Library

Notes: Abstract: The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAQ but not agonist (R-N6-−[3H]phenyl-isopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation. This is firm evidence that guanine nucleotides and NaCl must decrease agonist affinity by distinct mechanisms.

Notes: Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins (240, 105, and 65 kilodaltons) were eluted by an EGTA-containing buffer. 125I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common (65, 60, 53, and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.

Notes: Book reviewed in this articles: A History of Neurophysiology in the 19th Century by Mary A. B. Brazier. Psychopharmacology: The Third Generation of Progress edited by H. Y. Meltzer. Molecular Genetics of Neurological and Neuromuscular Disease (Advances in Neurology, Volume 48) edited by Stefano DiDonato, Salvatore DiMauro, Angelo Mamoli, and Lewis P. Rowland. Molecular Neurobiology in Neurology and Psychiatry (Association for Research in Nervous and Mental Disease [ARNMD], Research Publications, Volume 65) edited by Eric R. Kandel. Central Nervous System Disorders of Aging: Clinical Intervention and Research (Aging, Volume 33) edited by Randy Strong, W. Gibson Wood, and William J. Burk. Animal Models of Dementia: A Synaptic Neurochemical Perspective (Neurology and Neurobiology, Vol. 33) edited by Joseph T. Coyle.

Notes: Abstract: Expression of the D1.1 ganglioside was studied immuno-histochemically in developing cerebella from normal and weaver mutant mice. In the normal cerebellum at postnatal day 7 (P7), D1.1 expression was restricted to the external granule-cell layer (EGL). At later ages, D1.1 disappeared as the developing granule neurons ceased mitosis and began migrating toward the internal granule-cell layer. In the weaver cerebellum, D1.1 was expressed in the EGL in apparently normal fashion at P7, but failed to disappear at later ages. As late as P35, D1.1 immunoreactivity was observed throughout the weaver cerebellar cortex. The relative amounts of D1.1 ganglioside in weaver and normal cerebella were compared by thin layer chromatography of total gangliosides, followed by overlay of the chromatogram with anti-D1.1 and 125I-labelled second antibody. Autoradiograms showed that at P12 and P35 the weaver tissue contains six- to tenfold more D1.1 than normal tissue. These findings suggest that one result of the weaver mutation is prolonged expression of D1.1. We speculate that the D1.1 ganglioside might be involved in adhesive interactions that regulate the timing of granule-cell migration from the EGL. The prolonged expression of D1.1 could be responsible, in part, for the failure of granule-cell migration in the weaver cerebellum.

Notes: Abstract: Microtubules were prepared from head extracts of the adult fruit fly, Drosophila melanogaster, by one-step, taxol-assisted polymerization. The microtubular fraction displayed cyclic AMP-dependent protein kinase (protein kinase A) activity, as witnessed by endogenous protein phosphorylation and by protein kinase assay. Microtubule-bound protein kinase A amounts to 4–5% of total soluble kinase activity, which is almost an order of magnitude less than in mammals. The high-molecular-weight microtu-bule-associated protein-2 (MAP-2), the main binding species for protein kinase A in mammalian brain microtubules, is not detectable in the fly system by protein staining and immunoblotting with anti-pig MAP-2 serum, as well as by hybridization of fly DNA with a cDNA probe for human MAP-2. Cyclic AMP removes a major part of the regulatory (R) subunit of the enzyme from Drosophila microtubules, as demonstrated by enzyme assay, autophosphoryla-tion of R subunit, and quantitating cyclic AMP binding sites. It is proposed that permanently elevated cyclic AMP levels may elute protein kinase A from crucial intracellular binding sites, thereby interfering with signal transduction.

Notes: Abstract: The phorbol diesters 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate, but not 4–α-phorbol-didecanoate, inhibited the stimulation of inositol phospholipid hydrolysis by excitatory amino acids and carbamylcholine in primary cultures of cerebellar neurons. This inhibition was mimicked by the synthetic diacylglycerol 1,2-dioleoyl-rac-glycerol (DOG) and was selective for a specific glutamate-phosphoinositide receptor subtype (GP2 receptor) activated by glutamate and quis-qualate. TPA was nearly inactive in inhibiting the stimulation of inositol phospholipid hydrolysis by N-methyl-d-aspartate, a selective agonist of the GP1 receptor. Phorbol diesters and DOG attenuated the stimulation of inositol phospholipid hydrolysis by glutamate and quisqualate also in cerebellar slices from 9–15-day-old rats; however, using this preparation, their action was weak and required high concentrations (〉 1 μM). The inhibition of signal transduc-tion by phorbol diesters was not consequent to a reduced binding of glutamate to its membrane recognition sites. In fact, TPA induced only a small increase in the KD but no change in the Bmax of [3H]glutamate binding in cerebellar membranes. Phorbol diesters may act to inhibit specific GTP-binding proteins or particular molecular forms of phosphoinositidase C associated with GP2 or muscarinic cholinergic receptors.

Notes: Abstract: An antibody that inhibits over 95% of the cytosolic NADP+-dependent 7-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1–14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1–14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADPT and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-depen-dent cytosolic oxidoreductase which catalyzes this reaction.

Notes: Abstract: The contents of cystathionine and taurine, as well as cystathionine β-synthase activity, in various cerebellar regions and pineal body of normal and dl-propargylgly-cine-treated rats were measured. The contents of cystathionine and taurine were found to be distributed unevenly in cerebellar regions of brain of both normal and dl-propargylglycine-treated rats. The content of cystathionine in each cerebellar region and pineal body increased gradually when the dose of DL-propargylglycine was increased from 10 mg to 30 mg per 200 g body weight. On the other hand, taurine content in each cerebellar region and pineal body decreased with the administration of 30 mg of dl-propargylglycine per 200 g body weight. The contents of cystathionine and taurine were greater in the pineal body than in various cerebellar regions. The activity of cystathionine β-synthase was also distributed unevenly in various cerebellar regions of normal rat brain, and was unaltered following treatment of rats with dl-propargylglycine.

Notes: Abstract: Stimulated overflow of dopamine (DA) into the extracellular fluid of the rat caudate nucleus was measured with fast-scan cyclic voltammetry. DA concentrations were sampled in less than 10 ms at 100-ms intervals with a Na-fion-coated, carbon-fiber microelectrode. Overflow of DA was induced by electrical stimulation of the medial fore-brain bundle with 300–μA pulses of various duration and frequency. Stimulated overflow was measured as a function of stimulus duration before and after administration of benztropine, bupropion, and amphetamine. These results were correlated with simulated curves based on a simple uptake/overflow model. The observed overflow was assumed to be a function of [DA]P, the concentration of DA which overflows per stimulus pulse, and the kinetics of cellular uptake of DA. Correlation of experimental with simulated results was obtained at the 95% confidence limit for the duration studies; however, it was not possible to distinguish between the effects of pharmacological agents on uptake and overflow. In contrast, modulation of stimulus frequency did permit such distinction. Simulations of an increase in [DA]P fit results following dihydroxyphenyl-alanine methyl ester at 95% confidence limits, whereas an equivalent change in the apparent Km did not fit. An increase in the apparent value of Km correlated with results obtained at different frequencies following nomifensine and bupropion administration at the 95% confidence limit, whereas an equivalent increase in [DA]P did not fit. The effects of GBR 12909 best correlated with an increase in the DA available for overflow.

