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Notes: Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.

Notes: Abstract: The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood–brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 μ1 containing [1-14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi-lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 ± 0.08 μmol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22–0.27 μmol/min/g. The Vmax for the hippocampus was intermediate at 0.37 ± 0.12 μmol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier.

Notes: Abstract: Intravenous administration of d-lysergic acid diethylamide (LSD) to rabbits results in a transient inhibition of brain protein synthesis in vivo and in vitro. A translational inhibitor that appears in the postribosomal supernatant fraction of cerebral hemispheres following LSD administration was partially purified by gel filtration on Sephadex G-150 and precipitation with 60% ammonium sulfate. This inhibitor, which was proteinaceous, reduced the translational capacity of an initiating cell-free protein synthesis system derived from brain. It also inhibited a messenger RNA-dependent reticulocyte lysate programmed with brain polysomes and a globin-synthesizing reticulocyte lysate system. Addition of the partially purified inhibitor to a brain cell-free protein synthesis system resulted in the decreased formation of ternary complexes as well as 40 and 80S initiation complexes, suggesting that the inhibitor affects an early step in the initiation of protein synthesis in brain.

Notes: Abstract: In an effort to gain a more complete understanding of the regulation of myelin basic protein phosphorylation, we have been interested in defining further the mode of regulation of the myelin protein kinase involved in this posttranslational modification. Here we report the partial purification of a protein kinase from rat brain myelin. By gel filtration, it was determined that the molecular weight of this enzyme was in the range of 70–80 × 103 daltons. Furthermore, it was established that at low calcium concentrations, this enzyme was markedly activated by phosphatidylserine in combination with either 4β-phorbol 12-myristate 13-acetate or diolein. The enzyme was not affected by cyclic AMP or by calcium, alone or in combination with calmodulin. On the basis of these findings this enzyme can be identified as a protein kinase C-like enzyme.

Notes: Abstract: To determine the site of action of imipramine, the subcellular distribution of [3H]imipramine in rodents was followed after both in vivo administration and in vitro incubation with tissue slices under “physiological” conditions. Total [3H]imipramine (10–1,000 nM) binding was associated with all primary fractions, but in particular with the nuclear (P1) and mitochondrial (P2) pellets and the synaptosomal (P2B) and myelin (P2A) fractions; Using an excess of imipramine to define any nonspecific interactions, a specific association was observed mainly in those fractions containing isolated nerve terminals and to a lesser extent with the purified myelin fraction. Preparation of subsynaptosomal fractions by osmotic lysis indicated that [3H]imipramine was associated with the synaptic vesicle and microsomal fractions and also with synaptosomal membranes. The degree of binding to the vesicular and microsomal fractions was increased with the length of preparation time, whereas there was an inverse relationship between the length of preparation and the amount bound to the synaptosomal membrane fraction. There was no evidence of an intrasynaptosomal accumulation of [3H]imipramine at concentrations up to 1,000 nM. [3H]2-Nitroimipramine, a slowly dissociating imipramine derivative, was exclusively located in synaptic membrane fractions. Prior treatment of rats with a combination of 5,7-dihydroxytryptamine and desipramine reduced 5-hydroxytryptamine levels and the levels of [3H]imipramine associated with the synaptosomal fractions to the same extent. It is concluded that imipramine is associated with a binding site localised on 5-hydroxytryptaminergic nerve terminals and that there is a redistribution to other sites (vesicular and microsomal) during the isolation procedure.

Notes: Abstract: CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.

Notes: Abstract: We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000–64,000 daltons.

Notes: Abstract: In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mMpotentiates the stimulation of phosphoinositide turnover elicited by carbachol and (±)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+ -deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.

Notes: Abstract: Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of γ-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 ± 12 μM and a Vmax of 1.3 ± 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]e) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of + 2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.

Notes: Abstract: A procedure is described for the establishment of stable primary cultures of bovine chromaffin cells on microcarrier beads. The cells flatten and send out processes with varicosities over a few days and maintain their catecholamine content for 2 weeks. The beads may be incorporated into a superfusion apparatus with a chamber volume of about 150 μl, enabling the efficient perfusion of a high density of cells. The response to the introduction of nicotine and high potassium into the perfusing medium is shown to be more rapid and more transient than hitherto described, with each secretagogue producing a different degree of preferential stimulation of noradrenaline-secreting cells.

Notes: Abstract: Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/μg of total protein, was ∼20-fold higher in whole homogenates of cerebral white matter (4.7 ± 0.60) than of peripheral nerve (0.12–0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/μg; PNS myelin, 0.37 ng/μg. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP. These quantitative biochemical studies support earlier immunocytochemical observations suggesting that the MAG level is reduced more than levels of other myelin proteins at the periphery of some multiple sclerosis plaques.

Notes: Abstract: The release of γ-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10–1,000 μM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.

Notes: Abstract: The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of δ, K, μ1, and μ2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5′-(β, γ-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of “half-of-the-sites” reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous K–δ or μ1–μ2 receptor complexes is required for stabilization of the high-affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association-dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl]-methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of –SH/-SS– bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.

Notes: Abstract: The influence of chlorpromazine, haloperidol, morphine, chloral hydrate, γ-butyrolactone, probenecid, kainic acid, oxotremorine, pargyline, yohimbine, (+)-amphetamine, and cocaine on the efflux rate of 3,4-dihydroxyphenylacetic acid (DOPAC) from four brain areas was studied. All drugs studied except pargyline and morphine had an effect on the transport of DOPAC and homovanillic acid (HVA) from the brain. Nine drugs inhibited the efflux of DOPAC and HVA, whereas (+)-amphetamine stimulated this transport. These data suggest that most centrally acting drugs can interfere with the elimination of 3,4-dihydroxyphenylethylamine (DA or dopamine) metabolites from the brain. These effects are heterogeneously distributed throughout the brain and are probably related to indirect nonspecific drug effects. This implies that drug-induced changes in DA metabolite concentrations, especially when these changes are slight to moderate, cannot directly be translated to changes in the production rate of these metabolites. By studying five control groups, we concluded that formation and transport of DOPAC are not synchronized in the various brain areas.

