-Methyl-d-aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l-Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l-glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d-[3H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.

Notes: Abstract: To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [γ-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti–B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was 〉95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4β-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4α-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.

Notes: Abstract: Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B-50. To investigate whether B-50 mediates the actions of PKC in neurotransmitter release, we have studied B-50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B-50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B-50 was determined. Chemical depolarization (30 μM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B-50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization-induced stimulation of B-50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B-50 may mediate the action of PKC in neurotransmitter release.

Notes: Abstract: The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist-induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5′-deoxy-5′-methylthioadenosine, an inhibitor of S-adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide-derived second messengers, may be important in the mechanism of action of NGF and EGF.

Notes: Abstract: Although peripheral-type benzodiazepine recognition sites have been demonstrated in the brain of various species, the precise identity and function of the peripheral benzodiazepine receptor have not been established yet. In light of the recent demonstration of the mitochondrial localization of this receptor and its potential role in intermediary metabolism, we investigated the relationship between the benzodiazepines and the enzyme pyruvate dehydrogenase (PDH), a component of the mitochondrial membrane. The results obtained in the present study demonstrate a specific interaction between PDH and the ligands for the peripheral-type benzodiazepine receptor, which might account for their effects on cell growth and differentiation.

Notes: Abstract: The solubility and reactivity of the Folch-Pi proteolipid from bovine CNS have been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate, isooctane, and water. Such a membrane-mimetic system resembles the aqueous spaces of the native myelin sheath in terms of its physicochemical properties. Although the proteolipid is completely insoluble in water, it can be inserted into the water-containing micellar system. In contrast, the lipid-depleted protein failed to be incorporated into these organized assemblies. The lipid requirements for insertion of the proteolipid were studied, therefore, after delipidation by several precipitations with isooctane, a nondenaturing solvent. Novel extraction procedures and quantitative analyses by HPLC of the protein-bound lipids revealed the persistence of a lipidprotein complex (6 ± 1 mol of lipid/mol of protein) displaying optimal micellar solubilization. Competition experiments carried out with brain lipids provide evidence for a preference of the myelin protein for sulfatide, phosphatidylinositol, and phosphatidylserine, in that order. The resulting proteolipid, although differing in relative composition, showed good solubility in the membrane-mimetic system. In contrast, reconstitution experiments carried out with the lipid-depleted protein resulted in weak lipid binding and poor micellar incorporation. These results suggest that the tightly bound acidic lipids may stabilize a protein conformation required for insertion into the micellar system.

Notes: Abstract: Synaptosomes exposed to anoxic insult produce lactate at a slow rate (measured over 60 min). No measurable damaging effects were produced by prolonged depolarisation, anoxic insult, or exogenous lactate (2–32 mM) either on the synaptic plasma membrane (as judged by release of lactate dehydrogenase and soluble proteins), or on synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Potassium-stimulated acetylcholine release was decreased by incubation in the presence of lactate (2–32 mM), as was potassium- and veratrine-stimulated calcium uptake and the calcium content of depolarised synaptosomes. The intrasynaptosomal pH was also reduced but there was no stimulation of oxygen radical production (as judged by H2O2 generation) by exogenous lactate. The role that lactic acidosis may play in giving rise to the altered calcium homeostasis and decreased acetylcholine release from synaptosomes exposed to anoxic insult is discussed.

Notes: Abstract: The present study was designed to investigate the direct response of fetal adrenomedullary cells to hypoxia, and the possible change in this responsiveness with maturation. Ovine fetal adrenomedullary cells, when exposed to 30 min of hypoxia induced by perfusing with Krebs–Henseleit solution equilibrated with 1% O2, released significantly greater amounts of total catecholamine into the perfusate, compared to basal conditions. After a 1-h control period, a second 30-min hypoxic episode stimulated a catecholamine response which was significantly smaller in magnitude than the first. Following the two hypoxic episodes, the cells were capable of responding to 50 mM KCl with a large increase in total catecholamine release. During the first hypoxic episode, the release of both norepinephrine and epinephrine was stimulated by equal magnitude. Fetal adrenomedullary cells obtained from fetuses at 100, 120, and 130 days gestation showed similar responsiveness to the same hypoxic stimulus, and these responses were not different from that observed in maternal adrenomedullary cells. On the contrary, responsiveness to KCl-induced depolarization was greatest in cells obtained from fetuses at 130 days gestation when compared to that in the younger fetuses. This increased responsiveness to KCl was accompanied by a greater catecholamine store in the adrenal medulla of the fetuses at this gestational age. These results suggest that ovine fetal adrenomedullary cells can respond directly to hypoxia by releasing catecholamines. This direct responsiveness became desensitized after repeated exposure. Finally, a decrease in direct responsiveness to hypoxia associated with maturation could not be demonstrated.

Notes: Abstract: The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4–56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7–32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7–18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7–32 days), α-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.

Notes: Abstract: The Torpedo californica electric organ synaptic vesicle glycoprotein ATPase was solubilized with octaethyleneglycoldodecyl ether and stabilized with phosphatidylserine. The complex was analyzed by size exclusion chromatography and band sedimentation velocity ultracentrifugation in water/glycerol and deuterium oxide/glycerol density gradients. The complex was found to have a Stokes' radius of 79 ± 0.7 Å, a sedimentation velocity coefficient at 20±C in water of 6.8 ± 0.2S, a partial specific volume of 0.81 ± 0.01 cm3/g, and a frictional coefficient of 1.6. The molecular weight of the solubilized complex was calculated to be 320,000 ± 7,000 and that of the protein 210,000 ± 9,000. The relationship of this latter value to the major transport ATPase types is discussed.