Notes: Abstract: We examined the effects of in vivo hypoxia (10% O2/90% N2) on the γ-aminobutyric acid (GABA)/benzo-diazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.

Notes: Abstract: Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel nitration. The purified preparation showed a single protein band on sodium dodecyl sul-fate-polyacrylamide gel electrophoresis using both a gradient gel (3.75–12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to close of porcine brain MAP2. Im-munoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.

Notes: Abstract: The regional and cellular distribution of four synaptic vesicle-associated proteins, synapsins Ia and Ib (Mr 86,000 and 80,000, collectively referred to as synapsin I) and proteins IIIa and IIIb (Mr 74,000 and 55,000, collectively referred to as protein III), has been compared in selected rat brain regions, using both radioimmunoassays and back-phosphorylation assays. Lesions of several neuronal populations in the basal ganglia (corticostriatal fibers, intrinsic striatal neurons, striatonigral fibers, nigrostriatal fibers) induced decreases in the levels of these various proteins that were highly correlated (r= 0.96–0.97). Moreover, the synaptic vesicle-associated phosphoproteins displayed a similar and widespread distribution throughout the CNS. This evidence for colocalization indicates that the majority of, and possibly all, CNS neurons and nerve terminals may contain both forms of synapsin I and both forms of protein III.

Notes: Abstract: Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as loss of righting reflex. Measurement of γ-aminobutyric acid (GABA) content of brain tissue from symptomatic pyrithiamine-treated (PT) rats revealed significant reductions in thalamus, cerebellum, and pons. GABA content of cerebral cortex, however, was unaltered. Activities of the thiamine-dependent enzyme α-ketoglutarate dehydrogenase (aKGDH) were reduced in parallel with the GABA changes. On the other hand, activities of the GABA-synthetic enzyme glutamic acid decarboxylase (GAD) remained within normal limits, with the exception of a small but significant decrease in thalamus of symptomatic PT rats. Affinities and densities of high-affinity [3H]muscimol binding sites on crude cerebral membrane preparations from symptomatic PT rats were unchanged. Thiamine administration to symptomatic animals resulted in correction of abnormal righting reflexes and in normalization of decreased GABA levels and reduced αKGDH activities in cerebellum and pons. Thalamic GABA levels and αKGDH activities, on the other hand, remained significantly lower than normal. These results suggest that the reversible symptoms of pyrithiamine treatment may result from impaired GABA synthesis in cerebellum and pons of these animals. Similar mechanisms may play a role in the pathogenesis of the reversible symptoms of Wernicke′s encephalopathy in man.

Notes: Abstract: Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as γ-glutamyl octopamine and γ-glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine-rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of γ-glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that γ-glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that γ-glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.

Notes: Abstract: A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid-binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus-induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain-Barre syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.

Notes: Abstract: Infusion of tetrodotoxin (TTX) through the dialysis membrane and perfusion with calcium-free Ringer solution (calcium depletion) were used to evaluate the dopamine release determined by in vivo brain dialysis. Several hours after implantation, the dopamine release recorded by the U-shaped cannula did not respond to calcium depletion and was only partly (˜50%) TTX dependent. The half-life of the TTX-independent dopamine overflow was determined to be 2 h. In contrast, when a transstriatal cannula was used, the dopamine output displayed calcium and TTX dependency. Differences in the dimensions of the two types of probes are a likely explanation for the observed effects. Twenty-four hours after implantation, both types of cannula fulfilled the criteria of calcium and TTX dependency. The results indicate that infusion of TTX-contain-ing or calcium-free Ringer solution can be used to estimate the functional damage caused by the implantation of the cannula.

Notes: Abstract: This work shows that unsaturated fatty acids enhance the epinephrine-stimulated adenylate cyclase activity in bovine retina. The modulating effect on the epinephrine-stimulated formation of cyclic AMP seems to be linked to the degree of unsaturation of the fatty acid. Treatment of the intact retina with docosahexaenoic acid in the concentration range 0.5 × 10-6-l × 10-3M does not affect the enzyme activity measured in the absence of the hormone but markedly increases the cyclase activity when the tissue is incubated in the presence of 0.1 mM epineph-rine. Docosahexaenoic acid enhances the maximal response to epinephrine without affecting the apparent ED50 value for this effector. Docosahexaenoic acid at 0.5 mM also increases the hormone-stimulated adenylate cyclase activity in retinal cell-free homogenate, whereas it has no effect on the epinephrine-sensitive enzyme solubilized from the membrane fraction with 1% Triton X-305. When docosahexaenoic acid-preincubated intact retina and cell-free Homogenate are incubated in the presence of defatted albumin, both the observed activating effect of the fatty acid on the epinephrine-stimulated adenylate cyclase activity and the enhancement of the enzyme response to the hormone significantly diminish.

Notes: Abstract: Stimulation of phosphoinositide (PI) hydrolysis by excitatory amino acids (glutamate and ibotenate) or nor-epinephrine was potentiated in hippocampal slices from rats trained in an eight-arm radial maze, used as a test of spatial learning. No difference in basal or carbamylcho-line-stimulated PI hydrolysis was found between control and trained animals. An increased PI response to excitatory amino acids and norepinephrine was not found in hippocampal slices prepared from animals trained in a shock conditioning avoidance test. These results suggest a possible involvement of specific glutamate receptors coupled with PI hydrolysis in the synaptic mechanisms underlying formation and/or storage of spatial memory.

Notes: Abstract: Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37°C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (〈4 kilodaltons) and large (〉8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaC12, zinc ions in the range 0.1–1 mM inhibited MBP-membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (〈0.1 mM) inhibited MBP-membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X-100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone-binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP.

Notes: Abstract: The distribution of thiamine, thiamine phos-phoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydro-lyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.