Notes: Abstract: Cholinergic processes were measured in motor cortex, hippocampus, and striatum of cats in the terminal stages of GM1 gangliosidosis and compared to those of control cats. The greatest difference observed was elevation in the rate of K+-stimulated release of acetylcholine (ACh) from brain slices prepared from affected cats. The K+-stimulated release of endogenous ACh was increased by 31–43% and of newly synthesized ACh by 19–80% in brain slices from different brain regions. All regions that were examined were affected but the greatest effects occurred in cortex. The rate of synthesis of ACh was elevated in cortical and hippocampal slices. Choline acetyltransferase activity in brain regions of cats with GM1 gangliosidosis was not significantly different from that in controls, whereas high-affinity choline transport in cortical synaptosomes was elevated. Muscarinic receptor binding sites were reduced in the cortex, hippocampus, and striatum of GM1 mutant cats, whereas the apparent affinity was not altered. These results indicate that there are major alterations of cholinergic function in the brains of cats with GM1 gangliosidosis.

Notes: Abstract: Rapid axonal transport of glycoproteins was examined in the retinofugal projections of hypothermic and normothermic adult male Long-Evans hooded rats previously receiving intraocular injections of [3H]fucose. The amount of retinal fucosylation appeared normal in the hypothermic animals 3.5 h after isotope injection, but glycoprotein transport was reduced relative to normothermic controls. This reduction was especially pronounced in the most distal structure of the retinofugal tract (superior colliculus). We conclude that rapid axonal transport decreases with reductions in mammalian body temperature. This finding emphasizes the importance of controlling body temperature in in vivo studies of mammalian axonal transport.

Notes: Abstract: The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions: this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.

Notes: Abstract: Concentrations of [14C]2-deoxy-D-glucose ([14C]DG) and of glucose were measured in plasma of arterial and sagittal sinus venous blood from awake Fischer-344 rats at 3, 12, and 24 months of age, during continuous intravenous infusion of [14C]DG and after a steady-state arterial plasma concentration of [14C]DG was reached. Brain extraction, i.e., the difference between arterial and venous plasma concentrations divided by the arterial plasma concentration, was calculated for both [14C]DG and glucose. Because exchange of both substances between rat plasma and erythrocytes is slow, the ratio of the brain extraction of [14C]DG to that of glucose is identical to the lumped constant in the deoxyglucose procedure of Sokoloff et al. [J. Neurochem. 28, 897–916, (1977)]. This ratio equaled 0.502 ± 0.015 (SEM) at 3 months, 0.456 ± 0.007 at 12 months, and 0.418 ± 0.006 at 24 months of age (n = 15); the means differed significantly from each other (p 〈 0.05). The results indicate that the lumped constant declines between 3 and 24 months of age in awake rats, and suggest that many reported age reductions in regional cerebral glucose utilization, of 15–25%, are artifactual.

Notes: Abstract: The binding of [3H]neurotensin to membranes from human brain at 0°C was specific, saturable, and reversible. In the frontal cortex, the equilibrium dissociation constant (KD) for [3H]neurotensin determined from the ratio of rate constants (k-1/k1), saturation isotherms, and inhibition binding experiments was 0.80, 2.0, and 2.0 nM, respectively, and the maximum number of binding sites (Bmax) from the saturation isotherms and the competitive binding experiments was 2.4 and 2.2 pmol/g of tissue, respectively. Hill coefficients for binding were equal to 1, indicating the presence of single, noncooper-ative binding sites. Inhibition of specific binding of [3H]neurotensin by several analogs of neurotensin showed that [Gln4]neurotensin and neurotensin(8–13) had the highest affinities for these binding sites in human frontal cortex, with each analog being ∼13-fold more potent than neurotensin. In addition, these data showed that the carboxy-terminal portion of neurotensin played an important part in the binding of this neuropeptide in human brain, a result described for other species. Regional distribution of binding sites was different from that reported for animal brains. Of the 33 different regions investigated, the uncus and substantia nigra showed the highest specific binding of [3H]neurotensin, whereas such areas as the pineal body, medulla, and corpus callosum had few binding sites.

Notes: Abstract: Using a specific and sensitive epidermal growth factor radioimmunoassay (EGF-RIA) we measured EGF concentrations in whole brain, cerebral cortex, and cerebral cortical synaptosomal (pinched-off presynaptic nerve terminals) fractions of 26-day-old mouse brain. The relative EGF concentration in synaptosomal fractions was significantly greater than the growth factor concentrations in whole brain or cerebral cortex. Intracerebral injection, in an amount of EGF, several-fold greater than whole brain EGF content, did not appreciably increase synaptosomal EGF concentration, suggesting that no artifact was involved. The high synaptosomal EGF content suggests a neurotransmitter or a neuromodulator role for EGF in the CNS.

Notes: Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [125I] Bolton-Hunter (BH) CCK 8 and [3H]pentagastrin (Boc-β-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (KD) of 1.9 nM and 1.4 nM for [3H]pentagastrin and [125I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.

Notes: Abstract: Various putative striatal transmitters and related compounds were studied for their effects on the release of γ-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal out-flow whereas oxotremorine caused no consistent change; the effects of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, α2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.

Notes: Abstract: Stable opioid receptor binding activity that retains distinct subtype specificities (μ, 6, and K) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the δ, μ, and K sites (the latter subtype measured by the binding of [3H]dynorphin1–8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii 〉 60 Å), whereas both those and several small species present (〈 60 Å) bind opiate alkaloids and dynorphin1–8.

Notes: Abstract: The potent marine toxin, maitotoxin, induced the release of γ-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10−6M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10−4M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10−6M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.

Notes: Abstract: A radioiodinated analogue of somatostatin 28, 125I[Leu8, D-Trp22, Tyr25]SS-28, was used to localize and characterize somatostatin binding sites in both human and monkey brain. High-affinity binding sites (approximately 1 nM) were found in cerebral cortex. The highest binding was in cerebral cortex with intermediate binding found in hippocampus, striatum, and amygdala and low binding in hypothalamus and brainstem. There was a rough correlation between somatostatin receptor binding and concentrations of somatostatin-like immunoreactivity (SLI) in human brain. Somatostatin receptors were stable for up to 24 h in an animal model simulating human autopsy conditions and there was no correlation between postmortem interval and receptor binding in human brain. Pharmacologic characterization in human cortex showed that there was a correlation between the inhibition of receptor binding by somatostatin analogues and their known abilities to inhibit growth hormone secretion. These findings demonstrate that a highly specific membrane-associated receptor for somatostatin is present in both monkey and human brain. Examination of somatostatin receptor binding in Alzheimer's disease and Huntington's disease may improve understanding of the role of somatostatin in both these illnesses.