Notes: Abstract: In order to explore the pathogenetic mechanism underlying the changes in blood-brain barrier sodium transport in experimental diabetes, the effects of hyperglycemia and of hypoinsulinemia were studied in nondiabetic rats. In untreated diabetes, the neocortical blood-brain barrier permeability for sodium decreased by 20% (5.6 ± 0.7 versus 7.0 ± 0.8 × 105 ml/g/s) as compared to controls. Intravenous infusion of 50% glucose for 2 h was associated with a decrease in the blood-brain barrier permeability to sodium (5.4 ± 1.2 × 105 ml/g/s), whereas rats treated with an inhibitor of insulinsecretion (SMS 201–995, a somatostatin-analogue) had normal sodium permeability (7.3 ± 2.0 × 105 ml/g/s). Acute insulin treatment of diabetic rats normalized the sodium permeability within a few hours as compared to a separate control group (7.7 ± 1.1 versus 6.9 ± 1.4 × 105 ml/g/s). To elucidate whether the abnormal blood-brain barrier passage is caused by a metabolic effect of glucose or by the concomitant hyperosmolality, rats were made hyperosmolar by intravenous injection of 50% mannitol. Although not statistically significant, blood-brain barrier sodium permeability increased in hyperosmolar rats as compared to the control rats (8.3 ± 1.0 and 7.0 ± 1.9 × 105 ml/g/s, respectively). It is concluded that either hyperglycemia per se or a glucose metabolite is responsible for the blood-brain barrier abnormality which occurs in diabetes. Further, we suggest that the specific decrease of sodium permeability could be the result of glucose-mediated inhibition of the Na+K+-ATPase localized at the blood-brain barrier.

Notes: Abstract: Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4.5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.

Notes: Abstract: The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.

Notes: Abstract: [3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8–12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1,3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50= 0.7–0.9 mM), N-ethylmaleimide, 5,5′-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.

Notes: Abstract: The release of endogenous glutamate from guineapig cerebrocortical synaptosomes evoked by dendrotoxin, β-bungarotoxin, and 4-aminopyridine is compared. Dendrotoxin and 4-aminopyridine cause Ca2+-dependent release, representing a partial depletion of the KCl-releasable transmitter pool. The decrease in the plasma membrane potential caused by 4-aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by β-bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of β-bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of β-bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.

Notes: Abstract: Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15°C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15°C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

Notes: Abstract: Protein kinase C (PKC), a calcium- and phospholipid-dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three-column procedure of diethylaminoethyl-cellulose, phenyl-Sepharose, and protamine-agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol-12,13-dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a β-type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78-kilodalton form of PKC. Micropunches (300 μm in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS–10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I-protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 ± 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 ± 0.02 and 0.085 ± 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.

Notes: Abstract: The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 × 106/dish) contained 100.4 ± 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 ± 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99–126) or ANP(1–126). These results indicate that ANPs are stored as ANP(99–126) and ANP(1–126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.

Notes: Abstract: Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22°C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22°C was 10 min; the dissociation rate constant of 1.1 × 10−2 s−1 was unaffected by a high concentration of unlabeled insulin (16.7 μM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka= 4.4 × 108M−1 and lower affinity Ka= 7.4 × 106M−1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 μM) for 2 h at 37°C increased [2-14C]uridine incorporation into nucleic acid by 62 ± 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 ± 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 ± 4% (n = 3) above basal]. Dipyridamole (50 μM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.

Notes: Abstract: The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1-14C]arachidonic acid into synapto-somal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration-dependent manner, with 50% inhibition occurring at less than 25 μM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non-competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.

Notes: Abstract: The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with l-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1 -receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with ki values of 1–6 × 10−8M. 11-[{2-t(Diethylamino)methyl]-1-piperidinyl}acetyl] -5,11 - dihydro - 6H -pyrido[2,3 - b][1,4]benzo - diazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of ∼2 × 10−7M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H-labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.

Notes: Abstract: Evidence was obtained for the release of amino acids by electrical stimulation of slices of regions of the rat medulla oblongata: rostral ventrolateral, caudal ventrolateral, and caudal dorsomedial. There was a Ca2+-dependent, tetrodotoxin-sensitive increase in the efflux of aspartate, glutamate, γ-aminobutyric acid (GABA), glycine, and β-alanine in all regions examined. There were distinct regional differences in the relative amounts of amino acids released. These results provide evidence for the possible neurotransmitter role of aspartate, glutamate, GABA, glycine, and β-alanine in these regions of the rat medulla oblongata.

Notes: Abstract: Staurosporine, which has a structure similar to that of K-252a, a potent protein kinase inhibitor that blocks nerve growth factor (NGF) action in PC12 and PC12h cells, is also known as a potent inhibitor of several protein kinases. This study shows that in PC12h cells staurosporine has a dual action: at lower concentrations than that required by K-252a, it is an inhibitor of NGF induction of neurite formation and of changes in the phosphorylation of specific proteins, whereasat concentrations higher than that required to inhibit NGF-induced neurite outgrowth, it rapidly enhances outgrowth by itself.