Notes: Abstract: We report an electrophoretic analysis of the hy-drophobic properties of the globular forms of acetylcholin-esterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. In charge-shift electrophoresis, the rate of electrophoretic migration of globular amphiphilic forms (Ga) is increased at least twofold when the anionic detergent deoxycholate is added to Triton X-100, whereas that of globular nonamphiphilic forms (Gna) is not modified. The G2a forms of the first class, as defined by their aggregation properties, are converted to nonamphiphilic derivatives by phosphatidylinositol phospholipase C (PI-PLC) and human serum phospholipase D (PLD). AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which also exists in very small quantities in detergent-solubilized extracts of electric lobes and spinal cord. They present different electrophoretic mobilities, so that each of these tissues contains a distinct “electromorph,” or two in the case of electric organs. The G2a forms of the second class (AChE in plasma, BuChE in heart), as well as G4a forms of AChE and BuChE, are insensitive to PI-PLC and PLD but may be converted to nonamphiphilic derivatives by Pronase.

Notes: Abstract: Glycine may be an inhibitory transmitter in the mammalian cochlear nucleus (CN). This study attempts to determine if cochlear and/or centrifugal projections to the CN use glycine as a transmitter. The high-affinity uptake and electrically evoked release of exogenous [14C]glycine were measured in vitro in the three major subdivisions of the guinea pig CN: the anteroventral, posteroventral, and dorsal cochlear nuclei (AVCN, PVCN, and DCN, respectively). [14C]Glycine (3.4 μM) was taken up by each subdivision, reaching tissue concentrations six to seven times that in the medium. Subsequent electrical stimulation evoked a Ca2+-dependent release of [14C]glycine from each subdivision. These activities were compared in subdivisions from unlesioned animals, and from animals with lesions of centrifugal or cochlear projections to the CN. Two knife-cut lesions were made to interrupt centrifugal projections to the CN lying in the right acoustic striae and trapezoid body. In one group of animals, centrifugal fibers projecting mainly to the right AVCN and PVCN were severed, which reduced [14C]glycine uptake and release by 44–53% in these subdivisions, but not in the right DCN. In another group of animals, fibers projecting mainly to the right PVCN and DCN were severed, which reduced [14C]glycine uptake and release by 33–47% in these subdivisions, but not in the right AVCN. In CN subdivisions contralateral to either lesion there was no significant change in [14C]glycine uptake or release. Neither of these lesions altered the uptake or release of D-[3H]aspartate in the right or the left CN. Ablation of the left cochlea, which presumably destroyed cochlear nerve fibers unilaterally, had no effect on [14C]glycine uptake and release. These observations suggest that centrifugal projections contribute a proportion of the glycinergic syn-aptic endings in the CN. In addition, some glycinergic endings probably arise from neurons intrinsic to the CN. The cochlear nerve contains very few, if any, glycinergic fibers.

Notes: Abstract: Binding of [3H](R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) to quisqualate receptors in the presence of SCN ions produced curvilinear Scatchard plots. Kinetic investigations of [3H]-AMPA binding showed that the curvilinearity cannot be explained by assuming binding to two separate binding sites or by considering it due to cooperative interaction. A more likely explanation is that the quisqualate receptors exist in two states, one with high and one with low affinity for [3H]AMPA. Chaotropic ions change the relaxation constant between the two states.

Notes: Abstract: Using the technique of transstriatal dialysis in halothane-anesthetized rats, we have studied the effects of intrastriatally infused N-methyl-D-aspartate (NMDA), kainate, and quisqualate on the liberation of endogenous striatal dopamine. The striatal infusion of NMDA (10-3-10-2M) or kainate (10-4-10-2M) but not of quisqualate (up to 10-2M) for one 20-min fraction provoked a dramatic increase in striatal dopamine efflux up to a maximum of 1,200 and 3,400% of basal levels for NMDA and kainate, respectively. NMDA (10-3M) evoked liberation of striatal dopamine was totally blocked by coinfusion of 2-amino-5-phosphonovalerate (2-APV; 5 × 10-4M) and by the systemic injection of phencyclidine (3 mg/kg i.p.). The effects of NMDA (10-3M) were also totally antagonized in a dose-dependent manner by the striatal coinfusion of atropine (10-7-10-4M), and abolished in rats that had received bilateral striatal ibotenate lesions (10μg/1μl) 1 week prior to implantation of the dialysis fiber. The striatal infusion of tetrodotoxin (10-6M) reduced basal dopamine efflux by 60–70% and abolished the NMDA (10-3M)-evoked liberation of striatal dopamine. The effects of kainate (10-3M) on striatal dopamine efflux were only partially reduced by doses of 2-APV or atropine that totally blocked the NMDA response, and were also partially resistant to tetrodotoxin. The intrastriatal infusion of carbachol (10-3M) for one 20-min fraction was without effect on striatal dopamine efflux; 10-2M carbachol produced a 50% increase in dopamine release in the fraction following carbachol application. The present results indicate that the effects of NMDA on striatal dopamine release are likely to be mediated via an active cholinergic striatal neuron which seems to play a permissive rather than an active role in this effect. The effects of kainate may involve a direct action on dopa-minergic terminals as well as activation of polysynaptic pathways or liberation of an endogenous NMDA receptor agonist.

Notes: Abstract: Tetanus exotoxin inhibited Ca2+-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca2+. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca2+ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca2+-dependent exocytotic pathway to inhibit secretion.

Notes: Abstract: Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to “high-affinity” and “low-affinity” binding sites. High-affinity quisqualate sites were termed “quisqualate-sensitive L-[3H]glutamate” binding sites. Quisqualate-sen-sitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and4,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 μM, kynurenate, L-aspartate, (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]-glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 μM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4°C compared with 37°C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.

Notes: Abstract: Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxy-phenolic compounds, and by hemin in a concentration-dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased.

Notes: Abstract: Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IPO in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 μM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3 Thi5,8, D-Phe7)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine Hi receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IPi accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carba-chol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, brady-kinin-induced [3H)IPi accumulation was found to require much less lithium. In the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-in-duced phosphoinositide breakdown was inhibited by 4β-phorbol 12, 13-dibutyrate, but was unaffected by the biologically inactive 4β-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the brady-kinin-induced [3H]IP1 accumulation, without affecting basal activity. These data might suggest that more than one type of GTP binding protein is involved in the accumulation of IP1. Preincubation of bradykinin (400 nM) with cells resulted in a time-dependent loss of the ability of bradykinin to stimulate phosphoinositide hydrolysis; more than 50% of the activity was lost after 45 min exposure at 37°C. The bradykinin response was markedly attenuated by prestimulation with bradykinin; this desensitization was time-dependent with a maximal effect observed after about 1 h prestimulation. Thus, the robust response of bradykinin on phosphoinositide turnover in NCB-20 cells may serve as another interesting model in the study of its role in some bradykinin-mediated physiological events.

Notes: Abstract: We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2′-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyrylcyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.

Notes: Abstract: Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.

Notes: Abstract: Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.

Notes: The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein ki-nase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphoryla-tion of both the α- and β-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothe-lial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.

Notes: Abstract: The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5′-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands. In particular, molecules carrying (an) intact, probably intramolecular, disulfide bond(s) show high affinity for agonists, whereas those carrying the reduced state of the relevant thiol group(s) show low affinity for agonists.