Notes: Abstract: In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent-disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose-6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.

Notes: Abstract: We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 μM GTP, 100 μM guanyl-5′-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two-to fourfold) in the EC50 value for forskotin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM–100 μM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194–1,825%, 122–1,141%, and 208–938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4-dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 μM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 μM) and SKF38393 (1 μM). At higher concentrations of forskolin (10–100 μM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine. In contrast to the effects observed with GTP, in the presence of 100 μM Gpp(NH)p there was no significant stimulation by dopaminergic agonists in the absence or presence of forskolin. These data demonstrate that in rat striatal homogenates forskolin can stimulate adenylate cyclase by (1) its direct action at the catalytic subunit, (2) by potentiating the stimulation elicited by the guanine nucleotide regulatory protein (Ns), and (3) by markedly potentiating the stimulation elicited by partial and full dopaminergic agonists acting at D-1 dopamine receptors.

Notes: Abstract: Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.

Notes: Abstract: Arginine-vasopressin (AVP), injected into the carotid artery in physiological concentration together with L-leucine, changed kinetic constants of the blood—brain barrier (BBB) transport of this neutral amino acid without changing the cerebral blood flow (CBF). The maximum velocity of transport (Vmax), the half-saturation constant (Km), the nonsaturable transport constant (KD), and CBF were estimated in nine brain regions of male Wistar rats anesthetized with ether. In cerebral hemisphere, Vmax decreased from 21 nmol min−1. g−1 (control) to 7.6 nmol. min−1. g−1 (AVP). Km decreased from 0.11 to 0.029 mM. Regional differences of the kinetic constants were found in controls as well as in AVP-treated animals. In all regions, the calculated constants Vmax and Km of animals coinjected with AVP were significantly decreased when compared to controls. A direct or indirect interaction of AVP with the transport system of large neutral amino acids is suggested.

Notes: Abstract: [3H]Cocaine dissociates from its binding sites in the mouse cerebral cortex with a half-time of 25 s. The dissociation kinetics in the striatum is consonant with the presence of two populations of sites with dissociation half times of 2 s and 27 s, comprising 88% and 12%, respectively, of the total binding sites. On the basis of previous pharmacological characterization of [3H]cocaine binding, we propose that the slowly dissociating component represents the sites associated with 5-hydroxytryptamine (serotonin) uptake, and the rapidly dissociating component the 3,4 dihydroxyphenylethylamine (dopamine)-related sites. Evidence is presented that the extremely high dissociation rates do not preclude the measurement of [3H]cocaine binding by rapid filtration. The dissociation of [3H]cocaine from cerebrocortical membranes is slowed to a small but statistically significant extent by serotonin.

Notes: Books review in this article: Photoreceptors (NATO AS1 Series A. Life Sciences, Vol. 75) edited by A. Borsellino and L. Cervetto Hormonal Control of the Hypothalamo-Pituitary-Gonadal Axis edited by K. W. McKerns and Z. Naor International Review of Neurobiology, Vol. 25 edited by C. C. Pfeiffer and J. R. Smythies. The Neurobiology of Zinc edited by C. J. Frederickson, G. A. Howell, and E. J. Kasarskis, Alan R. Liss

Notes: Abstract: Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass-fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 ± 38 pmol ml-1. In contrast, the concentration in control patients affected by different pathologies was 22 ± 7 pmol ml-1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 ± 0.6 versus 0.80 ± 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.

Notes: Abstract: Arachidonic acid is transiently accumulated in the brain as a result of a variety of pathological conditions. The synthesis and release of some of its metabolites, namely, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1α (6-keto-PGF1α) from cortical slices of mice were studied following exposure to 6 min of hypoxia (7% O2), 45 s of anoxia, and 5 min-4 h of reoxygenation following anoxia. Hypoxia induced a slight increase in the rate of TXB2 release and a slight decrease in the rate of PGE2 release, whereas 6-keto-PGF1α was unaffected. Anoxia (45 s) followed by reoxygenation induced a transient increase in the release of PGE2 and of 6-keto-PGF1α with a return to the normal rate at 30 min and 2 h of recovery, respectively. However, the rate of TXB2 synthesis and release reached its peak (twofold increase) after I h and remained significantly higher than the control rate even after 4 h of normal air breathing. Our results demonstrate that hypoxia and anoxia, even of short duration, selectively trigger the activity of thromboxane synthetase and that this elevated rate of synthesis and release persists long after normal oxygen supply is restored. We suggest that enhanced thromboxane synthesis, with normal prostacyclin levels, might have a role in the pathophysiology of ischemic cell damage.

Notes: Abstract: The relationships between presynaptic acetylcho-linesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: (a) determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; (b) determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; (c) evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and (d) comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 μM [N-methyl-3H] ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was of the amount passing through the synapto-somal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.

Notes: Abstract: Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]α-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an α-bungar-otoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatogra-phy step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]α-bungaro-toxin to P2 membranes was inhibited by (-)-nicotine (Ai= 9 × 10-6 M), and this sensitivity was preserved after affinity purification. It is proposed that α-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]α-bungarotoxin displayed little stereoselectivity. In this respect, the brain α-bungarotoxin binding site resembles the nicotinic acetyl-choline receptor from Torpedo electric organ.

Notes: Abstract: Three-week-old rats were made hypocalcemic or hypercalcemic by being fed diets low or high in Ca. Both total and ionized [Ca]s in the plasma decreased about 40% and remained depressed for 4 weeks in rats fed a low-Ca diet. Plasma [Ca]s in rats fed a high-Ca diet increased by 30% and remained elevated for 7 weeks. After 8 weeks on the diets, cerebrospinal fluid (CSF) [Ca] changed by 〈30% whereas brain [Ca] changed by 〈20% of the chronic changes in plasma ionized [Ca]. Assuming a brain extracellular volume of 20% and noting that brain extracellular volume equilibrates freely with CSF, the findings demonstrate only small perturbations in the Ca content of the brain cellular compartment during sustained hypo or hypercalcemia. Partial regulation of CSF and brain extracellular Ca suggests a role for the blood-brain barrier in regulating CNS [Ca] during chronic changes in plasma [Ca].

