Library

Notes: Abstract: To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi·mmol−1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27–39 pmol·g−1·min−1 and 2.6–4.8 μM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol·g−1· min−1, thus representing a significant component of thiamine supply to nervous tissue.

Notes: Abstract: The binding of [3H]neurotensin(8–13) to membranes from human frontal cortex at 0°C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant (KD) of 0.52 nM, and the maximal number of binding sites (B max) was 3.5 pmol/g original wet weight of tissue. Scat-chard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [3H]-neurotensin(8–13) bound to single, noncooperative sites. The KD values of several analogs of neurotensin determined in competition with [3H]neurotensin(8–13) were similar to those previously determined in competition with [3H]-neurotensin. The regional distribution of binding sites for [3H]neurotensin(8–13) was also similar to that for [3H]-neurotensin. These results suggest that [3H]neurotensin(8–13) binds to the same sites as [3H]neurotensin and that [3H]neurotensin(8–13) has a higher affinity than [3H]-neurotensin for these sites in human brain.

Notes: Abstract: A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C-terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.

Notes: Abstract: The calcium-dependent, energy-independent incorporations of 14C-labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+-ATPase, a plasma membrane marker. This strongly suggests that the base-exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 ± 0.09, 1.60 ± 0.17, and 0.56 ± 0.06 nmol/mg of protein/h; the respective Km(apparent) values were 241 ± 29, 65 ± 18, and 77 ± 22 μM. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 ± 8, 78 ± 28, and 639 ± 106 nmol/g, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids. Moreover, that endogenous choline levels are well below those needed for enzyme saturation suggests that the incorooration of choline into phosphatidylcholine via this pathway may be sensitive to transitory increases in free choline concentration (such as occur intrasynaptically after acetyl-choline is released and hydrolyzed). Various polyvalent cations (Mg2+, Ba2+, Ni2+, Co2+, and La3+) inhibited synaptosomal base-exchange activity with a rank order of potency similar to their ability to block calcium channels.

Notes: Abstract: The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100. 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.

Notes: Abstract: It has been found previously that the ratio of aspartate to glutamate released and retained by brain slices reversibly changes with changing glucose concentrations in the medium. To find out whether increased neuronal activity also results in changes in the ratio of aspartate to glutamate, in this study electrical-field stimulation was applied for 10 min to hippocampal slices in the presence of 0.2–5 mM glucose. In 5 mM glucose, the ratio of aspartate to glutamate released did not change during stimulation, but the amount of aspartate retained at the end of stimulation was reduced. In contrast, in 1 mM or less glucose, the ratio of aspartate to glutamate released increased progressively and the rate of increase was inversely proportional to the glucose content of the medium. The evoked release of aspartate and glutamate both in low and high glucose was nearly suppressed in low (0.1 mM) Ca2+ or by tetrodotoxin. In low glucose, the ratio of aspartate to glutamate contained in the slices also increased as a result of stimulation. This increase was reduced only a little in low Ca2+, but was nearly eliminated by tetrodotoxin. Results suggest that increased neuronal activity causes a shift in the ratio of aspartate to glutamate released in the presence of glucose concentrations similar to those found in the brain in normoglycemic rats. This shift, due to an increased energy demand, probably originates from terminals which release aspartate and glutamate in different proportions.

Notes: Abstract: The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 × 10−5M for ATP and 10−6M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1. and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.

Notes: Abstract: We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 μg of N-acetylneuraminic acid/ mg of protein, respectively), owing to the low protein-to-lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 μg, of N-acetylneuraminic acid/ mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled-pore glass-bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N-acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.

Notes: Book reviewed in this article: Synaptic Function edited by G. M. Edelman, W. E. Gall, and W. M. Cowan. A Neuroscience Institute Publication.

Notes: Abstract: A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.

Notes: Abstract: Previous studies indicated that DL-buthionine sulfoximine (dl-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of l-BSO with marked declines (79.6–86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8–29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.

Notes: Abstract: The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence represents residues (72–92) of γ-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-γ, lacks residues (3–17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of β- and γ-preprotachykinins in the rabbit gut are different.

Notes: Abstract: Nutritional iron deficiency induced in rats causes a significant reduction in level of brain nonheme iron and is accompanied by selective reduction of dopamine D2 receptor Bmax. Our previous studies have clearly demonstrated that these alterations can be restored to normal by supplementation with ferrous sulfate; however, neither brain nonheme iron level nor dopamine D2 receptor Bmax can be increased beyond control values even after long-term iron therapy. The possibility that iron deficiency can induce the breakdown of the blood-brain barrier (BBB) was examined. A 70 and 100% increase in brain uptake index (BUI) for l-glucose and insulin, respectively, were noted in iron-deficient rats. However, the BUI for valine was decreased by 40%, and those for l-norepinephine and glycine were unchanged. In addition, it was demonstrated that in normal rats insulin is transported into the brain. The data show that iron deficiency selectively affects the integrity of the BBB for insulin, glucose, and valine transport. Whether the effect of iron deficiency on the BBB is at the level of the capillary endothelial cell tight junction is not yet known. However, this study has shown that an important nutritional disorder (iron-deficiency anemia) has a profound effect on the BBB and brain function.

Notes: Abstract: We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2′:3′-cyclic nucleotide 3′-phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 μg of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein-primed SJL/J mice, after selective expansion with antigen and in-terleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.

Notes: Abstract: The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radio-ligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomor-phine gave a dose-dependent inhibition (IC50= 1 μM) of 45Ca2+ uptake stimulated by either nicotine (10 μM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (—)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.

Notes: Abstract: The effect of oral administration of ammonium acetate for 2, 15, 30, and 100 days on protein synthesis in rat brain was investigated. Although protein synthesis changes were modest, i.e., maximal increase of 24%, there was induction of synthesis and accumulation of a protein with an Mr of 55,000. We show, on the basis of its position on two-dimensional electrophoresis and its immunological reactivity, that this protein is tubulin. Its content increased by 33% as determined by isolation of tubulin after 15 days of oral administration of ammonium and to 49% after 100 days as determined by quantitative immunoblotting.

Notes: Abstract: Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3–[3–(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2–(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r= 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5′-guanylylimidodi-phosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (∼60 kilodaltons) and one G protein (∼80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5–HT1A receptors.

Notes: Abstract: The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57–67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.

Notes: Abstract: Polyclonal antibodies against Ca2+/calmodulin-de-pendent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both sub-units of the enzyme with MrS 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neu-ronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cer-ebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as as-trocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.

Notes: Abstract: The effect of the calcium channel blocker nimodipine on the previously described regional cerebral acidosis accompanying thiamine deficiency was investigated. Local cerebral pH (LCpH) and blood flow (LCBF) were separately determined autoradiographically in normal and 16-day thia-mine-deficient rats administered the calcium antagonist drug and compared to appropriate controls. Nimodipine did not modify LCpH in normal brain. In thiamine deficiency, nimodipine significantly raised LCpH in 5 of 17 structures evaluated, two of which, the medial dorsal nucleus of the thalamus and the mammillary body, are vulnerable to the development of histological lesions in this condition. Although the calcium blocker augmented LCBF in normal brain, it had no effect on the hyperperfusion already present by day 16 of thiamine deprivation. Thus, the pH changes we are reporting are probably not related to an effect on cerebral perfusion, but could have resulted from an improved ability of the brain to reduce its proton load in the presence of nimodipine. These results may have wider therapeutic implications than in thiamine deficiency alone.

