bluuebunny/crossref_metadata_2025 huggingface dataset:
Substance P Synthesis and Transport in Explants of Nodose Ganglion/Vagus Nerve: Effects of Double Ligation, 2‐Deoxyglucose, Veratridine, and Ouabain
Authors: MacLean David B.
Published: October 2006

Abstract: Substance P (SP), the widely distributed undeca‐peptide, is synthesized in cell bodies of vagal sensory ganglia and transported bidirectionally toward the CNS and thoracic and abdominal viscera. In explants of the guinea pig inferior (nodose) vagal sensory ganglion and attached 2 cm of distal vagus nerve, SP is synthesized within the ganglion and transported predominantly distally. The quantity of distal transport is similar to that observed in vivo and provides an index of ongoing synthesis within the ganglion. In this report, the model is further characterized. Double ligation of the explant distal to the ganglion demonstrates that all the transported peptide is derived from the ganglion; there is no evidence of intraaxonal processing of peptide precursor. Approximately 50% of the peptide is in a rapid transport vs. an apparent stationary compartment. Not only transport, but also synthesis, of SP was blocked by 20 mM colchicine. Ongoing SP biosynthesis is dependent on a nutrient medium [medium 199 (M‐199)] and is partially inhibited with added fetal bovine serum (FBS; 10%): total ex‐plant content in M‐199/FBS vs. M‐199, 1, 785 ± 101 (n = 8) vs. 2, 254 ± 123 pg (n = 9); p < 0.02. Addition of 2‐deoxyglucose (2‐DG) decreased both total SP synthesis and transport (total explant content for 2‐DG vs. control, 986 ± 94 vs. 1, 391 ± 111; p < 0.05). Medium supplemented with glucose to a final concentration of 600 mg/100 ml or with glucose (300 mg/100 ml) with or without insulin (50 ng/ml) did not alter explant SP content or transport. Veratridine (5 ± 10−−6M) inhibited both SP synthesis and transport; ouabain (10−−4M) also inhibited synthesis, but less so transport. Tetrodo‐toxin reversed the effects of veratridine. These studies demonstrate the usefulness of this model, which can examine factors regulating both synthesis and transport of sensory neuropeptides in vitro. The results suggest that SP synthesis/transport may be under tonic inhibition, perhaps by both neural and humoral mechanisms.
Oligodendroglial Differentiation in Glial Primary Cultures: Requirement for Mevalonate
Authors: Langan Thomas J. , Volpe Joseph J.
Published: October 2006

Abstract: The oligodendroglial enzyme, 2′,3′‐cyclic nucleo‐tide 3′‐phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibu‐tyryl‐3′,5′‐cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three‐ to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by >95% by placing the cultures in 10% lipoprotein‐poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 μM), a specific inhibitor of 3‐hy‐droxy‐3‐methylglutaryl coenzyme A reductase, the rate‐limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low‐density lipoprotein sufficient to increase the cholesterol content of the medium 80‐fold failed to correct mevi‐nolin's inhibition of the induction of CNP. Thus, these results demonstrate that the presence of mevalonate is obligatory for the induction of CNP in primary cultures of developing glia, and suggest that a critical nonsterol derivative of mevalonate is required for this expression of oligodendroglial differentiation.
Specific Antisera Against the Catecholamines: L‐3,4‐Dihydroxyphenylalanine, Dopamine, Noradrenaline, and Octopamine Tested by an Enzyme‐Linked Immunosorbent Assay
Authors: Mons Nicole , Geffard Michel
Published: October 2006

Abstract: Antisera were raised against L‐3,4‐dihydroxyphe‐nylalanine (L‐DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme‐linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catechol‐amine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the non‐conjugated compound was poorly recognized. The nonre‐duced conjugates of L‐DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross‐reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L‐DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti‐NA and anti‐OA antibodies, the lateral chain is important.
Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid
Authors: Atack John R. , Perry Elaine K. , Bonham James R. , Perry Robert H.
Published: October 2006