Notes: Abstract: The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37°C of [monoiodo-Tyr3 neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preprations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1 (R, S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p-aminobenzoate. The secondary processing occurring on neurotensin degradation products are: (1) a bestatin-sensitive aminopeptidasic conversion of neurotensin, 11-13 to free Tyr11, and (2) a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.

Notes: Abstract: Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 ± 11 fmol/mg protein and a KD value of 1.4 ± 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 μM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: (1) preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; (2) concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and (3) NT lost its capacity to release PRL in the absence of extracellular calcium. The significant correlation of the effective concentrations of NT and its analogs for binding and biological activities indicates that occupancy of NT receptors in anterior pituitary modifies calcium channel permeability of the cell membranes, resulting in an accumulation of intracellular calcium that participates in the cascade of events that are ultimately expressed as an enhanced release of PRL.

Notes: Abstract: The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated (1) by the order of potency of agonists in inhibiting PRL release and calcium influx (DA 〉 epinephrine 〉 norepinephrine isoproterenol); (2) by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and (3) by the high degree of stereoselectivity of sulphide. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells. The putative correlation between DA suppressing activity of the NT effects and the adenylate cyclase system was verified in pertussis toxin-treated pituitary cells. At the time that DA receptors negatively coupled to inhibition of adenylate cyclase were not operative, DA was still fully effective in preventing NT stimulation of PRL release. The lack of effect of low concentrations of DA on both basal adenylate cyclase activity and cyclic AMP content in pituitary cells, together with their capacity to inhibit the NT effects in pertussis toxin-treated cells, suggest that DA may act independently of cyclic AMP in the control of PRL secretion. These observations lead to the hypothesis of the existence of two inhibitory mechanisms for DA in mammotrophs.

Notes: Abstract: Soluble extracts from nerve growth factor (NGF)-stimulated PC 12 cells prepared by alkaline lysis show a two-to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4–7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5′-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.

Notes: Abstract: Twenty-day-old mice received a single tail vein injection of [guanido-14 C]arginine. The cytoskeleton was extracted from the spinal cords at varying lengths of time thereafter. Glial fibrillary acidic protein (GFAP) formed a distinct, broad band that was widely separated from other protein bands in one-dimensional polyacrylamide gels. The purity of the GFAP band was verified by Western blot analysis of one- and two-dimensional electrophoretic patterns. In addition, enzyme-linked immunosorbent assay and quantitative Western blot analysis indicated that 95% of the total spinal cord GFAP was extracted in the cytoskeletal preparation. The specific activity of GFAP was obtained by eluting the protein from the cytoskeletal GFAP band in preparative one-dimensional gels. Specific activity reached a peak 2 h after injection with [14C]arginine. Forty percent of the incorporated radioactivity was still present in cytoskeletal GFAP at 9 weeks, indicating that a significant proportion of glial filaments turns over relatively slowly in vivo.

Notes: Abstract: Studies were conducted on the properties of 125I-labeled α-bungarotoxin binding sites on cellular membrane fragments derived from the PC 12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC 12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC 12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.

Notes: Abstract: The concentration of gangliosides in the Snell dwarf mouse cerebrum was monitored from postnatal day 5 to day 40. In the dwarf cerebrum, the concentration of total gangliosides increased up to postnatal day 20 and then stopped, whereas in the control cerebrum, it continued to increase up to postnatal day 40. At postnatal day 40, the ganglioside level in the dwarf cerebrum was 70% of that in the control cerebrum. Among the ganglioside species, the concentrations of GM4, GM2, GM1, GD1a, GD3, GD1b, GT1b, and GQ1b were significantly lower in the dwarf cerebrum than in the controls at postnatal day 40. The reduced concentrations of ganglioside species GM2, GD1a, GD3, GD1b, and GQ1b, were completely restored by administration of bovine growth hormone (GH) during the first 20 days of postnatal life. The reduced concentration of the GM1 and GM4 species were most efficiently restored by administration of bovine GH plus thyroxine (T4) during the second 20 days of postnatal life. These results indicate that the lower ganglioside concentrations in the dwarf cerebrum can be elevated by hormone therapy and that there exist distinct GH and T4 actions on the enzymes participating in ganglioside metabolism.

Notes: Abstract: Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magno-cellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on re-combinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was 〈1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdis-sected supraoptic nucleus (SON) mean (SEM) basal levels of 1.370.18pgVP mRNA and 1.950.I4pgOT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 ± 0.02 pg VP mRNA and 1.77 ± 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85-fold increase in VP mRNA levels of the SON and a 1.6-fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.

Notes: Abstract: Angiotensin-converting enzyme (ACE) activity of Neuro-2A mouse neurobiastoma cells was found predominantly in particulate fractions. Density gradient centrifugation of the particulate fractions showed ACE activity in light fractions of the gradient, a result suggesting a plasma membrane localization. This was confirmed using the aqueous two-phase polymer system of plasma membrane isolation. The rapid and energy-independent hydrolysis of exogenous substrate by ACE of intact cells and the sensitivity of the enzyme of intact cells to proteases indicate further that the active site of ACE is oriented extracellularly.

Notes: Abstract: The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1–0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguano-sine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleo-sides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood-brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.

Notes: Abstract: Cells of the rat pheochromocytoma line PC 12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of β and iso-forms of nonmuscle actin in extracts of these differentiating cells showed that the β: ratio decreased from 1.30 ± 0.05 to 0.99 ± 0.05 after 6 days of NGF treatment. Cells treated with. N6, O2-dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of β: isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the β: actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension-grown PC 12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a “second messenger” for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC 12 cells.

Notes: Abstract [3H]Adenosine was taken up and metabolized by isolated ganglia of the marine mollusc Aplysia californica. After 2 h, most of the radioactivity was recovered as metabolites, including ATP, ADP, and AMP, as well as the deaminated products, inosine, hypoxanthine, and uric acid. Little remained in the form of adenosine. These pathways were not uniformly distributed among various tissue elements. In most individual neurons, inosine and its breakdown products were the principal metabolites of [3H]adenosine, whereas ATP and other nucleotides predominated in the connective tissue sheath. Endogenous levels of ATP, ADP, AMP, and adenosine in ganglia, sheath, and individual neurons were also determined using a fluorimetric-HPLC assay. The concentrations of the nucleotides were quite uniform in sheath and among the individual neurons assayed (1–5 pmol/μg of protein); however, concentrations of adenosine were considerably higher in neurons than in the sheath.