Notes: Abstract: The effects of nerve growth factor (NGF) on the intracellular content of acetylcholine (ACh) in cultured septal neurons from developing rats have been examined. The content of ACh could be measured by using HPLC and electrochemical detection (HPLC-ECD), coupled with an immobilized enzyme column. This method of determination is very simple and rapid, and is highly sensitive. The content of ACh and the activity of choline acetyltransferase (ChAT) in cultured postnatal day 1 (PI) septal neurons grown on an astroglial “feeder” layer was increased during the period of cultivation by the addition of NGF. The activities of ChAT and the content of ACh increased in a dose-dependent manner in direct relationship to the different amounts of NGF employed. These effects of NGF, i.e., elevating the intracellular content of ACh, accompanied by an increase in activity of ChAT, also were confirmed in the PI septal organotypic cultures. Additionally, embryonic day 17 (E17) septal neurons in a serum-free medium displayed a similar responsiveness to NGF with respect to the elevation in the content of ACh and the increase in activity of ChAT. These results suggest that intracellular levels of ACh are likely to be regulated by NGF in a fashion similar to that of the activity levels of the biosynthetic enzyme.

Notes: Abstract: In the present work, we have studied the effect of ruthenium red (RuR), La3+, and 4-aminopyridine (4-AP) on the specific binding of (+)-[3H]PN200–110 to synaptosomes, as well as the effect of nitrendipine, nifedipine, and BAY K. 8644 on γ-[3H]aminobutyric acid ([3H]GABA) release induced by potassium depolarization and by 4-AP in synaptosomes. Scatchard plots indicated that neither RuR nor 4-AP modifies the KD and Bmax of [3H]PN200–110 specific binding, whereas La3+ decreased the Bmax by about 25%; when the effect of the drugs on the total binding of PN200–110 was studied, a similar inhibition by La3+ was found. The calcium antagonists, nitrendipine and nifedipine, did not affect at all the potassium-stimulated release of [3H]GABA nor its release induced by 4-AP. The calcium agonist BAY K 8644 failed to affect both the spontaneous and the potassium-stimulated GABA release. Our results suggest that the binding sites of dihydropyridines in presynaptic membranes are not related to the calcium channels involved in neurotransmitter release with which RuR, La3+, and 4-AP interact.

Notes: Abstract: The direct effects of chronic ethanol administration on adenylate cyclase, Na,K-ATPase, and Mg-ATPase activities in a cell containing neuronal characteristics were investigated using PC 12 pheochromocytoma cells. Exposure of PC12 cells to0, 75, and 150 mM ethanol for 4 days caused a dose-dependent increase in the stimulation of adenylate cyclase by in vitro ethanol without altering activation of the enzyme by GTP, NaF, MnCl2, or 2-chloroadenosine. Conversely, a 4-day treatment with 150 mM ethanol increased Na,K-ATPase and Mg-ATPase activities without altering the inhibitory effects of in vitro ethanol. The increase in Na,K-ATPase activity was associated with an increase in Vmax without any change in the Km for KC1. Chronic ethanol exposure also increased the amount of [3H]ouabain specifically bound to PC 12 cell membranes. Except for the increase in Mg-ATPase activity, the above results were also observed when chronic ethanol treatment was carried out in the presence of pyrazole. Although ethanol slowed PC 12 cell growth, observed changes were not due to an ethanol-induced reduction in cellular density. A 4-day exposure of a nonneuronal cell line (Madin Darby canine kidney cell) to 150 mM ethanol did not alter adenylate cyclase or ATPase activities. The present study indicates that the direct effects of chronic ethanol exposure of a neuronal-like cell involve an increase in the density of sodium pumps per cell and an enhanced sensitivity of adenylate cyclase to activation by ethanol.

Notes: Abstract: LiCl stimulated the formation of inositol mono-phosphate in PC 12 cells that had been exposed to nerve growth factor (NGF) for 4–5 days. Half-maximal accumulation was observed at ∼8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inos′itol trisphosphate was half-maximal at ∼ 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at ∼0.7 mM and the second half-maximal at −15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from ∼200 to ∼70 μM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.

Notes: Abstract: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an ∼50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for ∼23 days resulted in an ∼80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.

Notes: Abstract: Both ammonia and β-methylene-dl-aspartate (β-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and β-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mMβ-MA resulted in a decreased production of 14CO2 from [U-14Clglucose, but did not affect 14CO2 production from [2–14C] pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2–14C] pyruvate, but 14CO2 production from [U-14C] glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C] glucose or [2–14]pyruvate by neurons. However, incubation of neurons with β-MA or β-MA plus ammonium chloride resulted in a 45% decrease of 14CO2 production from both [U-14C] glucose and [2–14C] pyruvate. A 2-h incubation of astrocytes with β-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in 〉50% decrease in ATP, but had little effect on phosphocreatine. β-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards β-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by ∼45% in astrocytes and by ∼55% in neurons.

Notes: Abstract: Enzyme-linked immunosorbent assays for acetylcholinesterase (AChE) and for butyrylcholinesterase (BuChE) were markedly more specific than conventional assays using selective enzyme inhibitors. The new assays were used with blood and brain samples containing traces of one enzyme dominated by large amounts of the other. The results showed that human plasma does contain AChE (8 ng/ml), even though its major cholinesterase is BuChE (3,300 ng/ml). BuChE immunoreactivity was not detected in human red blood cells but occurred in all brain regions. The cerebellum was the richest region tested (540 ng of BuChE/g of tissue), whereas the cerebral cortex was the poorest (240 ng of BuChE/g). However, because of the small local AChE content (99 ng/g), BuChE was the major cortical cholinesterase. The picture was reversed in the putamen, where BuChE immunoreactivity (340 ng/g) was far outweighed by that of AChE (6,100 ng/g).

Notes: Abstract: The brindled mouse (Mobr/y) carries an X-linked mutation that produces severe copper deficiency. Affected males suffer profound deficits in oxidative metabolism. We have examined astrocyte pathology in Mobr/y during development and have found marked changes in the metabolism of glial fibrillary acidic protein (GFAP). Immunocytochemistry with anti-GFAP antisera revealed a marked increase in staining at postnatal day 12 (P12), compared to heterozygous female and unaffected male littermates, particularly in neocortex and thalamus. Septum, hypothalamus, and striatum showed little change. Western blot analysis revealed increased levels of GFAP in Mobr/y forebrain and cerebellum. Levels of GFAP mRNA were determined by Northern blotting with a mouse GFAP cDNA probe. At P10, mRNA levels were normal, but increased to 8–10 times normal by P12. Levels at P15 remained similarly elevated. Thus, immunostaining and protein determinations correlate with mRNA elevations. Astrocytes can alter GFAP mRNA and protein levels over a relatively short time. Counts of neocortical cells did not reveal differences in cell numbers between Mobr/y and controls, indicating that the observed changes reflect increased cellular levels and not a large increase in the numbers of astrocytes.

Notes: Abstract: Partially purified preparations of GABAa/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABAa/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.

Notes: Abstract: This work was carried out to evaluate the importance of glial cells in providing precursors for the in vivo synthesis of γ-aminobutyric acid (GABA). Fluorocitrate, which selectively inhibits the tricarboxylic acid cycle in glial cells, was administered locally in rat neostriatum. Inhibition of the glial cell tricarboxylic acid cycle led to a decrease both in glutamine level and in γ-vinyl GABA (GVG)-induced GABA accumulation, an observation indicating reduced GABA synthesis. The role of glutamine, which is synthesized in glial cells as a precursor for GABA, was further investigated by inhibition of glutamine synthetase with intrastriatally administered methionine sulfoximine. In this case, the glutamine level was reduced to near zero values, and the GVG-induced GABA accumulation was only half that of normal. The results show that glutamine is an important precursor for GABA synthesis, but it cannot be the sole precursor because it was not possible to depress the GVG-induced GABA accumulation completely.