Notes: Abstract: Tonic-clonic seizures elicited in convulsionprone gerbils resulted in a large increase in immunoreactive prostaglandin (PG) F2α and in a smaller increase in immunoreactive leukotriene (LT) C4-like material in brain tissue. Brain tissue contents of both eicosanoids were found to reach a maximum at 6 min after the onset of seizures and were still elevated at 54 min after the beginning of convulsions. By reversed phase HPLC the immunoreactive LTC4-like material was identified as LTC4 and LTD4 at 6 min after the onset of convulsions, whereas at 54 min after the onset, transformation of LTD4 to LTE4 could be detected as well. In gerbils showing only weak seizure activity a small increase in PGF2α but no increase in immunoreactive LTC4-like material could be detected at 6 min after the onset of convulsions. Pretreatment with indomethacin abolished the formation of PGF2α but significantly enhanced the biosynthesis of immunoreactive LTC4-like material at 18 min after the beginning of seizures. The results demonstrate formation of cysteinyl-LT following tonicclonic convulsions in spontaneously convulsing gerbils which could be enhanced by inhibition of the cyclooxygenase pathway of arachidonic acid metabolism. Since cysteinyl-LT have potent biological actions in various organs this finding warrants further investigations on the potential role of cysteinyl-LT in the CNS.

Notes: Abstract: The binding of L-[3H]glutamate to membranes from human temporal cortex was studied in the absence of Na+, Ca2+, and Cl− ions. Pharmacological characterisation revealed that approximately 35% of specific binding at 50 nM L-[3H]glutamate was sensitive to a combination of kainate and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. The remaining approximately 65% of specific binding was to a single population of sites with a KD of 844 nM and a Bmax of 0.92 pmol/mg protein. The pharmacological characteristics were consistent with an interaction at the N-methyl-D-aspartate subclass of excitatory amino acid receptor. The inclusion of Cl− ions revealed additional glutamate binding; this was sensitive to quisqualate and DL-2-amino-4-phosphonobutyrate, but not to kainate, DL-2-amino-7-phosphonoheptanoate, or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.

Notes: Abstract: Two τ cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the τ mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6-kb mRNA was detected with the two probes. In the astrocytes a 6-kb mRNA hybridized clearly only with the 1.6-kb probe. (2) During brain development the abundance of τ mRNA increases from a late fetal stage (— 4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of τ mRNA parallels that of α-tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) τ mRNAs of the same size encode several τ proteins differing in molecular weight: several τ proteins are expressed either during early stages of development (juvenile τ proteins of 48 kilodaltons) or in adulthood (mature τ proteins of 50–70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and τ proteins, seems to be developmentally coordinated.

Notes: Abstract: Chronic in vivo exposure of rats to ethanol in a complete liquid diet for 14 or 21 days produced a behavioral tolerance to the acute injection of ethanol. After 21 days, but not 14 days, of chronic exposure, there was a significant increase in the maximum density of striatal D1 and D2 dopamine receptors without a change in these receptors' affinities. A 24-h withdrawal from the 21-day exposure did not alter the observed increase in density. Both the level and duration of ethanol exposure appear to be important variables for demonstration of an increase in striatal D1 and D2 dopamine receptors.

Notes: Abstract: Pretreatment of synaptosomal membranes with a diazo-coupling reagent and the presence of CI− ions were used to differentiate high- and low-affinity populations of postsynaptic γ-aminobutyric acid (GABAA) receptors. The super-low-affinity GABAA receptors were characterized by the enhancing effect of GABA on [3H]diazepam binding. The GABA antagonists 2–(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride (SR 95103) and 3-α-hydroxy-16-imino-5β-17-aza-androstan-11-one (R 5135) shifted and suppressed the dose-response curve of GABA on diazepam binding. SR 95103 displaced the lower affinity [3H]GABA binding with higher potency. Dissociation of the binding of the antagonist 2–(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) was polyphasic. Displacing potencies of SR 95531 and GABA were examined on the major (85%) rapid and minor slower phases of dissociation separated kinetically. The slower phase corresponded to higher affinity binding of SR 95531 which was displaced by GABA with about 10 times less potency. Photoaffinity labeling with muscimol decreased the number of [3H]muscimol binding sites by 27%. It decreased the displacing potency of GABA by 72%, but not that of bicuculline methiodide. These findings can be explained by a preferential binding of antagonists to hydrophobic accessory sites around low-affinity GABAA receptors.

Notes: Abstract: The main objective of the present study was to determine whether cholinergic markers (choline acetyl-transferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N-[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2-like muscarinic sites were studied using [3H]-acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2-like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.

Notes: Abstract: In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.

Notes: Abstract: Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), β-nucleoside triphosphate (βNTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue Pco2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by‘H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.

Notes: Abstract: Inositol-1,4,5-trisphosphate, produced in cells as a breakdown product of phosphatidylinositol-4,5-bisphosphate, induces, in many cell types, release of calcium from intracellular stores. In murine striatal neurons, differentiated in primary culture, carbachol, norepinephrine, glutamate, and neurotensin stimulate 3H-labeled inositol phosphate (3H-IP) production. The glutamate response was recently characterized as being mediated primarily by receptors of the quisqualate subtype. In the present study, we found that major differences exist between glutamate-stimulated 3H-IP formation and those stimulated by the other neuromediators. The maximal response to glutamate occurred before and during synaptogenesis and declined thereafter, whereas the maximal response to either carbachol or norepinephrine required complete neuronal differentiation. Although the glutamate response appears to be mediated exclusively by direct interaction with the neuro-transmitter receptors, responses to carbachol, norepinephrine, and neurotensin were partially or completely blocked by tetrodotoxin.

Notes: Abstract: We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aIdosterone ([3H]ALDO) in a time-and temperature-dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0°C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22°C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 °C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0°C on the subsequent binding of [3H]ALDO yielded an inverse U-shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity. In view of these findings, and proposed mechanisms of action of polyand monohydric compounds, we suggest that isolation of type I receptors into aqueous brain cytosol leads to a spontaneous infolding of hydrophobic domains on the receptor surface and a concomitant reduction in ligand binding capacity.

Notes: Abstract: Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20–1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5′-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.