Notes: Abstract: A solid-phase radioimmunoassay, specific for the monomeric form of human Thy-1, was developed and used for quantitation of the Thy-1 antigen in human brain tissue. Determination of Thy-1 in homogenates of 12 anatomically defined brain regions showed that Thy-1 is present throughout the human brain. However, significant variation was found in the expression of the glycoprotein in different regions. Thy-1 appears to be generally enriched within gray matter: caudate nucleus, cerebral cortex, and putamen were found to contain the highest Thy-1 concentration (approximately 2.5 μg Thy-1/mg protein). Interestingly, the cerebellar cortex contained only 25% of the Thy-1 concentration of cerebral gray matter. Cerebral subcortical white matter contained half the amount of Thy-1 compared to cerebral cortex. Determination of Thy-1 in subcellular fractions prepared from human brain biopsy tissue indicated that the highest relative concentration of Thy-1 is associated with synaptosomal membranes and myelin/axonal membrane fractions.

Notes: Abstract: The effect of 4 weeks of spontaneous chronic ethanol intake in drinking water and then ethanol withdrawal on the γ-aminobutyric acid (GABA) steady-state levels and turnover rates was investigated in 15 brain areas of C57 B1/6J alcohol-preferring mice. These mice did not display typical ethanol withdrawal convulsions. There was no statistically significant difference in the brain GABA steady-state levels among the control group, chronic ethanol-treated mice, and mice after ethanol withdrawal. In contrast, chronic ethanol treatment induced significant variations in GABA turnover rate, as measured by gabaculine-induced accumulation of GABA, in eight of 15 areas examined versus a decrease in seven brain areas [cerebellum (–29%), amygdala (–28%), olfactory tubercles (–24%), septum (–24%), striatum (–53%), frontal cortex (–21%), and hippocampus (–24%)]; an increase in turnover rate in the posterior colliculus (100%) was observed. At 26 h after ethanol withdrawal, in the seven areas in which GABA turnover rate decreased after spontaneous chronic ethanol intake, a return to the initial control value was observed; in the posterior colliculus, the turnover rate did not change, remaining higher than the control value. This persisting alteration of GABA turnover rate may be related to the absence of the ethanol withdrawal syndrome in the C57 mouse strain.

Notes: Abstract: Putative nicotine receptors in the human cerebral cortex were characterized with l-[3H]nicotine. l-[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25°C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimer's disease were comparable to age-matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimer's disease had a 50% reduction in the number of l-[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimer's disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimer's disease.

Notes: Abstract Neuronal and glial cell-enriched nuclei were prepared from the brain cortex of rats of different ages for study of alterations in the synthesis of cellular RNA with age. RNA synthesis by isolated neuronal nuclei was substantially reduced in senescent rats, whereas that of glial nuclei remained constant throughout development. The nuclear content of polyadenylic acid-containing RNA in neurons was particularly decreased in old rats. The results show that the activity of the chromatin-bound neuronal RNA polym-erase declines during senescence, an observation indicating an age-related reduction in template activity in neuronal nuclei. The activity of nuclear poly(adenylate) polymerase also progressively decreases in aging neurons. The decrease in the transcription and polyadenylation of nuclear RNA may contribute to the decline in neuronal protein synthesis observed in old animals.

Notes: Abstract 3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnor-apomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of gua-nylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5′-(γ-thio)triphos-phate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.

Notes: Abstract It has been postulated that changes in the concentration of 3-methoxytyramine (3-MT) in the brain might reflect changes in the release of 3,4-dihydroxyphenylethylamine (DA, dopamine) and, therefore, might be used as an index of dopaminergic activity in the brain. 3-MT is known to accumulate rapidly after death. Killing by microwave irradiation (MWR) is considered to be the method of choice to obtain “undisturbed” 3-MT concentrations. We measured striatal 3-MT concentrations even lower than those following MWR when the brains were excised and frozen in dry ice very rapidly (typical time between decapitation and freezing of the brain 22 s). There was a linear increase in striatal 3-MT concentration when the time between decapitation and freezing was varied between 13 and 300 s. Extrapolation to time zero indicated negligible amounts of 3-MT at the time of decapitation. In addition, it was observed that DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid decompose during the cooling phase after heating the brain by microwave. It is concluded that MWR induces arti-factual changes in the postmortem levels of DA and metabolites. Consequently 3-MT cannot be considered to be a reliable indicator of DA release in the rat brain.

Notes: Abstract The effects of arachidonic acid on glutamate and γ-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 μmol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015–0.6 μmol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 μmol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.

Notes: Abstract: 7S-Nerve growth factor (NGF) and its α, β-NGF, and γ subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material, β-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.

Notes: Abstract: The effects of hypoxia on metabolism of 5-hydroxytryptamine (5-HT or serotonin) and 3,4-dihydroxy-phenylethylamine (DA or dopamine) were compared with those on open-field activity in male CD-1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5-hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (−41%) and [3H]Trp to [3H]5-HT (39%). Hypoxia also reduced dihydroxy-phenylacetic acid (DOPAC) levels (−27%) and DOPAC/DA ratios (−20%). Open-field behavior, as measured in an automated activity monitor, decreased in a dose-dependent fashion with 75–150 mg/kg of NaNO2 (−35 to −90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine.

Notes: Abstract: The kinetic constants Km and Vmax for the uptake of γ-aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie-Hofstee plots and computer analysis. The Km values were much greater in 0.1-mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the “Km level.” Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 μM, 43 μM, and 3.9 mM, respectively. In contrast, only two uptake systems for d-aspartate were detected, with km values of 1.8 μM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.

Notes: Abstract: Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules 〈 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4–5 times for Lys and 2–13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5–15 times more [3H]Lys and [3H]Leu into proteins than did the shafts. When optic axons were removed from the nerve by degeneration, most of the PTPM activity was lost, indicating a primarily intraaxonal locus for the components of the reaction. SDS-PAGE of 3H-modified proteins showed the presence of radioactivity in proteins of ∼13,000–18,000 38,000–46,000, and 53,000 daltons. These experiments indicate that the components necessary for PTPM by amino acids are transported axonally in regenerating optic nerves of goldfish and that the greatest activity is in the most advanced tips of the growing nerve.