Abstract: The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120–500‐fold) between total BChE activity in the CSF and the BChE‐rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF—both lumbar and ventricular—was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.
Glycoprotein as a Constituent of Purified γ‐Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain
Authors: Kuriyama Kinya , Taguchi Jun‐ichi
Published: October 2006

Abstract: The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified γ‐aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydro‐chloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N‐ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cys‐tine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but SH and S‐S‐groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A‐Sepharose eluted protein having [H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glyco‐peptidase A, β‐galactosidase, and α‐mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with β‐galactosidase resulted in the disappearance of the low‐affinity site for [3H]muscimol binding and in an increase of Bmax of the high‐affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low‐affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with β‐galactosidase and glycopeptidase A were much higher than that with α‐mannosidase may also indicate a special importance of the β‐galactosyl residue in the inhibition of GABA receptor binding activity. Furthermore, the observation that the activation of high‐affinity [3H]muscimol binding by benzodiazepines disappeared following β‐galactosidase treatment suggests that the carbohydrate chain in the receptor complex may also be involved in the functional coupling between the GABA receptor and the benzodiazepine receptor.
Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover
Authors: Heacock Anne M. , Fisher Stephen K. , Agranoff Bernard W.
Published: October 2006

Abstract: The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo‐[2‐3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day‐18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1–10 mM) was at the adult level (i.e., threefold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose‐response curve was shifted to the left, giving a half‐maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine‐2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of “spare” muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.
Monoclonal Antibody Tor 23 Recognizes a Determinant of a Presynaptic Acetylcholinesterase
Authors: Kushner P. D. , Stephenson D. T. , Sternberg H. , Weber R.
Published: October 2006

Abstract: A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent‐treated electroplax homogenates and insoluble in high‐salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.
Discriminatory Effects of Forskolin and EGTA on the Indirect Cyclic AMP Responses to Histamine, Noradrenaline, 5‐Hydroxytryptamine, and Glutamate in Guinea‐Pig Cerebral Cortical Slices
Authors: Donaldson J. , Kendall D. A. , Hill S. J.
Published: October 2006

Abstract: The effects of forskolin (1 μM) and EGTA (5 mM) on indirect cyclic AMP responses in slices of guinea‐pig cerebral cortex were examined. Forskolin had little effect on the direct 2‐chloroadenosine‐stimulated cyclic AMP response. However, it completely abolished the glutamate‐induced augmentation of this response. In contrast, forskolin had very little effect on the indirect cyclic AMP responses to noradrenaline, 5‐hydroxytryptamine, and histamine. Conversely, rapid removal of extracellular calcium with EGTA 2 min before addition of the indirectly acting agent markedly reduced the augmentation responses produced by these latter agonists, but had little effect on the glutamate augmentation. When EGTA was added once a steady level of cyclic AMP had been achieved with the indirect agents, it was without effect on any of the responses. Thus, calcium appears to have a role in the early, but not the later, stages of the noradrenaline, 5‐hydroxytryptamine, and histamine responses. A role for protein kinase C in the glutamate augmentation response was suggested, because forskolin inhibited the augmentation of the 2‐chloroadenosine response produced by phorbol esters (which mimic the actions of diacylglycerol in activating protein kinase C). We conclude that there is more than one mechanism by which the augmentation of cyclic AMP responses can occur.
Apomorphine Does Not Alter Amphetamine‐Induced Dopamine Release Measured in Striatal Dialysates
Authors: Kuczenski Ronald , Segal David S. , Manley Leslie D.
Published: October 2006