Notes: Abstract: Daily injections of cocaine or morphine into rodents produces behavioral sensitization such that the last daily injection results in a greater motor stimulant effect than the first injection. To evaluate a role for brain dopamine in behavioral sensitization to cocaine and morphine, tissue slices from the ventromedial mesencephalon (containing dopamine cell bodies), the nucleus accumbens, and striatum (dopamine terminal fields) were obtained from rats pretreated with daily cocaine, morphine, or saline 2–3 weeks earlier. When the tissue slices were depolarized by increasing potassium concentration in the superfusate, the release of endogenous dopamine from the ventromedial mesencephalon of cocaine- and morphine-pretreated rats was significantly decreased. In contrast, the release of dopamine from the nucleus accumbens and striatum was either unaltered or slightly enhanced in rats pretreated with cocaine and morphine. When dopamine was released by amphetamine, a significant decrease in dopamine release from the ventromedial mesencephalon of cocaine-pretreated rats was measured. No other significant changes were measured after amphetamine-induced release. It is postulated that the decrease in dopamine release from the ventromedial mesencephalon of cocaine- and morphinesensitized rats results in less somatodendritic autoreceptor stimulation, and thereby produces an increase in dopamine neuronal activity.

Notes: Abstract: Kinetic analysis of the combination of rat brain apotransketolase with thiamine diphosphate suggested that the enzyme exists in more than one form. One part of the apoenzyme reacted rapidly with thiamine diphosphate to reconstitute the holoenzyme, but another part appeared to combine only relatively slowly. In addition, an apparently irreversible further change took place, the apoenzyme being converted progressively to a form which apparently could not be activated by thiamine diphosphate. The relative proportions of the three forms i.e., that reacting rapidly, slowly, or not at all with thiamine diphosphate, were a function of the duration and conditions of storage, with the proportion of the apoenzyme form which reacted rapidly with thiamine diphosphate decreasing progressively. The findings reported here provide a possible explanation for problems various workers have encountered in attempting to evaluate Michaelis constants for the reaction of thiamine diphosphate with apotransketolase. Key Words: Transketolase—Brain—Wernicke-Korsakoff syndrome—Thiamine. Jeyasingham M. D. and Pratt O. E. Rat brain apotransketolase: Activation and inactivation.

Notes: Abstract: β-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, “free” mitochondria, synaptosol, and synaptic mitochondria, with 2 mMβ-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of “free” brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in “free” mitochondria was also decreased in the presence of 2 mMβ-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.

Notes: Abstract: Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of ∼74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle-specific monoclonal antibody. The 40,000-Mr protein appeared to be identical to the 38,000-Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000-Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000-Mr protein is ∼5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these proteins.

Notes: Abstract: Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), τ factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, τ factor, and tubulin were compared. The Km values were in the ranges of 1.6–2.7 μM for MAP2 and τ factor. The Km value for tubulin decreased from 25 to 10–12.5 μM in the presence of 1.0 μM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, τ factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and τ factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.

Notes: Abstract: Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).

Notes: Abstract: Lesion of the nucleus basalis in the basal fore-brain of the rat results in the degeneration of the large cholinergic neurones which innervate the cortex. Parameters of cholinergic function, namely, acetylcholinesterase activity, muscarinic acetylcholine receptor number, and the depolarisation-induced release of acetylcholine, fall in ipsilateral cortex subsequent to lesion. These deficits are likely to reflect the loss of the presynaptic input to the cortex. A reversal in these deficits is seen 1 month after lesion, and a full recovery is seen after 150 days. This is thought to be due to a process of “spared axon sprouting” followed by the reestablishment of synapses. To examine the integrity of the cortical muscarinic receptor response following denervation, an assay of the polyphosphoinositide response was carried out. Cortical tissue slices, prela-belled with [3H]inositol, were incubated for 40 min with carbachol in the presence of Li+; the accumulation of [3H]-inositol monophosphate ([3H]IP1) was used as an index of this response. A 92% increase in the carbachol-stimulated production of [3H]IP1 was seen 5 days after lesion com pared to normal cortex. Sham-operated animals showed no change in [3H]IP1 accumulation at this time point. Dose-response experiments showed that this increase was due to an increase in the maximal response to carbachol after lesion with no change in EC50 values. Two weeks after lesion, this increased response was much attenuated; tissue slices from denervated cortex showing a strong acetylcholinesterase decrease (36–66%) showed an increase of just 30% above normal. There was no increase in the polyphosphoinositide response 50 and 118 days after lesion. The transient time course of this response to lesion suggests that it participates in or arises from processes of recovery from denervation and the reestablishment of synapses.

Notes: Abstract: We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphor ylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks. After 30 min, this decrease was greater (-34%, p 〈 0.05) and was significant. There was a concomitant but nonsignificant increase (+22%) in phosphorylation of a 45 kDa component in the SPM fraction. No significant changes in phosphorylation patterns were seen in PSD fractions or in SPMs and PSDs isolated from cerebella of chicks with passive avoidance training at any time.

Notes: Abstract: The characteristics of [3H]strychnine and t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine- and γ-aminobutyric acid (GABA)-gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine- and GABA-gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.

Notes: Abstract: It is shown here that glycolipids of the sulfoglucuronyl neolacto series (SGGLs) are present in the adult rodent cerebellum. SGGLs were not detected in the cerebellar murine mutants lurcher, Purkinje cell degeneration, and staggerer, in which Purkinje cell loss is the primary defect. SGGLs were present, however, in normal amounts in weaver and reeler mutants, in which there is a major and relatively specific loss of granule cells without obvious deficiency in Purkinje cells. In the myelin-deficient quaking mutant, the expression of SGGLs also was nearly normal. The loss of SGGLs in Purkinje cell-deficient mutants was specific, since most of the major lipids were not affected significantly and only the percentage composition of other lipids, such as sulfatides and gangliosides, was altered in the mutants. These and other results strongly suggest that SGGLs and other glycolipids of the paragloboside family are localized specifically in Purkinje cells and their arbors in the adult cerebellum. This is the first demonstration of the localization of a specific glycolipid and its analogs in a specific cell type in the nervous system.

Notes: Abstract: The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HC1. Cysteic acid thus produced was separated from taurine by ion-exchange chromatography using Biorad-AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.

Notes: Abstract: The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT, sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT, sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

Notes: Abstract: The effects of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg subcutaneously per day for 8 days) to C57BL/6N mice were studied on tyrosine hydroxylase (TH), l-3,4-dihydroxyphenylalanine decarboxylase (DDC), and monoamine oxidase (MAO) activities in the striatum, and TH, DDC, dopamine-β-hydroxylase (DBH), and MAO activities in the hypothalamus. Treatment with MPTP led to a large decrease in TH activity and a parallel decrease in DDC activity in the striatum, as compared with the saline controls. In contrast, MPTP administration did not cause a decrease of the activities of TH, DDC, and DBH in the hypothalamus. There was also no reduction in MAO activities of striatum and hypothalamus. These data indicate that MPTP administration to mice results in specific degeneration of the dopaminergic nigrostriatal pathway and that DDC in the mouse striatum may mainly be localized in the dopaminergic neurons with TH.