Notes: Abstract: Isolated rat neurointermediate lobes were incubated in vitro. The release of 3,4-dihydroxyphenylethylamine (dopamine, DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and methoxyphenylethanol (MOPET) was determined by HPLC with electrochemical detection. Under resting conditions, the outflow of metabolites was 35–50 times that of DA. HVA accounted for 50%, DOPAC for 45%, and MOPET for 5% of the metabolites. Although an equivalent of 40–50% of the tissue DA content was released per hour as metabolites, the tissue DA content was not reduced after 110 min of incubation. The spontaneous outflow of DA and its metabolites was not affected by the DA uptake inhibitor GBR 12921 (100 μM). Pargyline (10 μM) caused a time-dependent decrease of all metabolites (up to 90%). In the presence of GBR 12921 and pargyline, the spontaneous outflow of DA increased sevenfold. Removal of the intermediate lobe caused a 78% reduction in tissue DA content and a corresponding reduction of the outflow of metabolites. Electrical stimulation of the pituitary stalk (0.2 ms, 10 V, 15 Hz, three times for 1 min at intervals of 1 min) induced an increase in outflow of DA and all metabolites. DA accounted for 15%, HVA for 41%, DOPAC for 32%, and MOPET for 12% of the evoked release. The electrically evoked release of DA increased fourfold in the presence of GBR 12921 or pargyline and the effects of both drugs were additive. The evoked release of metabolites was not significantly affected by GBR 12921 but completely abolished by pargyline. In conclusion, oxidative deamination and O-methylation are important pathways for the catabolism of DA in the neurointermediate lobe. Most of the DA metabolites released spontaneously originate from DA that had been degraded intraneuronally without undergoing prior release. Reuptake and extraneuronal metabolism contribute to the inactivation of DA released during electrical stimulation.

Notes: Abstract: The effect of moderate insulin deficiency of 2 weeks in duration on hypothalamic catecholamine metabolism in food-deprived and meal-fed rats was evaluated. Hypothalamic tyrosine content in food-deprived (from 0700 to 1600 h), diabetic rats was normal. Also normal were the rates of 3,4-dihydroxyphenylalanine accumulation following aromatic amino acid decarboxylase inhibition, norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) clearance after tyrosine hydroxylase inhibition, and intraneuronal amine accumulation following monoamine oxidase inhibition. Differences in hypothalamic amine metabolism were apparent, however, when diabetic and normal rats were fed 2-g meals. The 3-methoxy-4-hydroxyphenylethyleneglycol sulfate accumulation rate was depressed in diabetic rats by the carbohydrate meal but was stimulated by the tyrosine-supplemented protein meal. In contrast, the tyrosine-supplemented diet had no effect on 3, 4-dihydroxyphenylacetic acid accumulation in diabetic animals, whereas the production rate in normal rats was increased. We conclude that normal responses occurring in hypothalamic, catecholamine metabolism after the consumption of a meal are modified by the presence of diabetes.

Notes: Abstract: It was suggested in a recent report by Phillips et al. [J. Neurochem.43, 479–486 (1984)] that the low-affinity binding of [3H]imipramme in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 μM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data), (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.

Notes: Abstract: A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1, 1′-(4-hydroxy-iminopyridinium)trimethylene (TMB-4) (I50= 8 μM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenyl-carboxypyridinium) dimethylether (HGG-12) (I50= 150 μM). As postsynaptic antagonists, the latter drugs are more potent (K1= 1.3-3.3 μM) than TMB-4 (K1= 50 μM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyper-activity of the cholinergic system.

Notes: Abstract: Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, d-3-hydroxybu-tyrate, and 14CO2 and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only ∼20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.

Notes: Abstract: Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons-medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 μg/ml). The 4-N, N-dimethylamino-azobenzene-4′-isothiocyanate (DABITC) derivatization method of Chang [Biochem. J.199, 537–545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur-containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+-dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur-containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur-containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.

Notes: Abstract: Ethanol increases the activity of “basal,” guanine nucleotide-and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2, D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.

Notes: Abstract The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihy-droxyphenylethylamine ([3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum ≥ cerebral cortex ≥ hippocampus, whereas for [3H]NE the order was hippocampus ≥ corpus striatum ≥ cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10-7M flu-oxetine whereas [3H]NE uptake was preferentially inhibited by 10-7M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.

Notes: Abstract: Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the photoaffinity-labeled benzodiazepine receptor in duck brain, two equally intensive bands of relative molecular masses of 53,000 and 54,000 were observed, thus being identical to the subunit pattern previously described for chicken brain. An attempt was made to chacterize further the avian subunits. Comparison of the fluorographic subunit pattern in cerebellum and telencephalon revealed a pronounced quantitative heterogeneity. Addition of ethyl β-carboline-3-carboxylate resulted in the selective inhibition of the 53,000 subunit. These results are compared with the mammalian subunit pattern in the corresponding brain regions. The findings are discussed in view of the apparent contradiction between the existence of regional heterogeneity when photoaffinity labeling is applied and its lack when monoclonal antibodies are used.

Notes: Abstract 22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K= 0.6–5.0 × 10−8M) and an insensitive population (Ki= 3.3–4.9 × 10−6M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki= 2.4 × 10−7M). A single population was also found in 6-day-old cultures (Ki= 5.3 × 10−7M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki= 1.3 × 10−9M and 9.6 × 10−7M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.

Notes: Abstract Sialyltransferase has been characterized in P2 pellets derived from animals of increasing age. The enzyme was found to be associated with the plasma membrane and to be developmentally regulated at times coincident with cell migration and fibre outgrowth. This regulation appeared to be due, in part, to an endogenous competitive inhibitor in the P2 pellet but not in the synaptosome. Optimal transfer of [14C]N-acetylneuraminic acid to endogenous synaptosomal acceptors was achieved only in the absence of detergent. Furthermore, the transferred sialic acid was found to be inaccessible to the action of membrane-bound sialidase. The significance of these findings is discussed.

Notes: Abstract The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenyl-ethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydrophenylalanine (l-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, l-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous l-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors par-gyline and (-)-deprenyl differentially affected the degradation of DA, since 100 μM pargyline was apparently more effective than 100 μM (-)-deprenyl. Reserpine and (±)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (±)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.