Abstract: Amphetamine facilitates the release of dopamine from nerve terminals, but the mechanisms underlying this effect have not been fully delineated. The present experiments were designed to test the extent to which amphetamine‐induced dopamine release is dependent on impulse flow and autoreceptor function in dopaminergic neurons. Rats were pretreated with a low dose of apomorphine (0.05 mg/kg) to inhibit dopamine neuronal activity, and the striatal dopaminergic response to amphetamine (0.5 mg/kg) was assessed by in vivo dialysis in freely moving animals. Consistent with previous results, apomorphine alone substantially decreased, whereas amphetamine increased, striatal dialysate dopamine concentrations. However, whereas apomorphine pretreatment decreased the locomotor response to amphetamine, the amphetamine‐induced increase in dialysate dopamine was unaffected. These results indicate that amphetamine‐facilitated dopamine release is independent of neuronal firing and autoreceptor regulation, consistent with the putative accelerative exchange‐diffusion mechanism of amphetamine‐induced dopamine release. Other possible mechanisms underlying the inhibitory effects of apomorphine on amphetamine locomotor activation are discussed.
Loss of Sulfoglucuronyl and Other Neolactoglycolipids in Purkinje Cell Abnormality Murine Mutants
Authors: Chou Denise K. H. , Flores Sally , Jungalwala Firoze B.
Published: October 2006

Abstract: A significant reduction in the content of two members of the sulfoglucuronyl‐neolacto series of glycolipids (SGGLs), 3‐sulfoglucuronyl‐lacto‐N‐neotetraosylceramide (SGGL‐1) and 3‐sulfoglucuronyl lacto‐N‐norhexaosylceramide (SGGL‐2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto‐N‐neotetraosylceramide (paragloboside) and lacto‐N‐norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X‐determinant fucoglycolipids and disialosyllacto‐N‐neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK‐1‐reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK‐1‐reactive glycoproteins were not affected in these mutants.
GABAergic Growth Cones: Release of Endogenous γ‐Aminobutyric Acid Precedes the Expression of Synaptic Vesicle Antigens
Authors: Taylor Joanne , Docherty Maureen , Gordon‐Weeks Phillip R.
Published: October 2006

Abstract: Growth cone fractions isolated from neonatal [postnatal day 3 (P3)] rat forebrain contain GABAergic growth cones as demonstrated by immunofluorescence staining with monospecific antibodies to γ‐aminobutyric acid (GABA). HPLC analysis shows that GABAergic growth cones release this endogenous GABA when stimulated with high K+. Endogenous GABA release is Ca2+‐independent and, in this respect, similar to that seen previously with [3H]GABA. Isolated growth cone fractions also exhibit a K+‐stimulated, Ca2+‐independent release of endogenous taurine. None of the other amino acids shown to be present in isolated growth cone fractions were released, including glutamate, aspartate, and glycine. A population of dissociated cerebral cortical neurones prepared from P1 rat forebrain were GABA‐immunoreactive after 1 day in culture. The cell body, neurites, and growth cones of these neurones were all stained with GABA antibodies. At this time in culture, neurones did not stain with either of two antibodies to synaptic vesicle antigens, i.e., p65 and synaptophysin. Growth cones isolated from P3 rat forebrain were also not immunoreactive with these antibodies. After about 8 days in culture, when neurones had established extensive networks of long, varicose axons and elaborately branched dendrites, many neurones and their neurites were immunoreactive for GABA antibodies. At this time in culture, p65 and synaptophysin antibodies did stain neuronal cell bodies and particularly their varicose axons. Dendrites were not stained with synaptic vesicle antibodies. These results suggest that GABAergic neurones synthesize GABA during neurite outgrowth and that GABA is present in, and can be released from, the growth cones of these neurones. The presence of GABA in GABAergic growth cones is not associated with synaptic vesicles, which explains the Ca2+ independency of both endogenous and [3H]GABA release from these growth cones.
Glutamate‐Evoked Release of Endogenous Adenosine from Rat Cortical Synaptosomes Is Mediated by Glutamate Uptake and Not by Receptors
Authors: Hoehn Katja , White Thomas D.
Published: October 2006

