bluuebunny/crossref_metadata_2025 huggingface dataset:
Nerve Growth Factor and Dexamethasone Modulate Synthesis and Storage of Catecholamines in Cultured Rat Adrenal Medullary Cells: Dependence on Postnatal Age
Authors: Müller Thomas H. , Unsicker Klaus
Published: December 2006

Abstract: Catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and noradrenaline N‐meth‐yltransferase (NMT) were measured in cultures of isolated adrenal medullary cells from newborn and young postnatal rats to study the effects of the differentiation factors glucocorticoids and nerve growth factor (NGF). During the 4‐day culture period the cellular catechol‐amine (CA) content and TH activity remained stable, whereas NMT activity dropped to about half of the initial level. In cells from 2‐ and 10‐day‐old rats 10 μM dexamethasone specifically prevented this loss in NMT activity. Furthermore, this glucocorticoid treatment increased, in a dose‐dependent manner, the total CA content by 50–100% over control levels without changes in the adrenaline (A) proportion or TH activity. In contrast, NGF did not affect NMT activities at all. In cells from 10‐day‐old rats 100 ng/ml NGF elevated TH activity and total CA content to about 160% of controls and did not change the proportion of A. This increase in total CA content was linear with the NGF dose and required >5 ng/ml NGF. In chromaffin cells from 2‐day‐old rats 100 ng/ml NGF affected neither TH activity nor the total content, whereas it significantly reduced the proportion of A by about 25%.
Bony Fish Myelin: Evidence for Common Major Structural Glycoproteins in Central and Peripheral Myelin of Trout
Authors: Jeserich G. , Waehneldt T. V.
Published: December 2006

Abstract: Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two‐dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc Comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IPO. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP. These results differ completely from those observed in mammalian myelins albeit “normal” features of the glia‐myelin morphologies in trout PNS and CNS tissues.
Changes in Regional Blood–Brain Transfer of l‐Leucine Elicited by Arginine‐Vasopressin
Authors: Brust Peter
Published: December 2006

Abstract: Arginine‐vasopressin (AVP), injected into the carotid artery in physiological concentration together with L‐leucine, changed kinetic constants of the blood—brain barrier (BBB) transport of this neutral amino acid without changing the cerebral blood flow (CBF). The maximum velocity of transport (Vmax), the half‐saturation constant (Km), the nonsaturable transport constant (KD), and CBF were estimated in nine brain regions of male Wistar rats anesthetized with ether. In cerebral hemisphere, Vmax decreased from 21 nmol min−1. g−1 (control) to 7.6 nmol. min−1. g−1 (AVP). Km decreased from 0.11 to 0.029 mM. Regional differences of the kinetic constants were found in controls as well as in AVP‐treated animals. In all regions, the calculated constants Vmax and Km of animals coinjected with AVP were significantly decreased when compared to controls. A direct or indirect interaction of AVP with the transport system of large neutral amino acids is suggested.
Effect of pH on Muscarinic Acetylcholine Receptors from Rat Brainstem
Authors: Anthony Betty L. , Aronstam Robert S.
Published: December 2006

Abstract: The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]‐methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1‐h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1‐h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide‐dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.
A Double‐Isotope Procedure for Examining Protein Microheterogeneity: Multiple Forms of Fast‐Transported Glycoproteins and Sulfoproteins Possess a Common Polypeptide Chain
Authors: Hammerschlag Richard , Stone George C. , Bolen Franci A.
Published: December 2006