Notes: Abstract: The properties of muscimol, β-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-β-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or[3H]flumazenil (Ro 15–1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5–α][1–4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is ∼20–25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly 〈1; the potency of the different BCs in displacing [3H]flumazenil is 25–50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5–4864 (4′-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of 〈 1 (nH= 0.4) by CL 218,872 (3-methyl-6–(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b]pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (∼80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominately of the central-type. BZD1 subclass. In both cerebellar and spinal cord membranes, micromolar γ-aminobutyric acid (GABA) enhanced the binding of [3H]FLU; however, this effect is less efficacious and less potent in the spinal cord, observations indicating two possibilities: (a) that in spinal cord some of the BZD2 binding sites are not coupled to the GABAA binding sites, or (b) that they are coupled in a GABAA/BZD2 receptor complex containing a large proportion of BZD2 binding sites associated with a relatively small number of GABAA binding sites.

Notes: Abstract: A newJy developed enzyme-linked immunosorbent assay for acetylcholinesterase (AChE) protein was combined with conventional measures of enzyme activity in a study of 15 brain regions from six control cases (non-neurological deaths), six cases of Alzheimer's disease, and six cases of Huntington's disease. In the control brains, the mean AChE activity varied 100-fold from region to region (cortex lowest, striatum highest). The variation in enzyme activity was exactly paralleled by a variation in protein immunoreactivity. Overall, the homospecific activity of AChE averaged 0.26 ± 0.007 mU/pg, close to the value for electrophoretically homogeneous enzyme isolated from red blood cells. Similar homospecific activities were observed in samples from Huntington's and Alzheimer's brains. Evidently, AChE that is immunoreactive but enzymatically inactive does not accumulate in any of the three conditions examined. Huntington's brain samples showed normal total contents of AChE, but Alzheimer's brains showed significant decreases of both enzyme activity and immunore-activity in all seven cortical regions and in two out of the eight subcortical structures examined, hippocampus and nucleus accumbens.

Notes: Abstract: The monoamine transporter of dopamine (DA), noradrenaline, and 5-hydroxytryptamine synaptic vesicles was assayed in rat and human brain homogenates by in vitro binding of [3H]dihydrotetrabenazine. [3H]Reserpine, a second ligand of the vesicular monoamine transporter, could not be used. [3H]Dihydrotetrabenazine binding in rat brain was stable after 72 h at 22°C postmortem. In major human brain regions, [3H]dihydrotetrabenazine binding was specific and saturable (KD, 2.7 nM). Displacement constants by substrates or inhibitors of vesicular monoamine uptake, and regional distribution in human brain were similar to those found in rodents. The highest densities of binding sites were observed in caudate nucleus, putamen, and accumbens nucleus. In caudate nucleus and in putamen from normal human subjects, [3H]dihydrotetrabenazine binding and homovanillic acid concentration were significantly or nearly significantly correlated. A weaker correlation was found between [3H]dihydrotetrabenazine binding and DA, in association with a higher variability of DA. [3H]Dihydrotetrabenazine binding in caudate nucleus and in putamen decreased significantly with age, unlike DA and homovanillic acid concentrations. The results establish [3H]dihydrotetrabenazine as a presynaptic monoaminergic ligand of interest for studies on postmortem human brain.

Notes: Abstract: The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol-specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane-bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The Upases uncover an immunological determinant that is found on other glycosylphosphatidylinositol-anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane-bound enzyme migrates faster during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.

Notes: Abstract: The effects of arachidonic acid and other polyunsaturated fatty acids (PUFAs) on both oxidative and metabolic perturbation were studied in primary cultures of rat cerebral cortical astrocytes. In the presence of 0.1 mM arachidonic acid, the rate of the reduction of nitroblue tetrazoiium (NBT) to nitroblue formazan (NBF) was stimulated from 0.65 ± 0.10 to 1.43 ± 0.15 and from 0.092 ± 0.006 to 0.162 ± 0.009 nmol/min/mg protein in intact and broken cell preparations, respectively. The rate of superoxide radical formation, as measured by the superoxide dismutase (SOD)-inhibitable NBT reduction was 0.042 nmol/mg protein in broken cells and was negligible in intact cells. The latter is due to the impermeability of SOD into the intact cell preparation. NBF formation in intact astrocytes stimulated by arachidonic acid was both time- and dosedependent. Other PUFAs, including linoleic acid, linolenic acid, and docosahexaenoic acid, were also effective in stimulating NBF formation in astrocytes, whereas saturated palmitic acid and monounsaturated oleic acid were ineffective. Similar effects of these PUFAs were observed in ma-londialdehyde formation in cells and lactic acid accumulation in incubation medium. These data indicate that both membrane integrity and cellular metabolism were perturbed by arachidonic acid and by other PUFAs. The sites of superoxide radical formation appeared to be intracellular and may be associated with membrane phospholipid domains, because liposome-entrapped SOD, which was taken up by intact astrocytes, reduced the level of superoxide radicals and lactic acid content, whereas free SOD was not effective.

Notes: Abstract: The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na++ K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37–110 μg) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 μM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of “high-affinity” binding sites with an apparent binding affinity (KD) of 64.7 ± 2.0 nM and a binding capacity (Bmax) of 10.1 ± 1.5 pmol/mg protein. In the presence of 100 μM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 ± 2.7 nM (p 〈 0.01), whereas the Bmax remained virtually unchanged (12.5 ± 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na++ K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.

Notes: Abstract: Nonsynaptic mitochondria isolated from rat brain hippocampus were compared with those obtained by means of the same preparative procedure from cerebral cortex and striatum. Protein recovery, marker enzyme activities (lactate dehydrogenase, citrate synthase, and acid phosphatase), state 4 respiration, and response to hypoosmotic shock showed no difference among the three cerebral regions, suggesting homogeneous behavior during the subfractionation procedure. Cholinergic markers—choline acetyltransferase, acetylcholinesterase activities, and high-affinity choline uptake—evaluated on synaptosomes showed the classic regional pattern with an enrichment in the striatum (striatum 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:00223042:JNC1233:ges" location="ges.gif"/〉 cortex 〉 hippocampus). The coupling state of the mitochondrial fractions was maintained (respiratory control ratios ranging from 3.62 to 5.08 with glutamate + malate as oxidizable substrates), showing a metabolic competence sufficient to perform metabolic studies. Regional differences were found in state 3, uncoupled state of respiration, and cytochrome oxidase activity. Hippocampus showed the lower values (hippocampus 〈 striatum 〈 cortex). A possible role of this lower capacity of mitochondrial energy metabolism in determining the sensitivity of hippocampal neurons to ischemia or epileptic seizures is suggested.

Notes: Abstract: The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for α-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a fivefold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10–13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10–20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.

Notes: Abstract: Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,d-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90– and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immu-nological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.