Notes: Abstract: A method for rapid, automated (〈5 min), and sensitive (detection limit 50 fmol/10 μl) determination of γ-aminobutyric acid (GABA) is described. The method is based on precolumn derivatiza-tion with o-phthaldialdehyde/t-butylthiol reagent and separation by reverse-phase HPLC with electrochemical detection under isocratic conditions. A 100 × 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3-mercaptopro-pionic acid is injected at a dose of 100 mg/kg i.p. There was a 15-fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.

Notes: Abstract: Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an rnRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr= 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6–5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.

Notes: Abstract: A chondroitin sulfate proteoglycan called PGM 1 has been isolated from the particulate fraction of adult rat forebrain. Delipidation of the material, solubilization of proteoglycans in guanidinium chloride, precipitation at low ionic strength, and final extraction at pH 5.0 were used for its isolation. Proteoglycans were subjected to further purification by diethylaminoethyl-cellulose chromatography. Individual components were separated by gel filtration. PGM 1 appeared to be a high-molecular-weight chondroitin sulfate proteoglycan, capable of strong interaction with hy-aluronic acid. It was finally isolated by gel filtration on Ultrogel AcA 22 in the presence of 4 M guanidinium chloride. Monospecific antibodies obtained in rabbits against the purified molecule did not cross-react with other brain proteoglycans. Immunocytochemical techniques revealed an almost unique association of this compound with axons, particularly those known to contain neurofilaments. However, not all these axons and all parts of these axons contained PGM 1. This component was not detectable in liver, intestine, spleen, kidney, lung, heart, skin, hair, lens, and muscle, a finding suggesting a specificity for the nervous tissue. This component is expressed in neural cell cultures. Despite the preservation of the neuronal specificity, it seems to lose its specific axonal localization in vitro.

Notes: Abstract: The B subunit of cholera toxin, which is multiva-lent and binds specifically to GM1 ganglioside on the cell surface, has previously been used as a ganglioside-specific probe to regulate DNA synthesis in thymocytes and fibro-blasts. To explore in more detail this growth-regulatory action of gangliosides, C6 glioma cells (which are GM1 ganglioside deficient) were used as a model system. When cultures of C6 cells were first treated with GM1, followed by exposure to the B subunit, proliferation was inhibited, as measured by 3H-labeled thymidine incorporation into DNA. Pretreatment of the cells with 50 μM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 μ/ml of B subunit for 21 h was sufficient to reduce DNA synthesis to 15% of control values (and confirmed by autoradiographic analysis), although maximal inhibition could be achieved with as little as 30 min exposure to B, followed by washing. Furthermore, the B subunit inhibited the response of the C6 cells to basic fibroblast growth factor only following GM1 pretreatment. The B subunit-induced inhibition of DNA synthesis was specific for the ganglioside GM 1, and was unrelated to increases of cyclic AMP. These results demonstrate that cell-incorporated GM1 ganglioside may act as a receptor capable of undergoing a specific ligand interaction, subsequently affecting molecular processes at the nuclear level.

Notes: Abstract: Intracranial microdialysis was used to measure changes in extracellular amino acids within the rat brain during local osmotic alteration of the extracellular micro-environment or during systemic water intoxication. Increased cellular hydration produced by either of these methods was accompanied by a marked increase in extracellular taurine levels without affecting the other amino acids measured. With local osmotic alteration, this increase was osmolarity dependent and reversible. The specificity, sensitivity, and reversibility of the increase in extracellular taurine strongly suggest a functional role in osmoregulation in the brain under normal as well as pathological conditions.

Notes: Abstract: The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by l-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of l-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergentsoluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Notes: Abstract: Severe hyperthyroidism from the time of birth causes a premature induction and termination of thymidine kinase activity in the cerebella of wild-type mice. This leads to elevated enzyme levels at postnatal days 5 and 6, with significantly lower levels by postnatal day 7 (which is actually the time of peak activity in normal animals). In this study, neonatal hyperthyroidism does not have significant effects on postnatal day 5, 6, or 7 enzyme levels in the neurological mutant staggerer. This is consistent with the hypothesis that thyroid hormone exerts its effects via the Purkinje cells, which are reduced in number and grossly stunted in the mutant.

Notes: Abstract: We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes from adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.

Notes: Abstract: D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 ± 0.027 nM) with a density (Bmax) of 35.7 ± 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 ± 0.007 nMand the density was 25.7 ± 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151–2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/ SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.

Notes: Abstract: Noradrenaline potently antagonizes the effects of N-methyl-D-aspartate (NMDA) (80 μM) on cyclic GMP production in immature rat cerebellar slices in vitro (IC50 = 0.6 μM). The effect is stereospecific (D-noradrenaline, IC50 = 100 μM), and also observed with adrenaline (IC50= 0.5 μM) and isoprenaline (IC50= 1.2 μM). The α1-adrenoceptor agonists methoxamine or phenylephrine or the mixed α/α2 agonists oxymetazoline or xylometazoline (100 μM) do not block the effects of NMDA, but the α2-adrenoceptor agonist clonidine is weakly active (IC50 = 200 μM). Salbutamol and terbutaline were also inactive except at high concentrations (300 μM), as were a number of other catechol and phenylethylamine derivatives. The antagonistic effects of noradrenaline on the NMDA response were insensitive to phentolamine, atenolol, or propranolol (up to 100 μM), but were blocked by the α2 antagonist idazoxan (1–10 μM). The Na+,K+-ATPase inhibitor ouabain (0.1–10 μM) markedly potentiates the effects of NMDA in this model, and also antagonizes and reverses the ability of noradrealine (10 μM) to block the effects of NMDA. The results suggest that noradrenaline and Na+,K+-ATPase activity have potent modulatory effects on the NMDA response.

Notes: Abstract: The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two-to fourfold in the presence of PDB or TPA. PDB at 1–100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the paniculate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the paniculate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the paniculate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.

Notes: Abstract: The primary structure of neuromedin U from the rat ileum was established as: Tyr-Lys-Val-Asn-Glu5-Tyr-Gln-Gly-Pro-Val10-Ala-Pro-Ser-Gly-Gly15-Phe-Phe-Leu-Phe-Arg20-Pro-Arg-Asn-NH2. There was no evidence for microheterogeneity. This amino acid sequence contains two deletions and nine substitutions compared with the neuromedin U-25 from the pig. In particular, the replacement of the Arg'6-Arg17 processing site in the porcine peptide by Gly14-Gly15 in the rat means that a peptide corresponding to neuromedin U-8 was not found in the rat intestine.

Notes: Abstract: Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.