Notes: Abstract Human myelin basic protein was subjected to ionexchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106–116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.

Notes: Abstract RNA isolated from goldfish retinas before and during optic nerve regeneration, when translated in vitro, directed the synthesis of neurofilament proteins that are normally found in high levels in the optic nerve. The major neurofilament proteins of the goldfish optic nerve comprise a group of four isoelectric variants of molecular weight 58,000 (58K) which we have identified previously as ON1-ON4. The levels of ON1 and ON2 within the optic nerve had been shown to decrease shortly after optic nerve crush and then increase to precrush levels during the regeneration process. Employing two-dimensional electrophoretic analysis of in vitro translation products and immunoprecipitations with antibodies specific for the ON proteins and an anti-intermediate filament monoclonal antibody, we show that ON1 and ON2 are encoded by mRNA synthesized in the retinas. The synthesis of ON3 and ON4 by retina RNA was undetected. This confirms data from previous ex vivo experiments that indicated that ON1 and ON2 are of neuronal origin whereas ON3 and ON4 are nonneuronal. ON1 and ON2 synthesis increases dramatically during optic nerve regeneration to levels 10- and 30-fold over precrush levels, respectively. In addition to ON, and ON2, the synthesis of a previously unidentified 52K protein is observed at relatively high levels 20 and 32 days after optic nerve crush, but is unobserved before regeneration. Thus, optic nerve regeneration can be correlated with specific changes in intermediate filament gene expression within the retina.

Notes: Abstract In the human fetus, obtained postmortem at estimated gestational ages of 8–22 weeks, biochemical activities of cortical choline acetyltransferase (ChAT) and acetylcho-linesterase (AChE) were comparable to those of adult brain tissue. In contrast cholinergic receptor binding, including muscarinic Ml and M2 subtypes (measured by displacement of [3H]N-methylscopolamine with, respectively, pirenzepine and carbachol) and [3H]nicotine (putative nicotinic) binding were undetectable before 13–14 weeks and even at 22 weeks were substantially (three- to fourfold) below the respective adult values. Cortical ChAT activity decreased significantly with gestational age whereas binding to the three receptors, including the proportion M1/M2, increased significantly. AChE was present at all ages investigated as the two molecular monomeric (G1) and tetrameric (G4) forms. The proportion of G4, which was much more soluble in fetal compared with adult cortex, increased approximately threefold. Histochemically AChE, although intense in the nucleus of Meynert, was generally confined to subcortical white matter at early fetal developmental periods, appearing later in the cortex localized to nerve fibres and occasional cell bodies. These observations suggest that during the second trimester of human fetal development, cortical cholinergic function may be preceded by relatively high ChAT activity and paralleled not only by increasing receptor binding but also by a proportional increase in the tetrameric form and histochemical reactivity of AChE.

Notes: Abstract High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, β-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.

Notes: Abstract We have isolated PC12 cell variants deficient in transporter-mediated uptake of 3,4-dihydroxyphenylethylamine (dopamine). The variants either were obtained nonselectively, or they were selected by resistance to guanethidine or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Dopamine uptake into guanethidine-resistant cells occurred with a decreased Vmax; the Km for dopamine and inhibition by guanethidine were normal. MPTP-resistant cells lacked the capacity to take up dopamine. Most of the variants resembled wild-type PC12 in their response to nerve growth factor and the storage and secretion of dopamine. MPTP-resistant cells exhibited several deficiencies in addition to dopamine transport, i.e., no measurable storage of dopamine or acetylcholine and no observable response to nerve growth factor. Wild-type and variant cells were compared with respect to the labeling of cell proteins with [3H]xylamine, which binds covalently to certain proteins apparently only after entering PC12 via the catecholamine transporter. When intact variant cells were used, there was markedly reduced labeling of the proteins by [3H]xylamine. Almost all of these proteins were readily labeled when cell homogenates were exposed to [3H]xylamine. However, MPTP-resistant cells were missing three of these proteins. Northern blot analysis with cDNA clones revealed that the MPTP-resistant cells had markedly reduced levels of several specific mRNA species.

Notes: Abstract The effect of the presynaptic neurotoxin β-bungarotoxin (β-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50–60% stimulation of the vesicle proton-pumping ATPase activity. The β-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by β-BuTx, which results in uncoupling of the ATPase and ACh transporter systems.The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more β-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of β-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of β-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.

Notes: Abstract: Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R. I., Epstein P. M., Andrenyak P. M., and Feinstein M. B. (1981) Proc. Natl. Acad. Sci. USA 78, 795–799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.

Notes: Abstract: The synaptosomal effluxes of d-aspartate and γ-aminobutyric acid (GABA) induced by a substitution of the Cl- ions with propionate in the incubation medium were measured in preparations of hippocampal tissue homogenates. The efflux of aspartate was significantly higher than spontaneous efflux at 125 mM Cl- (normal = 144 mM) and was increased with decreasing Cl- concentration. GABA efflux was much less sensitive to a reduction in Cl concentration than D-aspartate. The efflux was Ca2+-dependent in both cases.

Notes: Abstract: We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of l-glutamate (l-Glu). Oxidation of l-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from 〈 0.5 to 500 pmol of l-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (〉 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.

Notes: Abstract: The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.

Notes: Abstract: Nicotine evokes the release of catecholamines from bovine adrenal glands perfused with oxygenated Krebs-bicarbonate solution. Two 2-min pulses of 5 μM nicotine, at 40-min intervals (S1 and S2), gave net catecholamine outputs of 45.2 ± 3.6 and 29.1 ± 3.5 μg/8 min, respectively. Apomorphine (1 or 10 μM) markedly inhibited catecholamine release during S2 to 9.1 ± 2.2 and 0.5 μg/8 min, respectively. Haloperidol (0.5 μM) reversed the inhibitory effects of apomorphine. Haloperidol alone enhanced catecholamine release induced by nicotine to 67.9 ± 7.9 μg/8 min. [3H]Spiperone binds to adrenomedullary membranes with a KD of 0.24 nM and a Bmax of 117 fmol/mg of protein. Whereas spiperone and haloperidol potently displaced such binding, 3,4-dihydroxyphenylethylamine (dopamine) and sulpiride were poorer displacers, and SCH23390, prazosin, phenoxybenzamine, propranolol, BAY-K-8644, and nitrendipine did not displace [3H]spiperone bound. These data strongly suggest that, as in the cat, the bovine adrenal medulla chromaffin cell contains a dopaminergic receptor that modulates the catecholamine secretory process triggered by stimulation of the nicotinic cholinoceptor. Such a receptor seems to be of the D2 type and might be involved in a sympatho-adrenal cooperative mechanism contributing to the maintenance of cardiovascular homeostasis during stressful situations as well as to the pathogenesis of hypertension. If so, selective dopaminergic agonists might prove clinically useful in the treatment of hypertension.