Notes: Abstract: The effect of induction of adrenal tyrosine hydroxylase (TH) by various centrally acting drugs on catecholamine levels in adrenal and plasma was investigated in rats. All the drugs tested, namely oxotremorine, Piribedil, B-HT 920, and HA-966, produced significant increases in adrenal dopamine content and plasma epinephrine level. Denervation of the adrenal abolished the increase in adrenal dopamine as it did the induction of tyrosine hydroxylase. The results suggest that the induced increase of adrenal TH activity, as mediated by certain drugs, results in an elevation of the plasma epinephrine level and that the adrenal dopamine content is a better indicator of the catecholamine-synthesizing capacity of the adrenal medulla than are the other catecholamines.

Notes: Abstract: We obtained evidence that amiloride specifically potentiates 125I-labeled α-rat atrial natriuretic peptide (1-28) [atrial natriuretic peptide (ANP)-(99-126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/ mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10−4M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10−7M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCI in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride-sensitive Na+ transport. Thus, the possibility that ANPs control amiloride-sensitive Na4+ transport at the blood-brain barrier by interacting with specific receptors has to be considered

Notes: Abstract: A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37°C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fi-bronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced, (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 ± 10−9M, depending on ihe collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo

Notes: Abstract: The immunoreactivity of atrial natriuretic factor (ANF) was studied in the rat olfactory mucosa (OM). Endogenous immunoreactive ANF (IR-ANF) was purified from OM using Vycor glass beads for extraction and reverse-phase HPLC: two of three IR-ANF peaks, identified by retention time, were identical to both the circulating form of ANF (Ser99-Tyr126) and the ANF pro-hormone (Asn1-Tyr126). A radioreceptor assay, employing rat renal glomerular membranes, revealed that endogenous IR-ANF competed with radiolabeled ANF. IR-ANF was localized by immunocyto-chemistry in secretory cells of Bowman's gland and in some cells of the epithelial layer. The relatively low concentration of IR-ANF in the OM (2.5 ng/mg protein) suggests a local role of ANF in this tissue. This hypothesis is supported by the presence in OM of ANF-binding sites, characterized by a KD of 95 pM and a Bmax of 130 fmol/mg protein. We propose that ANF could be released from the OM and act throughout in a paracrine (if not autocrine) manner on some yet-unidentified targets containing ANF-binding sites.

Notes: Abstract: The effects of excitatory amino acid agonists and α-amino-ω-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, l-glutamate, l-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, l-homocys-teate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by l-2-amino-4-phosphonobutyrate (10−3M), but this antagonist had no effect on l-glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazoIepropionic acid, or kainate. At 10−3M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited l-glutamate stimulation, and had no effect on α - amino -3- hydroxy -5- methyl -4- isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobuty-rate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphono-propionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.

Notes: Abstract: The ATP-dependent uptake of l-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the pep-tide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested; the IC5o was de-termined to be 22 μM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate trans-locator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.

Notes: Abstract: An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cho-linergic-specific immune lysis of mammalian brain synap-tosomes and recognizes a group of minor gangliosides in mammalian brain. These minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyl transferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides

Notes: Abstract: The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KC1 at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 ± 13 n M above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 ± 0.35 nmol/ mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 ± 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl. and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin. Addition of 30 mM KCl to 4-ami-nopyridine-pretreated synaptosomes caused a large transient spike in [Ca2+]c and further release of glutamate. 4-Aminopyridine failed to increase [Ca2+]c in low-Na+ media or after addition of low concentrations of veratridine. It is proposed that an unstable membrane potential in the presence of 4-aminopyridine is amplified by repetitive firing of Na+ channels and that this leads to random opening of transient Ca2+ channels in the synaptosomal population. In contrast, KCl would induce a synchronous activation of transient Ca2+ channels followed by a plateau of low residual channel conductance. By mimicking repetitive stimulation in vivo, 4-aminopyridine may more closely model physiological excitation than does an elevated KCl level. Our results are difficult to reconcile with a major role for Na+/Ca2+ exchange in the elevation of [Ca2+]c and contradict proposals that 4-aminopyridine induces release of transmitter from isolated nerve terminals by a mechanism not linked to Na+-channel firing orCa2+ entry

Notes: Abstract: Biogenic amines in bovine retina have been identified and quantified by an extraction-derivatisation procedure involving their reaction with 3,5-di(trifluoromethyl)benzoyl chloride (DTFMBCl) in the aqueous phase followed by extraction into an organic solvent, hydrolysis of phenolic esters, and conversion of free hydroxyl groups to trimethylsilyl ethers. Subsequent analysis of these DTFMB-trimethylsilyl derivatives by gas chromatography-negative ion chemical ionisation mass spectrometry revealed that the molecular ion carried most (〉60%) of the ion current, which made the method highly specific and gave a potential limit of detection below the picogram level. This method establishes unequivocally that the principal amines in bovine retina are p-tyra-mine, dopamine, and 5-hydroxytryptamine

Notes: Abstract: The technique of estimating γ-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats. but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/ kg i.p., 5–30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation

Notes: Abstract: α1-Adrenergic receptor subtypes were differentiated by their affinities for the competitive antagonist WB 4101 and their sensitivities to inactivation by chlorethylclonidine (CEC) in eight rat brain regions. WB 4101 showed low Hill coefficients for inhibition of specific 125I-[2-β-(4-hydroxy-phenyl)ethylaminomethyl]tetralone (125IBE) binding in all regions. Nonlinear regression analysis showed that there were two binding sites with different affinities for WB 4101 in each region. The proportions of these sites varied among regions, although the affinity of WB 4101 for each site remained constant. Thalamus and cerebral cortex had the highest proportion of low-affinity sites, whereas hippocampus and pons-medulla had the highest proportion of high-affinity sites. Pre-treatment with CEC in hypotonic buffer significantly reduced the density of 125IBE binding sites in all brain regions. Cerebral cortex and cerebellum had the highest proportion of CEC-sensitive sites, whereas hippocampus and spinal cord had the highest proportion of CEC-insensitive sites. There was a significant correlation between the proportion of binding sites with a low affinity for WB 4101 and those sensitive to inactivation by CEC

Notes: Abstract: The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with β,β′-iminodipropionitriIe (IDPN). A maturational increase in the relative binding of phosphor-ylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phos-phorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal tran; port of NF proteins

Notes: Abstract: Adenosine formation and release were studied in 48-h-old cultured ciliary- ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxygIucose and 2 μg/ml oli-gomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15–40% of the fall in ATP. Dilazep (3 × 10−6M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80–90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of α,β-methylene ADP that inhibited ecto-5′-nucleotidase by 80–90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto5′-nucleotidase was excluded.

Notes: Abstract: Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24–96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.

Notes: Abstract: The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108-15 cells preincubated with myo-[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15-60 min) of incubation in the cells. PGD2 and PGF2adid not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP1) formation at a late phase of incubation. The maximum stimulation was obtained at 〈10−7M concentrations of these PGs. the levels being three-and twofold for IP2 and IP1 respectively. 9α, 11 β-PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2α have no effect up to 10−6M. The effects of PGD2 and PGF2α were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation.Inositol 1-monophosphate was mainly identified in the stimulation by 10−5 M PGD2 and 10−5MPGF2a, whereas both inositol 1-monophosphate and inositol 4-monophosphate were produced in the stimulation by 10−5M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2α and late-phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late-phase effects. These results suggest that PGD2 and PGF2α specifically stimulate hydrolysis of inositol phospholipids.

Notes: Abstract: The neural cell adhesion molecule LI is a phos-phorylated integral membrane glycoprotein that is recovered from adult mouse brain by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, 80, and 50 kilodaltons (LI-200, LI-180, Ll-140, LI-80, and LI-50, respectively). In the present study, we show that two kinase activities are associated with im-munopurified L1: One specifically phosphorylates L1-200 and L1-80 but not L1-180, L1-140, or L1-50. This pattern of hosphorylation corresponds to the one described for L1 after metabolic phosphate incorporation into cultures of cerebellar cells. In both cases, serine is the main amino acid that is labeled by radioactive phosphate. The kinase activity is not activated by Ca2+, calmodulin, phosphatidylserine, diolein, cyclic AMP, or cyclic GMP, a result suggesting that the enzyme is distinct from Ca2+/calmodulin-dependent kinases, from protein kinase C, or from cyclic AMP/cyclic GMP-dependent kinases and may belong to the independent kinase group. The other kinase phosphorylates only casein but not L1, utilizes GTP as well as ATP, and is strongly inhibited by heparin. Because the primary structure of the L1 protein does not contain consensus sequences characteristic for known kinases, we believe that the catalytic activities detectable in immunopurified L1 are due to kinases that are strongly enough associated with L1 to withstand the stringent purification procedures.

Notes: Abstract: Lactate and pH were measured in frontal and temporal cortex, cingulate gyrus, and caudate nucleus in brains from sudden infant death syndrome (SIDS) cases, control infants, and control adults. Both the lactate levels and the pH values were significantly correlated (p 〈 0.001) between the four brain areas, whereas lactate and pH values were significantly correlated within each brain area (p 〈 0.001) with a value of pH 7.2 for zero lactate. The lactate concentration in heart blood was significantly correlated with brain lactate (p 〈 0.001). Adult sudden death cases (heart attacks) had low lactate and high pH values, whereas agonal state cases had high lactate and low pH values. Control infants who had died because of accidents also had low lactate and high pH values, but infants who might have been exposed to hypoxia before death had high lactate and low pH values. SIDS cases fell into two groups: the first, consisting of all victims over 30 weeks of age and about one-half to two-thirds of those aged less than 30 weeks, had low lactate and high pH values; the second group, consisting of about one-third to one-half of those less than 30 weeks old, had high lactate and low pH values. The changes in lactate levels and pH values indicate that the majority of SIDS cases had died suddenly, but that a sizeable minority had been exposed to hypoxia prior to death

Notes: Abstract: A method for the isolation of γ-aminobutyric acid-ergic (GABAergic) and glutamatergic terminals from crustacean muscle was developed, using differential centrifugation and sucrose density gradient centrifugation. Individual fractions were assessed using a variety of markers. One fraction was isolated which showed 40-fold purification of glutamate decarboxylase with a yield of 12%. This fraction was enriched in GABA, glutamate, glutamate dehydrogenase, and 5′-nu-cleotidase, but not in NADPH cytochrome c reductase. This fraction possessed an uptake system for GABA and glutamate with apparent kinetic constants of Km= 50 μM, Vmax= 250 pmol/min/mg of protein and Km - 183 μM, Vmax= 219 pmol/min/mg of protein, respectively. Electron microscopy showed nerve terminal profiles and a heterogeneous population of membrane vesicles. This fraction contained 3.4 nmol ATP/mg of protein which was stable for 30 min at 12°C, and was also able to synthesise ATP from exogenous adenosine. The terminals released labelled GABA and glutamate in a Ca2+-dependent fashion on depolarisation. No release of ATP was detected. It is concluded that viable nerve terminals have been isolated which could be used as model systems for the study of GABAergic and glutamatergic neuro-chemistry.