Notes: Abstract: In an experimental model of perinatal hypoxic-ischemic brain injury, we examined quisqualic acid (Quis)-stimulated phosphoinositide (PPI) turnover in hippocampus and striatum. To produce a unilateral forebrain lesion in 7-day-old rat pups, the right carotid artery was ligated and animals were then exposed to moderate hypoxia (8% oxygen) for 2.5 h. Pups were killed 24 h later and Quis-stimulated PPI turnover was assayed in tissue slices obtained from hippocampus and striatum, target regions for hypoxic-ischemic injury. The glutamate agonist Quis (10-4M) preferentially stimulated PPI hydrolysis in injured brain. In hippocampal slices of tissue derived from the right cerebral hemisphere, the addition of Quis stimulated accumulation of inositol phosphates by more than ninefold (1,053 ± 237% of basal, mean ± SEM, n = 9). In contrast, the addition of Quis stimulated accumulation of inositol phosphates by about fivefold in the contralateral hemisphere (588 ± 134%) and by about sixfold in controls (631 ± 177%, p 〈 0.005, comparison of ischemic tissue with control). In striatal tissue, the corresponding values were 801 ± 157%, 474 ± 89%, and 506 ± 115% (p 〈 0.05). In contrast, stimulation of PPI turnover elicited by the cho-linergic agonist carbamoylcholine, (10-4 or 10-2M) was unaffected by hypoxia-ischemia. The results suggest that prior exposure to hypoxia-ischemia enhances coupling of excitatory amino acid receptors to phospholipase C activity. This activation may contribute to the pathogenesis of irreversible brain injury and/or to mechanisms of recovery.

Notes: Abstract: An enzyme-linked immunosorbent assay (ELISA) to determine the level of galactosylceramide (GalC) in biological fluids is described. The assay uses GalC-coated plastic microtiter plates, with binding of an antibody to GalC detected by a peroxidase-labeled second antibody. The GalC level was directly estimated in the biological samples, without prior extraction, by competition with the coated hapten. This method allows the detection of 62 pmol of GalC (1.2 nmol/ml). Results using this procedure revealed positive sera only among patients suffering a myelin-destructive process: either primary, as in multiple sclerosis, or secondary to brain damage, as during ischemic strokes.

Notes: Abstract: This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angio-tensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H-labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.

Notes: Abstract: The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenal-ectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenyl-acetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-meth-oxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5–2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.

Notes: Abstract: The actions of excitatory amino acids on the release of previously incorporated γ-[3H]aminobutyric acid ([3H]GABA) were examined in purified (〉93%) striatal neurons derived from the fetal mouse brain and differentiated in primary culture. Glutamate, KC1, and veratrine evoked a dose-dependent, saturable, and reversible release of [3H]GABA from striatal neurons. Glutamate actions were not reduced in the absence of calcium, and were insensitive to tetrodotoxin. The dose-response relationships of excitatory amino acids demonstrated the following rank order of potency: glutamate 〉 aspartate =N-methyl-d-aspartate 〉 kainate ≧ quisqualate. Kainate, however, was the most effective agonist, evoking an eightfold increase over baseline levels of [3H]GABA release. Aspartate- and N-methyl-d-aspartate-evoked release was abolished in the presence of either 2-aminophosphonovaleric acid or γ-d-glutamylglycine. Release due to glutamate and kainate was partially or ineffectively attenuated by these agents. Glutamate-, aspartate-, and N-methyl-d-aspartate-evoked GABA releases were augmented when calcium was omitted from the bathing medium and reduced when sodium was replaced with choline or lithium. Kainate-evoked release was unaffected when calcium was omitted, virtually unchanged when choline replaced sodium, and markedly potentiated when lithium was substituted for sodium. These findings suggest that at least two distinct receptor systems for excitatory amino acids mediate the evoked release of [3H]GABA from striatal neurons in primary culture. These two systems, aspartate/N-methyl-d-aspartate- and kainate-prefer-ring, are distinguishable on the basis of their pharmacological and ionic properties.

Notes: Abstract: 125I-SCH 23982, an antagonist with high affinity and selectivity for the D-l subtype of dopamine receptors, has recently been synthesized. Densities of D-l receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal ho-mogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in 5max values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.

Notes: Abstract: Brain extraction of a tricyclic antidepressant, imipramine, was investigated using the carotid injection technique in the rat. The extent to which drug binding to plasma proteins and erythrocytes could inhibit the brain extraction was measured. Equilibrium dialysis showed that imipramine is highly bound to human serum albumin (HSA), α1-acid glycoprotein (AAG), lipoproteins, and erythrocytes. The free dialyzable drug fraction was inversely related to the protein concentration. Despite this degree of binding, no significant reduction in the brain extraction of the drug was observed in the presence of HSA, lipoprotein, or erythrocytes. Only AAG reduced the brain transport of this drug in a ratio related to the protein concentration. However, the rat brain extraction was higher than expected from the in vitro measurement of the dialyzable fraction. These data indicate that the amount of circulating imipramine available for penetration in brain exceeds widely the dialyzable fraction of the drug as measured in vitro.

Notes: Abstract: The main objective of this study was to test the hypothesis that the chronic administration of choline supplements a bound pool of choline from which free choline can be mobilized and used to support acetylcholine synthesis when the demand for precursor is increased. For these experiments, brain slices from rats fed diets containing different amounts of choline were incubated in a choline-free buffer and acetylcholine synthesis was measured under resting conditions and in the presence of K+-induced increases in acetylcholine synthesis and release. Rats fed the choline-supplemented diet had circulating choline levels that were 52% greater than the controls, and striatal and cerebral cortical slices from this group produced significantly more free choline during the incubation than slices from the controls. However, the synthesis and release of acetylcholine by these tissues did not differ from those by controls, during either resting or K+-evoked conditions. In contrast, acetylcholine synthesis and release by striatal and hippocampal slices from choline-deficient rats, animals that had circulating choline levels that were 80% of control values, decreased significantly; the production of free choline by these tissues was also depressed. Results indicate that, despite an increased production of free choline by brain slices from choline-supplemented rats, the synthesis of acetylcholine was unaltered, even in the presence of an increased neuronal demand. In contrast, the choline-deficient diet led to a decreased release of free choline from bound stores and an impaired ability of brain to synthesize acetylcholine.

Notes: Abstract: In vitro quantitative autoradiography of high-affinity [3H]imipramine binding sites was performed on 16 human brains postmortem. The densities of binding sites were highest in the hypothalamus. Next, in descending order, were the basal and lateral nuclei of the amygdala; substantia innominata; insular cortex; the central nucleus of the amygdala; the anterior nucleus of the thalamus; the head of the caudate nucleus; portions of the frontal, parietal, and temporal cortex; claustrum; the granular layer of the dentate gyrus; substantia nigra; the pyramidal layer of CA fields; globus pallidus; red nucleus; and white matter. Imipramine binding was found to increase with age in a region-specific manner. The presence of alcohol had a similar effect, which was most pronounced in the hippocampus. Sex and time from death to autopsy did not affect imipramine binding, in our sample.

Notes: Abstract: The intrasynaptosomal free calcium concentration ([Ca2+]i) was measured in quin2-loaded synaptosomes prepared from rat cerebral cortex. Membrane-permeant cyclic adenosine-3′,5′-monophosphate (cAMP) analogues [8-bromo-cyclic adenosine-3′,5′-monophosphate (8-Br-cAMP) and dibutyryl-cyclic adenosine-3′,5′-monophosphate (db-cAMP)] increased [Ca2+]i in a dose-dependent manner; The maximal increases were ˜50% for 8-Br-cAMP and 35% for db-cAMP and occurred at ˜10 μM with both analogues. Clonidine (1 μM) alone reduced [Ca2+]i by 26.5%; db-cAMP and 8-Br-cAMP attenuated this reduction to 14.2 and 8.2%, respectively. In contrast, the reduction (19.9%) in [Ca2+]i induced by the preferential k-opiate agonist dynorphin A(1–13) was not attenuated by the cAMP analogues; in fact, db-cAMP and 8-Br-cAMP potentiated the effect of dynorphin A(1–13) (1 μM), producing decreases in [Ca2+]i of 33.6 and 29.6%, respectively. We conclude that although aradrenergic and k-opiate receptors both reduce [Ca2+]i, the α2-adrenoceptor-mediated response and the k-opiate receptor-mediated response involve different effector mechanisms. It appears that pre-synaptic α2-adrenoceptor agonist effects are linked to reductions in adenylate cyclase activity and cAMP production and a resultant increase in Ca2+ sequestration, Ca2+-channel blockade, or both. On the other hand, the k-opiate-mediated effects possibly involve an increase in cAMP production and a blockade of Ca2+ entry.