Abstract: Several fast‐transported proteins that appear as single bands after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis resolve into multiple spots during isoelectric focusing. A method was devised for determining if such microheterogeneity in net charge indicates that individual polypeptides have been posttranslationally modified to differing extents. Dorsal root ganglia were pulse‐labeled with [35S]methionine and either [3H]leucine or [3H]proline, proteins fast‐transported into peripheral sensory axons were separated by two‐dimensional gel electrophoresis, and isotope incorporation ratios of proteins associated with individual gel spots were determined. When four microheterogeneous glycoproteins were analyzed, each protein “family” showed markedly similar isotope ratios for its three to seven characteristic spots. Such ratios differed between families by almost twofold. In addition, a group of nonglycosylated, sulfate‐containing proteins was identified as a family on the basis of the similar isotope incorporation ratios of its component spots. These results suggest that protein microheterogeneity can result from variable sulfation of tyrosine residues as well as from variation in sialic acidcontaining oligosaccharide side‐chains. More generally, the method can be utilized to test for protein microheterogeneity in cases where the amounts of protein are too low to permit peptide mapping analysis and where the nature of the charge‐altering modification is unknown.
In Vitro and In Vivo Irreversible Blockade of Cortical S2 Serotonin Receptors by N‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline: A Technique for Investigating S2 Serotonin Receptor Recovery
Authors: Battaglia George , Norman Andrew B. , Newton Patricia L. , Creese Ian
Published: December 2006

Abstract: N‐Ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquin‐oline (EEDQ) treatment, both in vitro and in vivo, results in an irreversible blockade of cortical S2 5‐hydroxytryp‐tamine (serotonin) receptors. Incubation of rat cortical homogenates with EEDQ in vitro results in a concentration‐dependent (EC50∼5 μ.M) and time‐dependent decrease in the Bmax of [3H]ketanserin‐labeled S2 serotonin receptors. Extensive washing of the homogenate following in vitro or in vivo EEDQ treatment does not result in an increase in the amount of [3H]ketanserin binding, indicating that EEDQ acts to modify irreversibly cortical S2 serotonin receptors. That the modification of S2 receptor binding by EEDQ occurs at the recognition site of the receptor is indicated by the finding that coincubation with the S2 receptor antagonist ketanserin, but not the D2 3,4‐dihydroxyphenylethylamine (dopamine) receptor antagonist domperidone, selectively protects against the irreversible blockade of S2 serotonin receptors. Peripheral administration of EEDQ results in a dose‐dependent reduction in cortical S2 serotonin receptors with maximal effects (∼90% reduction) observed following 10 mg/kg (i.p.). Seven days following peripheral administration of EEDQ there is a recovery of S2 serotonin receptors back to 74% of the original receptor population. These data demonstrate that EEDQ in vitro and in vivo acts as an irreversible antagonist of S2 serotonin receptors and that it can be used to investigate the recovery rate of these receptors.
Further Evidence for an Intrinsic Neuraminidase in CNS Myelin
Authors: Yohe Herbert C. , Saito Megumi , Ledeen Robert W. , Kunishita Tatsuhide , Sclafani Joseoh R. , Yu Robert K.
Published: December 2006

Abstract: An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin‐associ‐ated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4‐4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF‐54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60°C, the myelin neuraminidase activity was more than sevenfold of that observed at 37°C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin‐associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres. Developmentally, the myelin‐associated neuraminidase activity in the cerebral hemispheres was also the highest at 20–30 days of age and declined to adult levels soon after. The combined evidence strongly indicates the presence of an intrinsic neuraminidase for gangliosides in CNS myelin and this neuraminidase may play an important role in determining the unique ganglioside pattern in the myelin sheath.
Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS
Authors: Simon J. , Benyhe S. , Abutidze K. , Borsodi A. , Szüs M. , Tóth G. , Wollemann M.
Published: December 2006

Abstract: Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3‐[(3‐cholamido‐propyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS). The yield of solubilization was 70–75% with digitonin and 30–35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of μ, δ, and κ opioid receptor binding sites were similar in membrane and digitonin‐solubilized fractions (48–50%μ, 35–37%κ, and 13–17%δ subtypes). The hydrodynamic properties of digitonin‐ and CHAPS‐solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose‐6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45Å and 65Å for the digitonin extract and 31Å and 76Å for the CHAPS‐solubilized preparation.
Release of Endogenous 3,4‐Dihydroxyphenylethylamine and Its Metabolites from the Isolated Neurointermediate Lobe of the Rat Pituitary Gland. Effects of Electrical Stimulation and of Inhibition of Monoamine Oxidase and Reuptake
Authors: Racké Kurt , Muscholl Erich
Published: December 2006