Notes: Abstract: Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopro-pyl)dimethylammonio]-l-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylami-no)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl2, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethyl-aminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1Abinding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium do-decyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.

Notes: Abstract: The relative expression of the immunoglobulin su-perfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC 12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.

Notes: Abstract: Acute excitotoxicity in embryonic chick retina and the ability of C1− channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of γ-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glu-tamate or aspartate. Concentrations inducing maximal γ-aminobutyric acid release were 50 μM quisqualate, 100 μM kainate, and 100 μM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant γ-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of γ-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of C1− completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the C1−/bicarbonate anion channel blocker 4,4′-di-isothiocyanatostilbene-2,2′-disulfonate at 600 μM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide. which blocks Na+/K+/2C1− cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of C1− is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl−flux may be involved.

Notes: Abstract: The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by ∼25%, whereas enzyme activity was increased in the particulate fractions by ∼20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant jdrugs, because representatives of several other classes of psychotropic drugs—namely, haloperidol, morphine, and diazepam—failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the blinical actions of these treatments.

Notes: Abstract: The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [3H]inpsitol monophosphate but suppressed the accumulation of the putative second messengers [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and f3H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P3showed similar inhibitory effects, which could be prevented by preincubation with myo-inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-midiated neurotransmission in brain.

Notes: Abstract: Tyrosine hydroxylase (TH) is selectively expressed in cat-echolaminergic neurons and in chromaffin cells of the adrenal medulla. Constructs in which 5’flanking sequences of the rat TH gene directed expression of bacterial chloramphenicol acetyltransferase (CAT) were transfected into cell lines and assayed for transient expression of CAT. In most nonexpressing cell lines, CAT levels were 〈 5% of that found in a TH-positive pheochromocytoma line (PC8b). In two lines described here, a rat anterior pituitary (pell line (GH4) and a rat fibroblast line (Fr3T3), CAT expression reached 12 and 20%, respectively, of the PC8b level. Greater than 90% of the PC8b activity was lost when sequences between -212 and - 187 (in relation to the transcriptional initiation site) were deleted. Further deletions that removed the cyclic AMP response element (CRE)|(-45) and the TATA box at -29 reduced transcriptional activity to background in all three lines. These data suggest that 212 nucleotides of the 5’sequence are sufficient for pheochromocytoma expression and that information between —212 and —187, which includes an API site (-206 to —200), is essential for full transcriptional activity. In addition, sites for other protein transcription factors (AP2, POU/Oct, SP1, and CRE) reside between -221 and -38 and are largely conserved between the human and rat gene.

Notes: Abstract: Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and normetane-phrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3-methoxytyramine (3-MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3-MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood-brain barrier, CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug-naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3-MT to define their correlational relationships. For the NE me-tabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806, p 〈 0.001), plasma MHPG and CSF NMN (r= 0.753, p 〈 0.001), and plasma and CSF MHPG (r= 0.776, p〈 0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady-state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3-MT correlating with plasma HVA (r= 0.301, p 〈 0.025). The results for the DA metabolites probably reflect regional differences in steady-state brain dopaminergic metabolism.

Notes: Abstract: Carboxypeptidase E (EC 3.4.17.10) is a carboxy-peptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete ∼73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCI. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCI. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.

Notes: Abstract: An antagonistic effect of calcium on the action of morphine was studied in rat hippocampal slices. The effect of repeated administration of morphine on γ-aminobutyric acid (GABA) release and binding of [3H]nitrendipine, a cal-cium antagonist, was also examined. (1) In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons, disinhibiting them through basket cells. When the calcium concentration was elevated, potentiation of the field potentials by morphine was reduced. Decrease of the calcium concentration, on the other hand, enhanced the potentiating effect of morphine. Following repeated administration of morphine, its enhancing effect on the field potentials in slices was not observed. (2) In hippocampal membrane fractions obtained from rats repeatedly treated with morphine, enhancement of [3H]nitrendipine binding was observed. (3) In hippocampal slice preparations from rats receiving morphine repeatedly, K+ (45 mM)-stim-ulated [3H]GABA efflux was enhanced. The above results indicate that morphine antagonizes calcium, thereby reducing the release of transmitters. Furthermore, increase in calcium channels following repeated treatment of rats with morphine may explain the mechanism underlying development of tolerance.

Notes: Abstract: This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with syn-aptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase. In the present study, we examined the high-affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 μM) increased uptake 1 by approximately 36%. This increase could be mimicked by aden-osine-5′-O-(3-thiotriphosphate) tetralithium salt (ATPγS), an analogue of ATP which can be utilized by protein kinases, and not by 5′-adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP. GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATPγS, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATPγS blocked the activation produced by ATPyS alone. Kinetic analysis showed that ATPγS pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase.