Abstract: L‐Glutamate (10 μM–1 mM) released endogenous adenosine from rat cortical synaptosomes. Studies with excitatory amino acid antagonists, (+)‐5‐methyl‐16, 11,dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate (MK‐801), 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), Mg2+, and agonists N‐methyl‐D‐aspartate (NMDA), kainate, and quisqualate, indicated that this release was not receptor mediated. D,L‐2‐Amino‐4‐phosphonobutanoic acid (APB) also did not affect glutamate‐evoked adenosine release. Inhibition of glutamate uptake by dihydrokainate or replacement of extracellular Na+ blocked glutamate‐evoked adenosine release. D‐aspartate, which is a substrate for the glutamate transporter but is not metabolized, also released adenosine, suggesting that release was due to amino acid transport and not to its subsequent metabolism. D‐Glutamate, a relatively poor substrate for the transporter, was correspondingly less potent than L‐glutamate at releasing adenosine. Glutamate‐evoked adenosine release was not Ca2+ dependent or tetrodotoxin sensitive and did not appear to occur on the bidirectional nucleoside transporter. Inhibition of ecto‐5′‐nucleotidase virtually abolished glutamate‐evoked adenosine release, indicating that adenosine was derived from extracellular metabolism of released nucleotide(s). However, L‐glutamate did not release ATP and did not appear to release cyclic AMP. Therefore, transport of glutamate into presynaptic terminals releases some other nucleotide which is converted extracellularly to adenosine. This adenosine could act at P1‐purinoceptors to modulate glutamatergic neurotransmission.
Reduction of Muscarinic Receptor Density and of Guanine Nucleotide‐Stimulated Phosphoinositide Hydrolysis in Human SH‐SY5Y Neuroblastoma Cells Following Long‐Term Treatment with 12‐O‐Tetradecanoylphorbol 13‐Acetate or Mezerein
Authors: Cioffi Catherine L. , Fisher Stephen K.
Published: October 2006

Abstract: The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH‐SY5Y cell line. Pretreatment of SH‐SY5Y cells with 50 nM 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N‐[3H]methylscopolamine and [3H]‐scopolamine binding, and a significantly larger reduction (48%) in agonist‐stimulated 3H‐inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4α‐phorbol 12,13‐didecanoate was without effect on both antagonist binding and agonist‐stimulated phosphoinositide (PPI) turnover. A decline (∼ 50%) in the agonist‐mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H‐inositol phosphate release was significantly reduced in SH‐SY5Y cells treated with these agents. Furthermore, pretreatment of SH‐SY5Y neuroblastoma cells with TPA or mezerein impaired 3H‐inositol phosphate formation induced by the addition of either guanosine 5′‐O‐(3‐thiotriphosphate) or carbamylcholine to digitonin‐permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum‐free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist‐stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist‐stimulated PPI hydrolysis and Ca2+ signaling in SH‐SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide‐stimulated PPI turnover.
Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin‐1
Authors: Cavanaugh Kathleen P. , Gurwitz David , Cunningham Dennis D. , Bradshaw Ralph A.
Published: October 2006

Abstract: When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process‐bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half‐maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin‐1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300–1,000‐fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase‐1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP‐induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.
Developmental Changes in the Activity of Enzymes of Purine Metabolism in Rat Neuronal Cells in Culture and in Whole Brain
Authors: Brosh Sara , Sperling Oded , Bromberg Yael , Sidi Yechezkel
Published: October 2006

Abstract: The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine‐nucleoside phosphorylase (PNP), IMP 5′‐nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine‐guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5′‐nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5′‐nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5′‐nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia‐producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.
Precursors and Metabolites of Norepinephrine in Sympathetic Ganglia of the Dog
Authors: Kristensen Eric W. , Chinnow Sharon L. , Montreuil Ramona S. , Tyce Gertrude M.
Published: October 2006