Notes: The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [l-14C]ara-chidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-Iabeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate. 36% in myo-inosi-tol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for α1-adrenergic receptors in this tissue, (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus. We conclude that IP3 accumulation and arachidonate release are produced in the iris muscle under in vivo conditions that produce contraction, i.e., electrical stimulation of the sympathetic nerve. These observations add further support to the concept that PIP2 and its derived second messengers are involved in excitation-contraction coupling in the smooth muscle cells of the iris.

Notes: Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which co-nigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.

Notes: [Arg8]-Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 ± 0.39 nM and 24.0 ± 7.01 fmol/mg of protein (mean ± SEM; n = 3) for the ventral septal area, 0.92 ± 0.13 nM and 47.0 ± 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 ± 0.14 nM and 25 ± 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1-like receptor for AVP. These data document the presence of a high-affinity, V1-like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.

Notes: Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and α-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either li-gand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for a-bungarotoxin. Agonist affinity for the a-bungarotoxin binding site was reduced bv treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetyl-choline. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.

Notes: We have examined some of the characteristics of phorbol ester- and agonist-induced down-regulation of astrocyte receptors coupled to phosphoinositide metabolism. Our results show that preincubation of [3H]inositol-labelled astrocyte cultures with phorbol 12-myristate 13-acetate (PMA) resulted in a time- (t1/2, 1–2 min) and concentration-dependent (IC50, 1 nM) decrease in the accumulation of [3H]inositol phosphates (IP) evoked by muscarinic receptor stimulation. Much longer (30–40 min) preincubation periods with higher concentrations (IC50, 600 μM) were required to elicit the same effect with the receptor agonist carbachol. Following preincubation, agonist-stimulated [3H]IP accumulation recovered with time; in both cases pretreatment levels of inositol lipid metabolism were attained within 2 days. Both phorbol ester and agonist pre-treatments were also effective in reversing the carbachol-evoked mobilisation of 45Ca2+ in these cells. However, their effects on phosphoinositide metabolism were found not to be additive. Although neither pretreatment affected the incorporation of [3H]inositol into phosphoinositides, both resulted in a loss of membrane muscarinic receptors as assessed by [3H]N-methylscopolamine binding. In washed membranes prepared from [3H]inositol-labelled cultures, the guanine nucleotide analogue, guanosine 5′-O-thiotri-phosphate (GTP-γ-S), caused a dose-dependent increase in [3H]IP formation. This response was enhanced when carbachol was also included in the incubation medium, although the agonist alone was without effect. Pretreatment with either PMA or carbachol had no effect on GTP-γ-S-stimulated [3H]IP accumulation but did reduce the ability of carbachol to augment this response. Similar findings were obtained when membranes were exposed directly to PMA. Phorbol ester pretreatment was also effective in reversing the increase in [3H]IP accumulation and 45Ca2+ mobilisation evoked by noradrenaline. However, following preincubation with carbachol there was no loss of nor-adrenaline-stimulated phosphoinositide breakdown although its ability to mobilise 45Ca2+ was blocked.

Notes: The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine ([3H]NMS) and [3H]qui-nuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki= 869–1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 {11–2[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} bound to the mAChRs with a very low affinity (Ki= 6.0 μM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki= 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK-N-SH mAChR (Mr= 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr=66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.

Notes: Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.

Notes: Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1 -phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [l-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (˜20 μM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.

Notes: Benzodiazepine-affinity chromatography, on a column of 1012S-Sepharose, resulted in the detection and purification of a binding protein (P36) from the cytosolic fraction of pig cerebral cortex. Purified P36 was enriched over 3,500-fold in a single step and was recovered with an efficiency of 50–60%. Analysis of the purified preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated a single polypeptide of Mr 36,000. TheStokes radius (3.44 nm) and sedimentation coefficient (4.43S) indicated that purified P36 is a dimeric protein. Analysis of the amino acid composition of P36 revealed a relatively high content of the hydrophobic amino acids, valine and leucine. Immunoblotting of several pig tissue preparations with an antiserum raised against purified P36 demonstrated approximately equal enrichment of P36 in cerebral cortex, cerebellum, and adrenal glands. Lesser enrichment was observed in kidney and liver, whereas a number of other tissues displayed no immunoreactivity. The γ-aminobutyrate/benzodiazepine receptor complex and P36 showed no immunological cross-reactivity. High-affinity binding activity for [3H]Ro 15–4513, [3H]flunitrazepam, or [3H]PK11195 was not detected in preparations of purified P36. However, the ability of the γ-aminobutyrate/benzodiazepine receptor inverse agonists, methyl- and ethyl-#bT-carboline-3-carboxylate, to inhibit the binding of P36 to 1012S-Sepharose at relatively low concentrations indicates that P36 exhibits a degree of binding specificity.

Notes: Developmental changes in protein N-glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4–6 days in culture), followed by a period of cellular differentiation. N-Glycosylation activity has been measured by assaying the incorporation of [2-3H]-mannose into dolichol-linked oligosaccharides and glyco-protein over a period of 1–25 days in culture. This study revealed a marked induction of N-glycosylation activity beginning at approximately 1 week of culture. [2-3H]-Mannose incorporation into the oligosaccharide-lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol-linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N-glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2′,3′-cyclic nucleotide 3′-phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N-glycosylation pathway and suggest a possible relationship between N-linked glycoprotein assembly and the growth and differentiation of glial cells.

Notes: Amphipathic compounds containing N-acetylneuraminic acid (sialic acid) [for example, D-N-acetylneuraminyl-(α2-1)-2S, 3R,4E-2-N-tetracosanoyl sphingenine, sialyl alkyl glycerol ethers, and sialyl cholesterols] induced neuritogenesis in a neuroblastoma cell line (Neuro2a). The sialic acid in the hydrophilic moiety of the compounds is specifically required for neuritogenesis. The requirement for molecular specificity of the hydrophobic moiety, however, is rather low. Regarding the hydrophobic moiety, no preference for cholesterol, alkyl glycerol ether, or ceramide residues was observed as to their neuritogenic activity. Sialyl compounds with α-ketosidic sialyl linkages were more active than the corresponding β-anomers. These sialyl compounds induced the growth of only one neurite, but a long one, from the cell body. This type of neuritogenesis is completely different from that induced by compounds capable of elevating the concentration of intracellular cyclic AMP, which induced the appearance of many neurites from a single cell body. Besides this morphological change, the active sialyl compounds also caused a change in the carbohydrate composition of the cell surface. Sialyl compound treatment drastically increased the concentration of peanut agglutinin binding sites.

Notes: Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 μM), ouabain (104 μM), atropine (10μM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 μM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 μM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1–3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin. Under the conditions used. the Ca2+-dependent K+-evoked release of ACh was not changed by 104 μM carbamoylcholine. It is apparent that the spontaneous Ca2+-independent liberation of ACh from Torpedo synaptosomes is not a simple diffusion and is mediated by a carrier. The carrier is inhibited by muscarinic activation; the molecular mechanism of the inhibition is unlikely to involve Ca2+ ions or hyperpolarization. During depolarization, muscarinic inhibition of ACh liberation is ineffective, but carbamoylcholine blocks the Ca2+-independent depolarization-stimulated ACh output, probably by a direct action on the carriers.