Notes: Abstract: To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degra dation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.

Notes: Abstract: Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 ± 0.33 ng g−1 and 12.45 ± 0.76 ng g−1 (mean ± SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (〈0.1%) were 12.46 ± 0.47 ng g−1 and 7.20 ± 0.37 ng g−1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and sub stance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide. Deamidation was ruled out on the basis of the unequivocal demonstration of both glutamine residues and a methionine amide residue on each metabolite. These data demonstrate the presence of the mammalian tachykinins substance P, neurokinin A, neurokinin B, and neuropeptide K in the bovine retina. The susceptibility of substance P to methionine oxidation has been confirmed by incubation with dispersed retinal cells, and this putative inactivation system may be of physiological relevance within the retina.

Notes: Abstract: The σ-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been sol-ubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective σ-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD= 28 ± 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(l-propyl)piperi-dine {(+)-[3H]3-PPP} (KD= 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a σ-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macro-molecular complex.

Notes: Abstract: A peptide corresponding to position 32-47 in tyrosine hydroxylase was synthesized (TH-16) and polyclonal antibodies against this peptide were raised in rabbits (anti-TH-16). The effects of anti-TH-16 on modulation of tyrosine hydroxylase activity were investigated. Anti-TH-16 enhanced the enzymatic activity in a concentration-dependent manner, and the antigen TH-16 inhibited the stimulatory activity of the antiserum in a concentration-dependent manner. The activated enzyme had a lower Kmapp for the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti-TH-16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of cate-cholaminergic neurons. Anti-TH-16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP-dependent protein kinase. Exposure of the enzyme to anti-TH-16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti-TH-16. The present study demonstrates the utility of anti-TH-16 in investigating the molecular aspects of the enzyme activation.

Notes: Book reviewed in this article: Basic Neurochemistry (Fourth Edition) edited by G. Siegel, B. Agranoff, R. W. Albers, and P. Molinoff. Affinity Labelling and Cloning of Steroid and Thyroid Hormone Receptors edited by H. Gronemeyer.

Notes: Abstract: Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are α-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.

Notes: Abstract: Isolated rat brain capillaries were incubated in the presence of high-density lipoprotein (HDL) containing [stearic acid-14C, (methyI-3H)choline]sphmgomyelim. This double-labeled sphingomyelin was taken up in a concentration-dependent manner. Cerebral capillary-associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hy-drolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of in-corporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of “pulse-chase” experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidylcholine of capillaries.

Notes: Abstract: We studied the hexose transporter prtotein of the frontal and temporal neocortex, hippocampui, putamen, cerebellum, and cerebral microvessels (which constitute the blood-brain barrier) in Alzheimer disease and control subjects by reversible and covalent binding with [3H]cytochalasin B and by immunological reactivity. In Alzheimer disease subjects, we found a marked decrease in the hexose transporter in brain microvessels and in the cerebral neocortex and hippocampus, regions that are most affected in Alzheimer disease, but there were no abnormalities in the putajnen or cerebellum. Hexose transporter reduction in cerebrajl microves-sels of Alzheimer subjects is relatively specific because other enzyme markers of brain endothelium were not significantly altered. The low density of the hexose transporter at the blood-brain barrier and in the cerebral cortex in Alzheimer disease may be related to decreased in vivo measurements of cerebral oxidative metabolism.

Notes: Abstract: The F3-87-8 glycoprotein was isolated from rat brain by immunoaffinity chromatography after biosynthetic labeling by intracerebral administration of [3H]glucosamine, and the oligosaccharide composition of pronase-derived gly-copeptides was determined by sequential lectin affinity chro-matography and alkali treatment. Triantennary complex oli-gosaccharides (65%) and O-glycosidic oligosaccharides (18%) were the predominant types present, accompanied by 7-10% each of biantennary and high-mannose oligosaccharides. Twenty-two percent of the complex oligosaccharides had a fucose residue linked to the proximal (N-acetylgluco|samine of the chitobiose units. No poly(N-acetyllactosaminyl) or hybrid oligosaccharides were detected. Immunocytochemical studies on the localization of this glycoprotein in developing rat brain demonstrated that in 1-week postnatal cerebellum, there is light staining of the internal granule cell layer and surrounding the Purkinje cells. By 2 weeks, an intense Staining of myelinating fiber tracts appears, accompanied by much lighter staining in the granule cell layer and at the base of the molecular layer. Staining of the white matter remains strong at 3 weeks postnatal, together with significant staining throughout the molecular layer, and then decreases in both areas by 1 month. In adult brain there is relatively uniform staining of approximately equal intensity in the white matter. granule cell layer, and molecular layer, whereas the Purkinje cell bodies appear unstained throughout development. In agreement with a previously reported immunochemical analysis, no staining was seen in other tissues, confirming the CNS-specific localization of this glycoprotein.

Notes: Abstract: The levels of the α-subunits of two GTP-binding proteins, Go and Gi2, were determined in neural and non-neural cloned cells by immunoassays. Goα was detected in all neural cells and some of nonneural cells, but not in HL-60 leukemic cells and PYS-2 teratocarcinoma-derived cells. The level of Goα was highest in the PC12 pheochroihocytoma cells. Gi2α was present in all types of cells tested, arid its level was highest in the HL-60 cells and relatively high in glioma ells. Treatment of PC 12 cells and neuroblastoma X glioma hybrid NG108-15 cells with nerve growth factor and forskolin, respectively, caused the extension of neuronal-like processes and increase in the level of Goα by 60-80%, but small changes in the levels of Gi2α.