Notes: Abstract: The lateral hypothalamus has an important role in regulating food and water intake. We have investigated the endogenous release of monoamines from the lateral hypothalamus during manipulations of plasma osmolality and circulating volume. Adult male Sprague-Dawley rats implanted with carbon paste in vivo electrochemical (EC) electrodes in the lateral hypothalamus were placed on a 72-h water deprivation schedule. Although the carbon paste EC electrode has an intrinsically ambiguous signal in which changes in ascorbic acid may appear as changes in catechol concentrations, pharmacologic studies in lateral hypothalamus indicated that the electrode most likely measured nor-epinephrine and possibly epinephrine. On the test day, the EC electrodes were scanned with linear sweep voltammetry from -0.2 to +0.4 V at a rate of 5 mV/s. Semiderivative signal processing showed catechol and hydroxyindole peaks at +0.11 and +0.23 V, respectively. Baseline recordings were made prior to rats drinking distilled water, 10% sucrose, 5% dextrose, 0.30% NaCl, 0.90% NaCl, or 10%D-mannitol. To control for the act of drinking, other implanted dehydrated rats were intraperitoneally injected with 5% dextrose, 0.30% NaCl, or 0.90% NaCl. To dissociate the effects of osmolality and circulating volume on the EC response, hydrated rats implanted with EC electrodes were subcutaneously injected with 12% NaCl or intraperitoneally injected with 35% polyethylene glycol. Other rats subjected to water deprivation and osmotic challenges were decapitated and trunk blood was collected for measurements of plasma osmolality and hematocrit. Similar experiments were conducted using homozygous Brattleboro rats which lack arginine vasopressin (AVP) but which preserve normal plasma osmolality with prodigious drinking. The use of these rats provided a way to dissociate monoamine changes related to AVP release from changes related to plasma osmolality. Results in Sprague-Dawley and Brattleboro rats showed that the EC catechol signal was lower in rats with high plasma osmolality and rose with drinking or intraperitoneal fluid administration as a direct function of the fall in plasma osmolality. Manipulations of plasma osmolality had a greater effect on the EC catechol signal than did manipulations of circulating volume. Thus, we conclude that changes in extracellular fluid catechol concentrations in the lateral hypothalamus linearly reflect changes in plasma osmolality.

Notes: Abstract: The binding of α-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 ± 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several otner excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, l-G1u, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intra-striatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2–3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopainergic pathway (using nigral injection of 6-hydroxydopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.

Notes: Abstract: Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (free mRNP particles) carrying messenger RNA has been characterized in rat brain. There were first-order kinetics for NAD with an apparent Km for NAD of 90.5 ± 0.70 μM and Vmax of 19.7 ± 2.8 pmol ADP-ribose incorporated min-1 mg protein-1. Five poly(ADP-ribose) protein acceptors were identified in the Mr 37,000–120,000 range. It is hypothesized that ADP-ribosylation of specific free mRNP proteins might play a role in the derepression and translation of the silent mRNAs of free mRNP particles.

Notes: The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in im-munoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2–3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.

Notes: Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20–30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-d-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][l-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD= 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki= 31 nM) and the anticonvulsant MK-801 (Ki= 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited-binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.

Notes: The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7–9-day-old rats was studied at 16–18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K.+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3–5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0–5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.

Notes: The changes in 16 cerebral metabolites produced by cardiac arrest and subsequent room temperature auto-lysis were studied using high-resolution proton nuclear magnetic resonance spectroscopy. Biopsies of rabbit cerebral cortex, cerebral white matter, and cerebellum were quantitatively analyzed for acetate, alanine, γ-aminobu-tyric acid, creatine, glutamate, glycine, inositol, lactate, N-acetylaspartate, phosphocreatine, succinate, taurine, and threonine. Of these, N-acetylaspartate and the total creatine pool are the best candidates for use as concentration reference standards linking in vitro to in vivo 1H nuclear magnetic resonance measurements. Both changed little immediately after death, and they varied in a distinctive way among cortex, white matter, and cerebellum.

Notes: Calf brain 3′-phosphoadenosine 5′-phosphosul-fate (PAPS):proteoheparan sulfate (PHS) N-sulfotransfer-ase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3′,5′-ADP-agarose as chromato-graphic supports. Sulfotransferase activity was followed by using 3′-phosphoadenosine 5′-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22– to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5–8. Apparent Km values for PAPS of 2.3 μM and 0.9 μM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and ly-sophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.

Notes: Kynurenic acid, a tryptophan metabolite able to antagonize the actions of the excitatory amino acids, has been identified and measured for the first time in the brain of mice, rats, guinea pigs, and humans by using an HPLC method. Its content was 5.8 ± 0.9 in mouse brain, 17.8 ± 2.0 in rat brain, 16.2 ± 1.5 in guinea pig brain, 26.8 ± 2.9 in rabbit brain, and 150 ± 30 in human cortex (pmol/g wet wt, mean ± SE). The regional distribution of this molecule was uneven. In rats, guinea pigs, and rabbits, the brainstem was the area richest in this compound. Tryptophan administration (100–300 mg/kg, i.p.) to rats resulted in a significant increase of the brain content of kynurenic acid. Similarly, 1 h after probenecid administration (200 mg/kg, i.p.), the brain content of kynurenate increased by fourfold, thus suggesting that its turnover rate is relatively fast.

Notes: Abstract: The levels of cholinergic, γ-aminobutyric acidergic (GABAergic), and excitatory amino acid neurotransmitter markers have been measured in 18 regions of the pigeon telencephalon as well as in supposedly homologous areas of the rat telencephalon. Among the basal telencephalic areas, some similar patterns of regional distribution were observed, with the noticeable exception of the ratio of levels of cholinergic markers between the striatum and globus pallidus, which was much larger in the rat than in the pigeon. In the rat cortical areas, some interesting differences were noticed among the archicortex, the paleocortex, and various parts of the neocortex. In particular, the area identified as prefrontal cortex by previous studies was significantly richer in cholinergic and excitatory amino acid markers and poorer in GABAergic activity than other neocortical regions. In the pigeon, presumedly neocortical equivalent areas—in particular, those constituting the dorsal ventricular ridge—were quite variable in levels of cholinergic markers, and some apparently well-established areas homologous to mammalian neocortex showed exceptionally low levels of cholinergic markers. The higher variability in levels of neurotransmitter-related markers shown by cortically equivalent areas of the avian dorsal ventricular ridge, as compared with the more uniform pattern present in basal telencephalic regions, may be the result of a greater plasticity of these structures during evolution, in response to different selective pressures.

Notes: Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to a-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.

Notes: Prostaglandins and Related Compounds (Advances in Prostaglandin, Thromboxane, and Leukotriene Research, Volumes 17A and 17B-Sixth International Prostaglandin Conference, Florence, Italy)Functional Mapping in Biology and Medicine: Computer Assisted Autoradiography (Volume 11, Experimental Biology and Medicine)Amino Acids in Health and Disease: New Perspectives (UCLA Symposia on Molecular and Cellular Biology, New Series, Volume 55)The Vertebrate Neuromuscular Junction edited by M. M. Salpeter.