Notes: 2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and mid-brain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.

Notes: Abstract: Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to ce-rebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of α-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of transstimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.

Notes: Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell-associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11°C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked- and control-degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann-cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann-cell premitotic response to axotomy.

Notes: The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3–[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0°C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 μM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 ± 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax= 31.3 ± 3.56 fmol/mg protein, KD= 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of 〉 1,000,000 and 55,000–60,000 (a corresponding Stokes radius of 35.5 nm).

Notes: Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the t MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.

Notes: Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [14C]octanoic and [14C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [14C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10--2× s-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10--2× s-1, respectively). Approximately 70, 30, and 25% of the [14C]myristic, [14C]octanoic, or [14C]linoleic acids, respectively, were extracted from the perfusate.

Notes: Book Review in this Article:Neuromethods Volumes 5: Neurotransmitter Enzyme edited by A. A. Boulton, G. B. Baker, and P. H. Yu, Human Press, Clifton

Notes: Glutamate appears to be the neurotransmitter of granule cells, the major neuronal population of the cerebellar cortex. To determine the role of astroglial cells in the synthesis of glutamate, we have measured the specific activity of glutamate dehydrogenase (GDH) in clonal cell lines that might be the in vitro equivalents of the different cerebellum astroglial cell types. In conditions where GDH operates in the direction of glutamate synthesis, the specific activity of GDH measured in the “Golgi-Bergmann”-like clone was 4–6 times higher than in the “velate protoplasmic”- or “fibrous-like” astrocytic clones. These data correlate well with the intense immunoreactivity to GDH in Golgi-Bergmann astrocytes in vivo that has been recently reported.

Notes: Abstract: Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co-release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P-40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co-release with acetylcholine or ATP.

Notes: Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10–15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl-5′-imidodiphosphate, cholera toxin) that act through the stimulatory GTP-binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30–40%. Opiate inhibition was maximal at low magnesium concentrations (〈5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with per tussis toxin lowered the apparent affinity for agonists in inhibiting activity. The δ opioid agonists were more potent than μ agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both μ and δ agonists (50–90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by μ opiate receptors, but whether classic high-affinity δ and k receptors are involved with this enzyme cannot be confirmed by these experiments.

Notes: To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100β polypeptide. The level of S100β in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about twofold. The subcellular distribution of S100β and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100– and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100β is the predominant S100 polypeptide in C6 cells and suggest that changes in S100β and S100β-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.

Notes: The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon. especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase. and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P: has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the “G16P2 system,” as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897–916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.

Notes: Abstract: Long-term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]d-Ala2, d-Leu5 enkephalin. This increase wm concentration and time dependent, with an EC50 of about 480 μM and a maximal effect detected after 3-day treatment. At saturating concentration of butyrate (1 mM) the increase was three- to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas α2-adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate-induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1-stimulated cyclic AMP levels but caused a three- to fourfold decrease in the IC50 of d-Ala2, d-Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long-term treatment of NCB-20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and α2-adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down-regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol-induced phosphoinositide breakdown, whereas the receptor upregulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid receptors and muscarinic cholinergic receptors in NCB-20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins.

Notes: Abstract: The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10−4M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 μM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.

Notes: Human myelin basic protein (MBP), a long-lived brain protein, undergoes gradual racemization of its amino acids, primarily aspartic acid and serine. Purified protein was treated at neutral pH with trypsin to yield peptides that were separated by HPLC using a C18 column. Twenty-nine peptides were isolated and analyzed for amino acid composition and aspartate racemization. Each aspartate and asparagine in the protein was racemized to a different extent, ranging from 2.2 to 17.1% D isomer. When the racemization was examined in terms of the β-structure model of MBP, a correlation was observed in which six aspartate/ asparagine residues assumed to be associated with myelin membrane lipids showed little racemization (2.2–4.9% D isomer), whereas five other aspartate residues were more highly racemized (9.9–17.1% D isomer). Although the observed aspartate racemization may be related to steric hindrance by neighboring residues and/or the protein secondary structure, interaction of aspartates with membrane lipids may also be a major factor. The data are compatible with a model in which each MBP molecule interacts with adjacent cytoplasmic layers of myelin membrane through a β-sheet on one surface and loops and helices on the other surface, thereby stabilizing the myelin multilamellar structure.

Notes: Abstract: The effect of bicuculline-induced convulsive seizures on lipid metabolism has been studied in four brain areas (cerebellum, cerebral cortex, hippocampus, and brainstem) using [2-3H]glycerol and [1,2-14C]ethanolamine as radioactive lipid precursors administered simultaneously with bicuculline. Twelve minutes after the administration, the uptake of radioactivity depended both on brain area and treatment, being generally higher in convulsing rats. The uptake of glycerol was influenced to a larger extent than that of ethanolamine and increased during convulsions, but its incorporation into lipids did not. In contrast, the amount of ethanolamine incorporated into lipids increased during bicuculline-induced seizures. The difference in behavior of glycerol and of ethanolamine is also indicated by the decrease of the 3H/14C ratio of phosphatidylethanolamine in various brain areas during convulsions. It is, therefore, evident that the metabolism of the two precursors is affected differently by seizures.

Notes: Abstract: A disorder of CNS myelination was found in paralytic tremor (“pt”) rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20–30% of control. Myelin isolated from 4-week-old “pt” rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+, K+-ATPase specific activity. 2′,3′-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in “pt” as in control myelin but differed by having greater sensitivity to detergent activation.

Notes: Abstract: The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13–14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recoveiry from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh. and thus we conclude that ACh synthesized in the presence of AH5183 is as releasable as preexisting ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]-choline): the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, α-latrotoxin, because an anti-α-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethyl-homocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of (3H]acetyldiethylhomo-choline efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.

Notes: Abstract: Static and superfused pineal slices (750 μm) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time-dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10−5M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12–18 min in response to NE stimulation. This reaches a 3.5–4.5-fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12–18 min. These time-course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]-tryptophan shows a linear increase with time. In the presence of NE (10−5M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N-acetyltransferase-independent mechanism of synthesis and release. In static incubations, propranolol (10−5M) inhibited NE-induced melatonin production by about 60%. but prazosin (10−5M) had no effect. As dibutyryl cyclic AMP (10−3M) stimulated melatonin production, it is concluded that β-receptors are of primary importance to the control of melatonin production, as in the rat. The role of α1-receptors is less clear, but the stimulatory action of phorbol 12-myristate 13-acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.