Notes: Abstract: Bradykinin, which activates polymodal nociceptors, increased cyclic GMP (cGMP) in a capsaiciiji-sensitive population of cultured sensory neurones from rat dorsal root ganglia (DRG) by stimulating guanylate cyclase, but had no effect on cyclic AMP (cAMP). In nonneunbnal cells from DRG, bradykinin increased cAMP, but not cpMP. The bra-dykinin-induced increase in cGMP in thej neurones was completely blocked by removal of extracellular Ca2+, or by incubation of the cells with the calcium channel blockers nifedipine and verapamil. Pretreatment of the| neurones with either dibutyryl cGMP or sodium nitroprusside (which elevates cGMP) inhibited bradykinin-induced formation of inositol phosphates. It is possible that cGMP could be involved in the regulation of polyphosphoinositide turnover in DRG neurones.

Notes: Abstract: Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of 〈 1 μM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to 〈1 μM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.

Notes: Abstract: The antagonistic effects of gallamine on muscarinic receptor-linked responses were investigated in N1E-115 neuroblastoma cells. M1 muscarinic receptor-mediated phos-phoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a Ki value of 33 μM. By comparison, gallamine was four- to fivefold less potent in blocking noncardiac M2 muscarinic receptor-mediated inhibition of cyclic AMP formation, with a Ki value of 144 μM. The resulting Arunlakshana-Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine's inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N-methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose-response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N-methyl-3H]scopolamime methyl chloride ([3H]NMS) or [N-methyl-3H]quinuclidinyl benzilate methyl chloride ([3H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor-bound [3H]NMS 〉 [3HJNMeQNB in a dose-dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine-mediated responses in N1E-115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS.

Notes: Abstract: α-Bungarotoxin (α-BGT), a snake venom polypeptide, interacts potently and specifically with a nicotinic receptor population in neuronal tissue. However, the identity of this site is unclear, because, unlike at the neuromuscular junction and in electroplax, in nervous tissue the toxin does not block nicotinic cholinergic responses. Therefore, we sought endogenous compounds other than acetylcholine that could interact with the neuronal α-BGT site. In the present experiments, thymopoietin, a polypeptide isolated from the thymus, is shown to inhibit potently α-BGT binding to brain membranes in a dose-dependent manner (IC50= 3.1 nM). This effect was not shared by a wide variety of other peptides, including thysplenin, a closely related polypeptide. Thymopoietin did not inhibit the binding of other radioligands known to interact with different populations of cholinergic receptors, such as [3H]nicotine and [3H]methylcarbachol, which bind to nicotinic receptors, or [3H]quinuclidinylbenzilate, which binds to muscarinic receptors. These results show that thymopoietin potently and specifically affects l25I-α-BGT binding to brain membranes and suggest that thymopoietin might be an endogenous ligand for α-BGT receptors in neuronal tissue.

Notes: A lysosomal preparation, obtained from brain ho-mogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40–120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GDla and fluorimetrically with 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma Membrane-bound enzyme. The apparent Km values for GDl a and MUB-NeuAc were 1.5 × 10-5 and 4.2 × 10-5M, respectively, for the lysosomal enzyme and 2.7 × 10-4 and 6.3 × 10-5M for the plasma membrane-bound one. Triton ×- 100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers. It is thus evident that the sialidase present in the lysosomal fraction is an authentic lysosomal enzyme distinct and different from the sialidase contained in the plasma membrane. The lysosomal sialidase affected other ganglio-sides, like GDlb and GM3. These data constitute the first direct evidence for the presence in brain lysosomes of a sialidase activity on gangliosides and contribute to a better knowledge of ganglioside breakdown and turnover in the brain.

Notes: The novel N-methyl-D-aspartate recjeptor channel ligand (+)-[3H]5-methyl-J0, l1-dihydro-5H-dibenzo[a, d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissiie. Characteristics of [3H]MK-801 binding to well-washeq membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of the and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding Was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.

Notes: In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immuno-histochemical staining with TH antibody and immunoblot-ting analysis. Several clones of the C6 transfectahts that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellulary due to the ajbsence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.

Notes: γ-Aminobutyric acidB (GABAB) receptor recognition sites that inhibit cyclic AMP formation, open potassium channels, and close calcium channels are coupled to these effector systems by guanine nucleotide binding proteins (G proteins). These G proteins are ADP-ribosylated by islet-activating protein (IAP), also known as pertussis tokin. This process prevents receptor coupling to these G proteins. In slices of cerebral cortex and hippocampus from rat, stimulation of GABAB receptors with baclofen, a receptor agonist, also potentiates the accumulation of cyclic AMP stimulated by β-adrenergic agonists. It was unknown whether those GA-BAB receptors that potentiate the β-adrenergic response were also sensitive to IAP. IAP was injected intracerebroventric-ularly into rats to ADP-ribosylate IAP-sensitive G proteins. Four days after the IAP injection, 38% and 52% of these G proteins from cerebral cortex and hippocampus, respectively, were ADP-ribosylated by the IAP injection. In slices of both structures prepared from IAP-treated rats, the GABAB receptor-mediated potentiation of the β-adrenergic receptor response was attenuated. Thus, many GABAB receptor-mediated responses are coupled to IAP-sensitive G proteins.