Notes: Abstract: An inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. α-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.

Notes: Abstract: Unilateral ligation of the left common carotid artery in anesthetized Mongolian gerbils resulted in a steep rise in extracellular dopamine in the ipsilateral striatum in 9 out of 19 animals. Extracellular dopamine was measured by cerebral dialysis in vivo and reached a peak of 0.19 mM at 40 min. At the same time, the level of homovanillic acid fell, whereas the levels of ascorbate and 3,4-dihydroxyphenylacetic acid remained relatively constant. In a separate group of animals studied with a combined dialysis/electrochemistry probe, a rise in the in vivo chronoamperometric signal in three out of six animals correlated with a rise in extracellular dopamine. The number of animals responding in these experiments (roughly 50%) corresponds to the frequency of incompetent Circle of Willis, as well as literature reports of the frequency of signs of stroke in unanesthetized gerbils. These results show a remarkable accumulation of dopamine in extracellular fluid in response to cerebral ischemia. Released dopamine appears to be responsible for the elevated in vivo electrochemical signal previously reported.

Notes: Abstract: A sodium-dependent high-affinity [3H]-hemicholinium-3 ([3H]HCh-3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh-3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh-3 binding to the solubilized preparation was both sodium- and chloride-dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh-3 binding to the solubilized preparation in a concentration-dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh-3 binding. These results suggest that the characteristics of the solubilized [3H]HCh-3 binding site are similar to those of the membrane-bound site.

Notes: Abstract: The binding of [125I]βh-endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl-5′-imidodiphosphate. In contrast to the binding of the μ-selective opioid agonist, [3H][d-Ala2,MePhe4,Glyol5]enkephalin, and the δ-selective opioid agonist, [3H][d-peniciIlamine2,d-penicillamine5]enkephalin, [125I]βh-endorphin binding was not affected by GTP or guanylyl-5′-imidodiphosphate in a concentration-dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl-5′-imidodiphosphate resulted in a concentration-dependent inhibition of [125I]βh-endorphin binding. This inhibition was significantly greater than the decrease in [125I]βh-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]βh-endorphin binding in the presence of sodium, inhibition of [125I]βh-endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]βh-endorphin, the binding of the putative σ receptor agonist, (+)-[3H]SKF 10,047, was not significantly altered by GTP or guanylyl-5′-imidodiphosphate in the absence or presence of sodium.

Notes: Abstract: Concentrations of the endogenous neurotoxic tryptophan metabolite, quinolinic acid (QA), were measured in postmortem brain tissue obtained from patients with Huntington's disease (HD) and matched controls, using a gas chromatography/mass spectrometry method. There was no significant difference in either the putamen or the frontal cortex between the HD and control groups. These results do not support the hypothesis that increased QA is responsible for neuronal degeneration in HD.

Notes: Abstract: Peptidyl-glycine α-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using d-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (〉30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10–30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1–10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no α-amidation activity (〈0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.

Notes: Abstract: Maternal alcohol consumption at a level that does not affect calorie intake increases cholesterol concentration and content as well as incorporation of labeled glucose into cholesterol in the brain and spinal cord of newborn rat pups. Continued consumption of alcohol during lactation also affects the galactolipid concentration in the brain and spinal cord of pups at 21 days of age, and this increase seems mainly to be due to an increase in content of myelin lipids. Analysis of myelin shows that the concentration of phospholipids also increases in this fraction. The increase in incorporation of labeled glucose into these membrane lipids suggests an increase in the synthesis of these lipids, which prevents fluidization of the membrane by alcohol. That in the brainstem the increase in levels of cholesterol and galactolipids is higher than in other regions and that there is also an increase in content of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine suggest that the brainstem needs better protection against fluidization.

Notes: Abstract: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions). However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 μM, respectively), whereas the number of uptake sites for MPP+is double (Vmax= 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented 〈10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures. These data support the view that MPTP is transformed into MPP+ by astrocytes and concentrated in DA neurons by a relatively specific uptake system, similar to that for DA.

Notes: Abstract: The main objective of these studies was to determine whether adenosine inhibits choline kinase in rat striata, leading to a decreased incorporation of choline into phosphorylcholine, a mechanism that may mediate seizure-induced increases in the levels of free choline in brain. Incubation of paniculate and soluble fractions of striatal synaptosomes with adenosine or its metabolically stable analogues significantly inhibited enzyme activity. The inhibition was noncompetitive versus choline and competitive versus MgATP. Inhibitor constants for adenosine, 2-chloroadenosine, and 2′,5′-dideoxyadenosine at the MgATP site were 94, 49, and 207 μM, respectively; these values were less than the Michaelis constant for MgATP (340 μM). To determine whether adenosine altered the phosphorylation of choline in an intact preparation, synaptosomes were in cubated with [3H]choline in the presence or absence of adenosine or its analogues and the amount of [3H]-phosphorylcholine formed from the [3H]choline taken up was measured. All compounds tested significantly reduced the synthesis of [3H]phosphorylcholine. Results suggest that following seizures or hypoxia, when levels of adenosine increase and the concentration of ATP decreases, inhibition of choline phosphorylation may be manifest, resulting in increased levels of free choline in brain.

Notes: Abstract: The contributions of five variables believed to influence the brain's metabolism of O2 during hypoxia [duration, PaO2, ΔCMRO2 (the difference between normal and experimental oxygen uptake), O2 availability (blood O2 content · CBF), and O2 deficit (ΔCMRO2· duration)] were assessed by stepwise and multiple linear regression. Levels of brain tissue carbohydrates (lactate, glucose, and glycogen) and energy metabolites [ATP, AMP, and creatine phosphate (CrP)] were significantly influenced by O2 deficit during hypoxia, as was final CMRO2. After 60 min of reoxygenation, levels of tissue lactate, glucose, ATP, and AMP were related statistically to the O2 deficit during hypoxia; however, CMRO2 changes were always associated more significantly with O2 availability during hypoxia. Creatine (Cr) and CrP levels in the brain following reoxygenation were correlated more to ΔCMRO2 during hypoxia. Changes in some brain carbohydrate (lactate and glucose), energy metabolite (ATP and AMP) levels, and [H+]i induced by complete ischemia were also influenced by O2 deficit. After 60 min of postischemic reoxygenation, brain carbohydrate (lactate, glucose, and glycogen) and energy metabolite (ATP, AMP, CrP, and Cr) correlated with O2 deficit during ischemia. We conclude that “O2 deficit” is an excellent gauge of insult intensity which is related to observed changes in nearly two-thirds of the brain metabolites we studied during and following hypoxia and ischemia.