Abstract: Isolated rat neurointermediate lobes were incubated in vitro. The release of 3,4‐dihydroxyphenylethylamine (dopamine, DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and methoxyphenylethanol (MOPET) was determined by HPLC with electrochemical detection. Under resting conditions, the outflow of metabolites was 35–50 times that of DA. HVA accounted for 50%, DOPAC for 45%, and MOPET for 5% of the metabolites. Although an equivalent of 40–50% of the tissue DA content was released per hour as metabolites, the tissue DA content was not reduced after 110 min of incubation. The spontaneous outflow of DA and its metabolites was not affected by the DA uptake inhibitor GBR 12921 (100 μM). Pargyline (10 μM) caused a time‐dependent decrease of all metabolites (up to 90%). In the presence of GBR 12921 and pargyline, the spontaneous outflow of DA increased sevenfold. Removal of the intermediate lobe caused a 78% reduction in tissue DA content and a corresponding reduction of the outflow of metabolites. Electrical stimulation of the pituitary stalk (0.2 ms, 10 V, 15 Hz, three times for 1 min at intervals of 1 min) induced an increase in outflow of DA and all metabolites. DA accounted for 15%, HVA for 41%, DOPAC for 32%, and MOPET for 12% of the evoked release. The electrically evoked release of DA increased fourfold in the presence of GBR 12921 or pargyline and the effects of both drugs were additive. The evoked release of metabolites was not significantly affected by GBR 12921 but completely abolished by pargyline. In conclusion, oxidative deamination and O‐methylation are important pathways for the catabolism of DA in the neurointermediate lobe. Most of the DA metabolites released spontaneously originate from DA that had been degraded intraneuronally without undergoing prior release. Reuptake and extraneuronal metabolism contribute to the inactivation of DA released during electrical stimulation.
In Vitro Release of Endogenous Excitatory Sulfur‐Containing Amino Acids from Various Rat Brain Regions
Authors: Do Kim Quang , Mattenberger Marianne , Streit Peter , Cuénod Michel
Published: December 2006

Abstract: Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons‐medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 μg/ml). The 4‐N, N‐dimethylamino‐azobenzene‐4′‐isothiocyanate (DABITC) derivatization method of Chang [Biochem. J.199, 537–545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur‐containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+‐dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur‐containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur‐containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.
Invalidity of Criticisms of the Deoxyglucose Method Based on Alleged Glucose‐6‐Phosphatase Activity in Brain
Authors: Nelson Thomas , Lucignani Giovanni , Goochee Janet , Crane Alison M. , Sokoloff Louis
Published: December 2006

Abstract: The observations made by Sacks et al. [Neurochem. Rea.8, 661–685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose‐6‐phosphate. predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze‐blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose‐6‐phos‐phate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose‐6‐phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose‐6‐phosphate in brain, that infusion of [12P]glucose‐6‐phosphate results in no labeling of brain, and that the blood‐brain barrier is impermeable to glucose‐6‐phosphate. Glucose‐6‐phosphate cannot, therefore, cross the blood‐brain barrier, and the observation by Sacks and coworkers [J. Appl. Physiol.24, 817–827 (1968); Neuro‐chein. Res.8, 661–685 (1983)J of a positive cerebral arteriovenous difference for [14C]glucose‐6‐phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose‐6‐phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity.
Extracellular Calcium‐Induced Neuroblastoma Cell Differentiation: Involvement of Phosphatidylinositol Turnover
Authors: Reboulleau Christian P.
Published: December 2006