Abstract: 3,4‐Dihydroxyphenylalanine, dopamine, epinephrine, 3,4‐dihydroxyphenylglycol, and 3,4‐dihydroxyphenylacetic acid as well as norepinephrine were measured in dog lumbar sympathetic ganglia. The responses of these compounds to several classes of stimuli were investigated using an isolated time‐resolved superfusion system. Nonselective (i.e., amphetamine and high K+) and receptor‐mediated selective (oxotremorine) stimuli were used to evoke releases. The overflows of all compounds were measured by HPLC with electrochemical detection. The efficiency of each stimulus was estimated by normalizing the amount of evoked release to the total neurotransmitter pool when the stimulus was applied; i.e., fractional release was calculated. Overflows of all compounds except 3,4‐dihydroxyphenylalanine were enhanced by a 10‐min 100 μM amphetamine stimulus, and each of the catecholamine pools (dopamine, norepinephrine, and epinephrine) was affected to the same degree. By contrast, the 3,4‐dihydroxyphenylalanine and dopamine pools were more readily releasable than the norepinephrine pool with a 10‐min 80 mM K+ stimulus, and these releases were Ca2+ dependent. Epinephrine was released in preference to norepinephrine by a 10‐min 1 mM oxotremorine stimulus. The data suggest the existence of at least three types of neurons in dog lumbar ganglia and are consistent with previous histological observations.
γ‐Aminobutyric AcidA Receptor α5‐Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology
Authors: Pritchett Dolan B. , Seeburg Peter H.
Published: October 2006

Abstract: A cDNA encoding a protein with 70% amino acid identity to the previously characterized γ‐aminobutyric acidA (GABAA receptor α‐subunits was isolated from a rat brain cDNA library by homology screening. As observed for α1‐, α2‐, and α3‐subunits, coexpression of this new α‐subunit (α5) with a β‐ and γ2‐subunit in cultured cells produces receptors displaying high‐affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing α2‐, α3‐ or α5‐subunits have low affinities for several type I‐selective compounds. However, α5‐subunit‐containing receptors have lower affinities for zolpidem (30‐fold) and Cl 218 872 (three‐fold) than measured previously using recombinantly expressed type II receptors containing either α2‐ or α3‐subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.
Potentiation by the Tetraphenylboron Anion of the Effects of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine and Its Pyridinium Metabolite
Authors: Heikkila Richard E. , Hwang John , Ofori Senyo , Geller Herbert M. , Nicklas William J.
Published: October 2006

Abstract: The 1‐methyl‐4‐phenylpyridinium species (MPP+) is the four‐electron oxidation product of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP‐induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase‐B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 μM TPB, the potentiation ranged from approximately 50‐fold to greater than 1,000‐fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above‐mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss‐Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP‐induced neurotoxicity.
Characterization of 3′‐Phosphoadenosine 5′‐Phospho[35S]Sulfate Transport Carrier from Rat Brain Microsomes
Authors: Vargas Froylan
Published: October 2006

Abstract: 3′‐Phosphoadenosine 5′‐phospho[35S]sulfate ([35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris‐maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 ± 0.06 μM and a Bmax of 20.8 ± 2.0 pmol/mg of protein. Low concentrations of Triton X‐100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 ± 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3′‐O‐phosphoryl group substitution on the ribose moiety. Thus, 3′‐phosphoadenosine 5′‐phosphate was more potent than 2′‐phosphoadenosine 5′‐phosphate in competing for [35S]PAPS specific binding. Adenosine 5′‐phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4‐Diisothiocyanostilbene‐2,2‐disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 ± 5.1 μM for inhibition of [35S]PAPS specific binding. 2,6‐Dichloro‐4‐nitrophenol had a low selectivity in competing for the [35S]PAPS binding site. The properties of the [35S]PAPS binding in rat brain tissue observed in this work correspond closely to the membrane transport carrier for the PAPS molecule, which is different from adenosine uptake sites or adenosine receptors.
Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken
Authors: Khaskiye Ahmad , Sine Jean‐Pierre , Colas Bernard , Renaud Didier
Published: October 2006