Notes: Abstract: Turnover rates of cerebral proteins were examined in control adult rats and in those subjected to prolonged in vivo treatment with “low” (0.02 mg/ml) or “high” (0.04 mg/ml) doses of nicotine (added to drinking water), using [14C]bicarbonate as the label. It was found that the turnover of proteins in various subcellular fractions consisted of two distinct components turning over at a “fast” or a “slow” rate and having relatively short or long half-lives, respectively. Thus in control animals the half-lives of the protein components turning over at a fast rate ranged from 1.31 to 3.61 days whereas for those turning over at a slow rate the half-lives ranged from 8.56 to 24.28 days. Treatment with low doses of nicotine resulted in a more rapid turnover of nuclear fast turning over component with a concomitant decreased turnover of homogenate, cytosol, mitochondrial, and microsomal proteins; in the synaptosomal membranes this component disappeared altogether. The half-lives of the slow turning over components decreased in general from 14.3 to 33.3% with the exception of the nuclear proteins, where the half-life increased by 71.1%. Turnover of microsomal proteins was not affected. When the animals were given a high dose of nicotine, the turnover of fast components became even more rapid for nuclear, myelin, and microsomal proteins with a decrease in half-life from 26.6 to 32.3%. By contrast, half-lives of synaptosomal and mitochondrial proteins increased by 16.1–89.3%. These changes were not reflected in the turnover rate of whole homogenate proteins. Turnover rates of even the slow components increased for cytosol, nuclear, homogenate, and myelin proteins with decrease in t1/2 from 22.9 to 60.2%. By contrast, t1/2 of microsomal and mitochondrial proteins increased by 51.1 and 66.4%. Turnover of synaptosomal proteins was not affected under these conditions. Treatment with nicotine also brought about a small but reproducible increase in the rate of turnover of liver homogenate proteins which is attributable almost completely to the nuclear proteins. With low doses of nicotine half-life of mitochondrial proteins increased by 7.2% whereas that of microsomal proteins decreased by 8.5%. The results thus emphasize that in vivo effects of nicotine are more specific for cerebral protein metabolism.

Notes: Abstract: A calcium/calmodulin-dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50-kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50-kD and 60-kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto-phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin-dependent to a calcium/calmodulin-inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin-independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.

Notes: Abstract: Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.

Notes: Abstract: The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 ± 1.4 × 106 M−1 min−1 and 1.1 ± 0.06 × 10−1 min−1, respectively. Saturation experiments revealed a single class of binding sites (nH= 1.1) for the radioligand with a total binding capacity of 160 ± 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency vas atropine sulfate 〉 pirenzepine 〉 AF-DX116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.

Notes: Abstract: Histamine stimulated the enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine in crude synaptic membranes of rat brain containing the methyl donor S-adenosyl-l-methionine (SAM). In the presence of, but not in the absence of SAM, histamine increased cyclic AMP accumulation at the concentrations that stimulate phospholipid methylation. S-Adenosyl-l-homocysteine, an inhibitor of phospholipid methyltransferases, inhibited histamine-stimulated phospholipid methylation and histamine-induced cyclic AMP accumulation in the presence of SAM in a concentration-dependent manner. Histamine-induced [3H]methyl incorporation into phospholipids exhibited a marked regional heterogeneity in rat brain in the order of cortex 〉 medulla oblongata 〉 hippocampus 〉 striatum 〉 midbrain 〉 hypothalamus. The regional distribution of histamine-induced cyclic AMP accumulation exactly paralleled histamine-stimulated [3H]-methyl incorporation in rat brain. Histamine-induced cyclic AMP accumulation was inhibited by the addition of cimetidine or famotidine, but not by mepyramine or diphenhydramine. The accumulation of cyclic AMP in the presence of SAM was observed by the addition of impromidine or dimaprit, but not by 2-pyridylethylamine. These results indicate that phospholipid methylation is induced by histamine and may participate in H2-receptor-mediated stimulation of adenylate cyclase in rat brain.

Notes: Abstract: The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 ± 2 nM for Percoll (C-band) synaptosomes, loaded at 30°C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37°C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30° or 37°C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin-2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37°C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes. The possible involvement of different synaptosomal pools to explain the divergence in [Ca]i between different preparations and the interpretation in physiological terms of [Ca]i measured in synaptosomes are discussed.

Notes: Abstract: Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 ± 0.05 μmol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (∼6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 μM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 ± 0.5 and 13 ± 1 μM for Ap4A and Ap5A, respectively.

Notes: Abstract: We determined the ontogeny of the GTP-binding protein Go in rat brain and heart by employing highly sensitive enzyme immunoassay methods. In the brain, the α subunit of Go (Goα) gradually increased and reached adult levels approximately 20 and 30 days after birth in cerebral cortex and cerebellum, respectively. Concentrations of β subunits, which were also quantified by the immunoassay, were almost equal to those of Goα in the brain of rats younger than 10 days, but were higher than those of Goα after 10 days. These results suggest the late development of GTP-binding proteins other than Go. Goα was immunohistochemically positive in neuropils and negative in cell bodies at any age tested. In the heart, the concentrations of Goα increased up to several times of the adult level just after birth, and then gradually decreased after the 20th postnatal day. The level of Goα in the liver, however, was very low and constant throughout ontogenic development. An immunohistochemical study indicated that Goα was positive in the cardiac muscle of young rat, but negative in that of adult rat. These results indicate that Goα exists in cells other than those of nervous tissues and neuroendocrine cells in some periods of ontogenic development.

Notes: Abstract: Glutamic acid decarboxylase (GAD), γ-[3H]-aminobutyric acid ([3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2–3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.

Notes: Abstract: P400 protein is a concanavalin A (Con A)-binding, 250–kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P400 protein were investigated, and complete solubilization of P400 protein from a submicrosomal fraction (P31 fraction) of mouse cerebellum was attained by the combination of 4% Zwittergent 3–14 and 4 M guanidinium chloride. The solubilized P400 protein was purified using Sepharose CL-4B and Con A-Sepharose chromatography. A monoclonal antibody (18A10) was prepared against P400 protein. Endo-β-N-acetylglucosaminidase F digestion of P400 protein revealed that P400 protein has a small number of asparagine-linked oligosaccharide chains and that the epitope that is recognized by 18A10 monoclonal antibody is not on the asparagine-linked oligosaccharide portion. Tissue distribution of P400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P400 protein was abundant in the cerebellum, but a very small amount of P400 protein or related antigen was also detected in other parts of the nervous system and in nonneural tissues. Immunohis-tochemical studies indicated that P400 protein was distributed abundantly in the soma, the dendritic arborization, and the axon of the Purkinje cell. No immunoreaction was observed in the other types of cells.

Notes: Abstract: NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 μM NaF. Addition of 10 or 100 μM A1C13 or aluminum maltol did not alter the effect of NaF, whereas at 500 μM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 μM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 μM phorbol 12–myristate 13–acetate, a phorbol ester, inhibited carbachol-induced, but not NaF-induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.

Notes: Abstract: Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP poly-peptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions. Purified MBP added prior to subcellular fractionation to ho-mozygous shiverer oligodendrocytes (which express no MBP endogenously) is recovered predominantly in the plasma membrane fraction, whereas purified MBP added to mock-transfected fibroblasts exhibits a dispersed distribution among subcellular fractions. On the basis of these results, we propose a model whereby localization of MBP to the plasma membrane in oligodendrocytes involves interaction between the newly synthesized MBP polypeptide and specific MBP receptors which are present in oligodendrocyte plasma membrane, but not in fibroblast plasma membrane.

Notes: Abstract: Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40–60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.

Notes: Abstract: In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50= 3–5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1–100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1–10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 μM). At this time, the SR 95191 -induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki= 68 μM). It is concluded that SR 95191 is a selective and reversible type A MAOI in vivo but not in vitro.

Notes: Abstract: The tissue and cellular distribution of a GTP-binding protein, Go, was investigated in the rat by immunochemical and immunohistochemical methods. Because the specific antibody for the α subunit of bovine Go (Goα) cross-reacted with rat Goα, an enzyme immunoassay method developed for bovine Goα was applied for measuring the tissue concentration of Goα in the rat. Goα was detected in all tissues examined except blood cells. The concentration of Goα was highest in the CNS (∼7.7 and 4.4 nmol/g in the cerebrum and cerebellum, respectively), followed by the pituitary gland and sciatic nerve. Among the other peripheral tissues, relatively high concentrations of Goα were observed in the urinary bladder, stomach, and intestines; however, these values were 〈2% of the concentration in the cerebrum. Goα in the intestine was located mostly in the muscle layer. Immunohistochemical study showed that Goα was associated mostly with the neural elements but not with cells particular to each peripheral organ. Goα was also present in the membranes of neuroen-docrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, islets cells in the pancreas, and parafollicular cells in the thyroid. These results indicate that Go is localized exclusively in the nervous tissues and neuroendocrine cells.