Notes: Abstract: Endogenous noradrenaline and 3,4-dihydroxyphenylethylamine (dopamine) levels were measured in different zones of the dog kidney following chronic unilateral renal denervation. In outer and inner renal cortex, and in outer medulla, 〉95% of the tissue content of both catecholamines was contributed by renal nerves, whereas in inner medulla only nonneuronal catecholamines were found. The amounts of neuronal dopamine present in outer renal cortex were greater than would be expected for a population of solely noradrenergic nerves.

Notes: Abstract: Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was detected in the cholinergic electromotor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP-like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle-specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP-like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (-50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP-like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse-phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP-like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.

Notes: Abstract: Continuous treatment (1–10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the β-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of β-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5′-yl-β-γ-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that β–receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of β-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.

Notes: Abstract: Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phospha-tase did not cause a dissociation of the 210–kD (NF-H), 160–kD (NF-M), or 70–kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.

Notes: Abstract: Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected sub fractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by “free” mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.

Notes: Abstract: We report here that anti-L1 antiserum, raised against material from embryonic brain, and anti-NILE antiserum, raised against purified NILE (nerve growth factor-inducible large external) glycoprotein of PC12 cells, immunoprecipitate from PC12 cells material of the same apparent molecular weight (230 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, each of these immune reagents has the capacity to clear from a PC12 cell extract all of the 230-kilodalton antigen recognized by the other antiserum. Finally, in immunohistochemicai staining of developing cerebellum the two antisera exhibit very similar staining patterns. We suggest that the NILE glycoprotein and the high-molecular-weight component of L1 antigen are closely related molecules, and probably the same.

Notes: Abstract: The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5′-N-Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2-fold). 2-Chloroadenosine (2-CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 μM dipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50 value for R-(-)-N6(2-phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to the K1 value for inhibiting the response to NECA. The EC50 value for adenosine was substantially higher than the K1 value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8-phenyltheophylline, 1,3-diethyl-8-phenylxanthine, and 8-p-sulfophenyltheophylline, with K1 values from 1 to 10 μM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low-affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor.

Notes: Abstract: The inhibition of the A and B forms of monoamine oxidase (MAO) inside and outside serotonergic, noradrenergic, and dopaminergic synaptosomes in homogenates of rat hypothalamus or striatum by clorgyline, a selective and irreversible MAO-A inhibitor, and selegiline, a selective and irreversible MAO-B inhibitor, was examined. Intrasynaptosomal deamination at low concentrations of the substrates [14C]5-hydroxytryptamine ([14C]5-HT; 0.1 μM), [14C]noradrenaline (0.25 μM), [14C]3,4-dihydroxyphenylethylamine ([14C]dopamine; 0.25 μM), and [14C]tyramine (0.25 μM), was hindered by selective uptake inhibitors (citalopram, maprotiline. and amfonelic acid) in the incubation media. Thus, the difference between the deamination of 14C-amine in the absence and presence of the appropriate selective uptake inhibitor provided a measure of deamination in the specific aminergic synaptosomes. This was verified by determining the loss of MAO activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine and p-chloroamphetamine. Results with the two inhibitors revealed that the A and B forms were responsible for 80 and 20%, respectively, of the deamination of [14C]5-HT within serotonergic synaptosomes from the hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes from the hypothalamus and that of [14C]dopamine and [14C]tyramine within the striatal dopaminergic synaptosomes were due to MAO-A. About 10% of the deamination of [14C]noradrenaline, [14C]dopamine, and [14C]tyramine outside the noradrenergic or dopaminergic synaptosomes was brought about by the B form, with the remainder being deaminated by MAO-A.

Notes: Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre- and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (〉 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age-related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.

Notes: Abstract: The originally described sequence of human myelin basic protein peptide 45–89 has recently been shown to contain two errors which have now been resolved. In the present study fast atom bombardment mass spectrometry was utilized to analyze the primary sequence of the other portions, peptides 1–44 and 90–170 of human myelin basic protein. The results obtained confirm the accuracy of the primary sequence published for both of these terminal peptides.

Notes: Abstract: Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented 〈 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma × glioma hybrid, NG 108–15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10−8M) for its receptors on striatal neurons and NG 108–15 whereas an apparent low affinity (EC50 7 × 10−6M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form.

Notes: Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [3H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [3H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [3H]uridine by 〉 75%. Cellular uptake of [3H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [3H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.

Notes: Book Reviewed in This Article: Advances in Neuroblastoma Research (Progress in Clinical and Biological Research, Vol. 175) edited by A. E. Evans, G. J. D'Angio, and R. C. Seeger. Alan R. Liss Neurochemical Techniques in Insect Research edited by H. Breer and T. A. Miller. Cell Culture in the Neurosciences edited by J. E. Bottenstein and G. Sato. The Developing Brain and Its Disorders edited by M. Arima, Y. Suzuki, and H. Yabuuchi. Neurobiology: Current Comparative Approaches edited by R. Gilles and J. Balthazart.

Notes: Abstract: A single slice of rat pons that contained the locus ceruleus (LC) or two slices of cerebellum were loaded with [3H]noradrenaline; superfusion with high (35 or 60 mM) potassium solutions evoked a release of 3H. In the presence of normorphine, the release of 3H evoked by 35 mM potassium and 60 mM potassium was reduced. In some of those experiments in which the release of 3H from the LC slice was measured, an intracellular microelectrode was used to measure membrane potential. This showed that solutions of increased potassium concentration depolarized the neurons to a potential at which inward calcium currents flowed (calcium action potentials occurred). Normorphine hyperpolarized the neurons; during this hyperpolarization the depolarization caused by 35 mM potassium did not reach the threshold for significant calcium entry. The results suggest that the inhibition by normorphine of transmitter release evoked by solutions of raised potassium concentration could result in part from the membrane hyperpolarization caused by the normorphine.