Abstract: The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast‐twitch posterior latissimus dorsi (PLD) and in slow‐tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5‐Hz frequency (slow rhythm) or 40‐Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.
Glucocorticoid Receptors and Enzyme Induction in the Spinal Cord of Rats: Effects of Acute Transection
Authors: González S. , Moses D. F. , De Nicola A. F.
Published: October 2006

Abstract: The spinal cord is a glucocorticoid‐responsive tissue, as demonstrated by hormonal effects on enzyme induction and by the presence of type II and type I glucocorticoid receptors in cytoplasmic extracts of this CNS region. Using microdissection techniques, we have found in the present investigation that glucocorticoid type II receptors are the most abundant class detected in gray (ventral and dorsal horns) and white (lateral funiculus) matter and that the distribution of type II sites among these regions was quantitatively similar. Type I sites were also quantified, with a slight prevalence in gray matter as opposed to white matter. Furthermore, stimulation of an inducible enzyme, ornithine decarboxylase (ODC), was found in ventral horn and lateral funiculus but not in dorsal horn after administration of dexamethasone (DEX), a type II receptor ligand. We also found that surgical transection of the spinal cord, while markedly increasing ODC activity per se, did not prevent the stimulatory effect of DEX administration on ODC activity measured in the lumbar enlargement of the spinal cord located below the surgical lesion. Taken together, the results suggest a direct effect of glucocorticoids on ODC activity in the spinal cord of rats, probably mediated by glucocorticoid receptors (type II) found in target cells of the ventral horn and lateral funiculus. The results also indicate that glucocorticoid receptors of the dorsal horn were not involved in ODC induction, and a function for these receptors awaits the results of further experimentation.
Separation of Brain Dolichol Kinase from Endogenous Activating Factors: Evidence That Phospholipid Enhances the Interaction Between Enzyme and Dolichol
Authors: Genain Claude P. , Waechter Charles J.
Published: October 2006

Abstract: Porcine brain dolichol kinase activity is effectively solubilized by extracting salt‐washed microsomes with 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS). When the detergent‐solubilized activity is chromatographed on Sepharose CL‐6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo= 1.9‐2.2) with the bulk of the membrane phospholipids. Although only ˜20% of the activity applied to the Sepharose CL‐6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat‐stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, ˜50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co‐factor requirement, the dolichol kinase activity recovered in the partially phospholipid‐depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme‐phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP. These results demonstrate that brain dolichol kinase has a strict phospholipid co‐factor requirement, raising the possibility that the phospholipid composition of the rough endoplasmic reticulum plays a role in the regulation of dolichol phosphorylation in nervous tissue.
Specific Antiserum and Monoclonal Antibodies Against the Taurine Biosynthesis Enzyme Cysteine Sulfinate Decarboxylase: Identity of Brain and Liver Enzyme
Authors: Remy Arnaud , Henry Sylvie , Tappaz Marcel
Published: October 2006

Abstract: Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800‐fold with a 1% yield from rat liver, where it was found to be 20‐fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfatepolyacrylamide gel electrophoresis and reverse‐phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51‐kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain–the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations–and CSD from liver: Both activities (a) were similarly eluted on ion‐exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of ˜63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD‐enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for the immunocytochemical visualization of CSD‐containing cells in the brain.
γ‐Aminobutyric Acid System in Developing and Degenerating Mouse Retina
Authors: Murashima Y. L. , Ishikawa T. , Kato T.
Published: October 2006