Notes: Abstract: Similar to metabolites of other aminergic transmitters, histamine metabolites of brain, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), could have a concentration gradient between rostral and caudal sites of CSF. To test this hypothesis, cisternal and lumbar CSF samples were collected in pairs from eight monkeys (Macaca mulatta), and levels of t-MH and t-MIAA were measured by gas chromatography-mass spectrometry. pros-Methylimidazoleacetic acid (p-MIAA), an endogenous isomer of t-MIAA that is not a histamine metabolite, was also measured. Cisternal levels (in picomoles per milliliter, mean ± SEM) of t-MH (9.9 ± 1.4) and t-MIAA (40.8 ± 7.6), but not of p-MIAA (9.7 ± 1.2), exceeded those in lumbar CSF (t-MH, 1.8 ± 0.3; t-MIAA, 6.8 ± 0.9; p-MIAA, 8.6 ± 0.6) in every monkey. The magnitudes of the mean cisternal-lumbar concentration gradients fort-MH(6.6 ± 1.1)and t-MIAA (6.5 ± 1.3) were indistinguishable. These gradients exceed those of metabolites of most other transmitters. There was no gradient for the levels of p-MIAA. The cisternal, but not lumbar, levels of t-MH and t-MIAA were correlated. There was no significant difference between the means of the metabolite concentration ratios (t-MIAAJ.t-MH) in cisternal (4.0 ± 0.4) and lumbar (4.4 ± 0.9) CSF. The steepness of these gradients suggests that levels of t-MH and t-MIAA in lumbar CSF might be useful probes of histaminergic metabolism in brain.

Notes: Abstract: Regional transport of 1-aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood-brain barrier was studied in pentobarbital-anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants of the frontal cortex equaled 9.7 × 10-−4μmol/s/g for Vmax, 0.054 μmol/ml for Km, and 1.0 × 10-−4 ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by 〉85% by either l-phenylalanine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one-fifth that for the more commonly used homologue, I-aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as l-methionine, l-isoleucine, and l-tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.

Notes: Abstract: In a postmortem study of nicotinic receptors in human brain, cigarette smoking was found to be associated with increased (−)-[3H]nicotine binding to membranes prepared from gyrus rectus (Brodmann area 11) (p 〈 0.001), hippocampal neocortex (Brodmann area 27), cerebellar cortex (p 〈 0.01), hippocampal formation (Ammon's horn + subiculum), and the median raphe nuclei of the midbrain (p 〈 0.05) but not the medulla oblongata. Analysis of the binding data suggested that the increased binding reflected an increase in the density of the receptors rather than a change in their affinity for (−)-nicotine. The effects of smoking were not influenced significantly by either the sex or age of the subject. It is concluded that smoking evokes an increase in high-affinity nicotine binding similar to that observed previously in animals treated chronically with nicotine and that the effect of smoking on these sites is probably caused by the nicotine present in the tobacco smoke.

Notes: Abstract: Hydrophobic interaction chromatography has been used to demonstrate an increase in the surface hydrophobicity of [3H]triamcinolone acetonide ([3H]TA)-labeled type II receptors in mouse brain cytosol following transformation of these receptor complexes to the activated DNA-binding form. After removing unbound [3H]TA and molybdate (which prevents activation) by gel nitration, [3H]TA-type II receptors were activated by incubation at 22°C for 20 min. Gel filtration was then used to remove newly dissociated steroid and to readjust the molybdate and/or KCl concentration. Unactivated and activated receptors were then added to propyl, butyl, pentyl, hexyl, octyl, decyl, and dodecyl alkyl agarose, phenyl agarose, or unmodified agarose columns equilibrated and eluted with buffers of various molybdate and KCl concentrations and/ or other additions, including glycerol, ethylene glycol, and urea. Under high-salt conditions, activated receptors were retained longer than unactivated receptors run on butyl, pentyl, hexyl, and phenyl agaroses. With the longer alkyl chain columns, essentially none of the [3H]TA was eluted in association with receptor macromolecules. Removal of the remaining steroid required receptor denaturation with urea. Under low-salt conditions, both receptor forms were retained more avidly on all alkyl agarose columns; however, on phenyl agarose only activated receptors displayed this increased retention. Further studies revealed that optimal separation and subsequent recovery of unactivated and activated [3H]TA-type II receptor complexes were achieved on pentyl agarose columns equilibrated and eluted with buffers containing 50 mM molybdate and 600–1,200 mM KCl. The activation-associated increase in surface hydrophobicity revealed in this study may reflect mechanisms underlying receptor subunit dissociation, increased permeation through the nuclear membrane, and/or increased affinity for DNA and other nuclear acceptors.

Notes: Abstract: The effect of continuous stimulation of splanchnic nerves at1, 3, and 10 Hz on the secretion of catecholamines from the isolated rat adrenal gland was examined. Secretion evoked at 10 Hz declined over 60% in 1 h, and by the end of 4 h the secretion was only 10% of the initial value. The secretion evoked at 3 Hz was unchanged in the first hour, but showed a gradual decline in subsequent hours. In contrast, secretion evoked at 1 Hz was well maintained for several hours. Even after 6 h of continuous stimulation, the decline was only about 35%. Atropine plus hexamethonium reduced the secretion evoked at 10 Hz by over 80%, but that evoked at 1 Hz was reduced by about 35%; addition of naloxone reduced it to 75%. When the secretion declined to very low levels after continuous stimulation at 10 Hz for 100 min, a change in frequency to 3 Hz or 1 Hz caused a sharp rebound in the secretory response. Returning the frequency back to 10 Hz led to a sharp drop in the secretion, whereas reducing the frequency to 1 or 3 Hz once again increased the secretion. The rebound in the secretory response after switchover of frequencies was observed in the presence of atropine plus hexamethonium, but was abolished by naloxone. Extensive stimulations, which caused large amounts of catecholamine secretions at each frequency, were not associated with any loss in tissue catecholamine contents. The major conclusion is that secretion of catecholamines is maintained uninterrupted for several hours when splanchnic nerves are stimulated at low frequency. Under these conditions, the release of noncholinergic transmitter(s) from splanchnic nerves is mostly responsible for inducing the secretion of catecholamines.

Notes: Abstract: We synthesized a number of fluorinated analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and tested their suitability as substrates for monoamine oxidase B in vitro and their dopaminergic neurotoxicity in vivo. Two of the compounds tested, 2′-F-MPTP and 2′-CF3-MPTP, were better enzyme substrates and possessed more potent neurotoxicity for nigrostriatal dopamine neurons than MPTP, especially 2′-F-MPTP. The results of the in vivo neurotoxicity experiments correlated well with the suitability of the compounds as substrates for monoamine oxidase. These findings could serve as a basis for the use of 18F-labeled analogs of MPTP for positron emission tomography studies of nonhuman primates for better understanding of the factors underlying MPTP toxicity. Furthermore, the discovery of two MPTP analogs with enhanced selective neurotoxicity to dopaminergic neurons may be an important clue in the continuing efforts to define the chemical structure-activity factors governing MPTP neurotoxic activation mechanisms.

Notes: Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spec-trophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [l-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units ± 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxy-lated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units ± 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units ± 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.