Notes: Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were Very low at birth. Excluding the cerebellum, the levels in these Regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions. These results suggest that (a) CCK gene expression, numbers of CCK neurons, or both may increase from 7 to 14 days of age and (b) differential regulation of CCK gene expression and posttranscriptional regulatory mechanisms may exist in the various brain regions during development.

Notes: K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC 12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factoi (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10–30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added tc the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at ±50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5′-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki± 500 μM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki= 50 μM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.

Notes: The regulation of the central σr-binding site was investigated using both in vitro and in vivo manipulations in conjunction with radioligand binding. The displacement of the binding of R(+)-[3H]3-[3-hydroxyphenyl][N-(l-pro-pyl)piperidine (R(+)-{3H]3-PPP} to cortical homogenates by a range of drugs was consistent with the site labelled being a σ-receptor. (+)-SKF 10,047, (-)-SKF 10,047, (±)-cyclazo-cine, phencyclidine, and dexoxadrol displaced R(+)-[3H]3-PPP with pseudo-Hill coefficients of less than . Further analysis employing nonlinear curve fitting techniques demonstrated that displacement data for these compounds were described better by a model whereby R(+)-[3H]3(-PPP was displaced from two discrete sites; approximately 6[5% of the total sites were in the high-affinity state. In the presence of 10 mM Mg2+ and 0.3 mM GTP, displacement curves for (+)-SKF 10,047 and (±)-cyclazocine were shifted to the right. These findings were due to the shift of some 15% of the high-affinity binding sites to a low-affinity state. Saturation experiments revealed that 0.3 mM GTP acted competitively to decrease the affinity of R(+)-[3H]3-PPP for the σ suites. The σ-binding site was thus likely to be linked to a guanine nucleotide regulatory (G) protein. Thus σ drugs could be subdivided on the basis of their GTP sensitivity and psdudo-Hill coefficients, and by analogy with other receptors R(+)-3-PPP. (+)-SKF 10,047, and (±)-cyclazocine, may be putative σ-agonists. l,3-Di(2-tolyl)guanidine (DTG), rimcazole, and haloperidol displaced R(+)-[3H]3-PPP with pseudo-Hill coefficients of approximately unity and thus may be σ-antagonists. Subchronic treatment with rimcazole was characterized by slight sedation and a concomitant up-regulation, with a decrease in the affinity, of σr-binding sites. The schedule of rimcazole also increased dopamine turnover in the nucleus ac-cumbens; both the concentration of 3,4-dihydroxyphenyl-acetic acid (DOPAC) and the DOPAC/dopamine ratio were elevated. DTG produced similar alterations to the binding parameters of the σ-binding site; however, changes were not observed in general behavior or accumbal dopamine turnover. (T-Receptors are likely to be linked to a G protein and are functionally involved in the CNS.

Notes: Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-indepen-dent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon auto-phosphorylation of both subunits of the enzynme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 ± 5% of the total activity within 1–2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophos-phorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the α- and β-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281–309 corresponding to a portion of the regulatory domain (residues 281–314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50= 3.6 ± 0.8 μM). Binding of Ca2+/CaM to peptide 281–309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281–309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.

Notes: Abstract: Isolated rat pineal glands were incubated in vitro in a medium containing [I4C]dopamine or [14C]tyrosine, and the tissue contents of l4C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [l4C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [l4C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [l4C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine β-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [l4C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [l4C]dopamine enhanced the accumulation of [l4C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [l4C]dopamine during incubation with [l4C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine β-hydroxylase, the two enzymes required for the synthesis of noradrenaline. The capacity of the pineal gland to synthesize dopamine and noradrenaline is enhanced during the night.

Notes: Abstract: Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine sej-um albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/0/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Pread-sorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.

Notes: Abstract: The brain uptake index (BUI) of L-jtryptophan and diazepam into the right and left hemispheres and the cerebellum has been measured after a bolus injection into the carotid artery of the anaesthetised rat. The effect of a range of albumin concentrations (38 μM to 1.4 mM; 0.25-9 g/100 ml) on the viscosity and osmotic pressure ot the bolus was studied as a preliminary to the brain uptake experiments. Dextran (Mr 60,000-90,000) was included in the injection to ensure constant viscosity and osmotic pressure. An increase in albumin concentration up to 2 g/100 ml substantially reduced the BUI of L-tryptophan, but a further increase in albumin concentration up to 9 g/100 ml respited in only a slow fall in the BUI of L-tryptophan which was not proportional to the larger fall in the concentration of unbound L-tryptophan. Furthermore, the use of norharmane as an inhibitor of L-tryptophan binding did not reveal a simple relationship between its unbound concentration and BUI. A decrease in the unbound concentration of diazepam also reduced its BUI, but again there was no straightforward relationship between this and unbound diazepam concentration. The differences observed in the BUI of inulin from solutions of either dextran or albumin indicate not only that each mac-romolecule may exert particular effects on the BUI, perhaps by an influence on cerebral blood flow, but also show how difficult it is to devise solutions for injection which differ in respect of only one variable, that of the unbound ligand concentration.

Notes: Abstract: A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5′-di-phosphoethanolamine at concentrations above 100 μM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.