Abstract: The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10‐25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium‐dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second‐messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6‐10 h after addition of calcium), and increase in intracellular calcium levels (12‐24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10‐25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic‐like junctions: this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.
Binding of [3H]Neurotensin in Human Brain: Properties and Distribution
Authors: Kanba Kiyoko S. , Kanba Shigenobu , Okazaki Harou , Richelson Elliott
Published: December 2006

Abstract: The binding of [3H]neurotensin to membranes from human brain at 0°C was specific, saturable, and reversible. In the frontal cortex, the equilibrium dissociation constant (KD) for [3H]neurotensin determined from the ratio of rate constants (k‐1/k1), saturation isotherms, and inhibition binding experiments was 0.80, 2.0, and 2.0 nM, respectively, and the maximum number of binding sites (Bmax) from the saturation isotherms and the competitive binding experiments was 2.4 and 2.2 pmol/g of tissue, respectively. Hill coefficients for binding were equal to 1, indicating the presence of single, noncooper‐ative binding sites. Inhibition of specific binding of [3H]neurotensin by several analogs of neurotensin showed that [Gln4]neurotensin and neurotensin(8–13) had the highest affinities for these binding sites in human frontal cortex, with each analog being ∼13‐fold more potent than neurotensin. In addition, these data showed that the carboxy‐terminal portion of neurotensin played an important part in the binding of this neuropeptide in human brain, a result described for other species. Regional distribution of binding sites was different from that reported for animal brains. Of the 33 different regions investigated, the uncus and substantia nigra showed the highest specific binding of [3H]neurotensin, whereas such areas as the pineal body, medulla, and corpus callosum had few binding sites.
Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse
Authors: Liles W. Conrad , Taylor Stephen , Finnell Richard , Lai Henry , Nathanson Neil M.
Published: December 2006

Abstract: The tottering mouse (tg/tg) is a single‐locusmutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalo‐graphic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tgltg) and coisogenic wild‐type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age‐matched control wild‐type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40‐60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.
Effects of Hypoxia and Anoxia on the Ex Vivo Release of Prostaglandins from Mouse Cortical Slices
Authors: Shohami E. , Gross J.
Published: December 2006

Abstract: Arachidonic acid is transiently accumulated in the brain as a result of a variety of pathological conditions. The synthesis and release of some of its metabolites, namely, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) from cortical slices of mice were studied following exposure to 6 min of hypoxia (7% O2), 45 s of anoxia, and 5 min‐4 h of reoxygenation following anoxia. Hypoxia induced a slight increase in the rate of TXB2 release and a slight decrease in the rate of PGE2 release, whereas 6‐keto‐PGF1α was unaffected. Anoxia (45 s) followed by reoxygenation induced a transient increase in the release of PGE2 and of 6‐keto‐PGF1α with a return to the normal rate at 30 min and 2 h of recovery, respectively. However, the rate of TXB2 synthesis and release reached its peak (twofold increase) after I h and remained significantly higher than the control rate even after 4 h of normal air breathing. Our results demonstrate that hypoxia and anoxia, even of short duration, selectively trigger the activity of thromboxane synthetase and that this elevated rate of synthesis and release persists long after normal oxygen supply is restored. We suggest that enhanced thromboxane synthesis, with normal prostacyclin levels, might have a role in the pathophysiology of ischemic cell damage.
Agonist‐Induced Phosphoinositide Hydrolysis in Choroid Plexus
Authors: Conn P. Jeffrey , Sanders‐Bush Elaine
Published: December 2006