Abstract: Freeze‐dried sections (14 μm thick) of retinal layers were prepared from mice with retinal degeneration (C3H strain) and control mice (C57BL strain). The weighed sections (2–30 ng dry weight) were analyzed using our microassay methods. In the control retina, γ‐aminobutyric acid (GABA) concentration and glutamate decarboxylase (GAD) activity, on a dry weight basis, increased from birth to 9 weeks of age and decreased slightly at 20 weeks. In the degenerated retina, the levels of GABA and GAD activity were higher at birth than in the control retina, and continued to increase until 20 weeks of age, at which time the GAD activity reached a markedly high level. This increase was found when the total GABA and GAD levels per retina were determined. In the normal retinal layers, GABA and GAD were confined primarily to the inner plexiform layer. In the degenerated retina, GAD activity gradually increased in the inner layers during postnatal development, but by 20 weeks the increase was most prominent in the inner part of inner nuclear layer and in the outer part of inner plexiform layer. GABA transaminase activity and its distribution were not much different in both normal and degenerated retinas during development.
Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation
Authors: Rapier Catherine , Lunt George G. , Wonnacott Susan
Published: October 2006

Abstract: Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (–)‐Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (–)‐nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro‐β‐erythroidine, mecamylamine, pempidine, and neosurugatoxin; α‐bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (–)‐[3H]nicotine and 125I‐α‐bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins α‐bungarotoxin and neosurugatoxin of the 125I‐α‐bungarotoxin and (–)‐[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (–)‐[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.
Photoaffinity Labeling of the Two Forms of Serotonin Binding Protein: Peptide Mapping of the Binding Sites
Authors: Liu Kuo‐Peing , Tamir Hadassah , Adlersberg Mella
Published: October 2006

Abstract: Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm‐derived cells that store 5‐hydroxytryptamine (5‐HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5‐HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56‐kDa form of SBP may be the precursor of the 45‐kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N‐(4‐azido‐2‐nitrophenyl)‐5‐hydroxytryptamine (NAP‐5‐HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV‐visible absorption spectra. A 1 M excess of NAP‐5‐HT inhibited the binding of [3H]5‐HT to SBP by 50%. NAP[3H]5‐HT was also synthesized and attached to both high‐ and lowaffinity binding sites on both forms of SBP. The high‐affinity binding constants for 45‐kDa and 56‐kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low‐affinity constants were 0.3 γM and 0.15 γM. When the high‐affinity site of partially purified SBP was photoaffinity‐labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15‐fold molar excess of 5‐HT. Drugs and reagents affected to the same degree the binding of [3H]5‐HT to SBP (45 kDa and 56 kDa) and the binding of NAP[3H]5‐HT to the two proteins. The 45‐kDa SBP and 56‐kDa SBP covalently labeled with NAP[3H]5‐HT were analyzed by partial proteolytic digestion with either Staphylococcus aureus V8 protease or proteinase K. The generated radiolabeled peptides, separated on SDS‐PAGE from both forms of the labeled SBP, exhibited a similar pattern, suggesting their close structural similarity. Structure‐binding requirements suggest that this probe will be also useful in studying other proteins that bind 5‐HT, such as carriers of 5‐HT in the plasma membrane and vesicles, as well as some serotonergic receptors (5‐HT1p).
Neurofilament Phosphorylation in Peripheral Nerve: Changes with Axonal Length and Growth State
Authors: Pestronk A. , Watson D. F. , Yuan C. M.
Published: October 2006

Abstract: We have previously reported that the intrinsic ability of motor axons to sprout can vary considerably from nerve to nerve with an inverse correlation to axonal length. In this study we asked whether this variation might be associated with differences in one axonal component, neurofilaments, near the site of outgrowth. The phosphorylation of epitopes on the heavy subunit of neurofilaments (NF‐H) was compared in normal and regenerating axons from long and short nerves in the rat. Quantitative determination of phosphorylation states on NF‐H epitopes was made by measuring immunoreactivity to monoclonal antibodies using an enzyme‐linked immunosorbent assay system. Our results showed a much higher degree of phosphorylation of epitopes on NF‐H in terminal portions of short compared to long axons. There was a significant inverse correlation between phosphorylation of NF‐H epitopes and axonal length. In newly formed sprouts NF‐H phosphorylation was reduced compared to normal. However, the absolute levels were related to the degree of NF‐H phosphorylation in the parent axons. The ability to phosphorylate axonal proteins near the site of outgrowth may correlate with the potential for plastic changes in the axon such as sprouting.