Abstract: 5‐Hydroxytryptamine (5‐HT, serotonin) stimulates phosphoinositide hydrolysis in choroid plexus by interacting with the 5‐HTlc site. In the present study, the effects of 5‐HT were compared with those of other agonists. 5‐HT stimulates a rapid release of all three inositol sugars in a mianserin‐sensitive manner. Inositol bisphosphate and inositol trisphosphate levels increase about twofold within 2.5 min, whereas inositol monophosphate levels are not appreciably elevated until 5 min. In contrast, glutamate, carbachol, histamine, substance P, and vasopressin, agents that increase phosphoinositide hydrolysis in other tissues, do not stimulate this response in choroid plexus. High concentrations of norepinephrine increase inositol phosphate release in choroid plexus, but this effect is apparently mediated by activation of the 5‐HTlc site. The depolarizing agents KCl and veratrine also fail to stimulate phosphoinositide hydrolysis in choroid plexus. These results, combined with the finding that the phosphoinositide response to 5‐HT is insensitive to tetrodotoxin, suggest that the effects of 5‐HT are not secondary to neurotransmitter release. Furthermore, an indirect effect mediated via arachidonic acid metabolism is unlikely, since inhibitors of cyclooxygenase and lipoxygenase do not reduce the 5‐HT response. We conclude, therefore, that phosphoinositide hydrolysis is the transducing mechanism of the 5‐HT 5‐HTlc receptor and that the choroid plexus will serve as a useful model system for studies of this receptor.
Estrogen‐Induced Up‐Regulation of γ‐Aminobutyric Acid Receptors in the CNS of Rodents
Authors: Maggi A. , Perez J.
Published: December 2006

Abstract: Previous studies have identified an effect of estrogen administration on the number of central GABAergic binding sites of rat. We have further characterized this effect by performing a series of experiments in vitro where we analyzed the changes of γ‐aminobutyric acid (GABA) binding in slices of nervous tissue incubated in a physiological medium in presence of estradiol. The tissues were dissected from ovariectomized rats. In such a system, estrogen augmented the amount of [3H]muscimol binding within 3 h of incubation. The effect was dose‐dependent and could be blocked by the addition of the anti‐estrogen tamoxifen. The increase in [3H]muscimol binding could not be observed by addition of estradiol to broken membranes or by incubation of the slices with steroids deprived of estrogenic activity. Furthermore, the estrogen‐induced increase of GABA binding sites could be prevented by addition of cycloheximide and α‐amanitin in the incubation medium. Our data indicate that the estrogen may increase the number of GABA binding sites by direct interaction with the GABA receptor gene or genes involved in the metabolism of GABA receptor.
Sexual Dimorphism in the Response of the GABAergic System to Estrogen Administration
Authors: Perez J. , Zucchi I. , Maggi A.
Published: December 2006

Abstract: Administration of estradiol benzoate to gonadectomized female rats results in up‐regulation of CNS γ‐aminobutyric acid (GABA) receptors. The increase of [3H]muscimol binding activity is observed in six of the seven brain areas examined. The same treatment, performed in castrated male or and rogenized female rats, induced an increase of [3H]muscimol binding only in the striatum. Evidence is provided suggesting that the dimorphic sensitivity of GABA receptor is not correlated with the difference in spontaneous motor activity reported between male and female rats.
Quantitation of Vasopressin mRNA and Oxytocin mRNA in Hypothalamic Nuclei by Solution Hybridization Assays
Authors: Burbach J. Peter H. , Van Tol Hubert H. M. , Bakkus Marleen H. C. , Schmale Hartwig , Ivell Richard
Published: December 2006

Abstract: Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magno‐cellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single‐stranded 35S‐labeled VP‐specific and OT‐specific DNA probes that were prepared by primer extension on re‐combinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was <1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdis‐sected supraoptic nucleus (SON) mean (SEM) basal levels of 1.370.18pgVP mRNA and 1.950.I4pgOT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 ± 0.02 pg VP mRNA and 1.77 ± 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85‐fold increase in VP mRNA levels of the SON and a 1.6‐fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.
Changes in the Expression of β and Actins During Differentiation of PC 12 Cells
Authors: Chang Andrew , Toloza Eric , Bulinski Jeannette Chloë
Published: December 2006

Abstract: Cells of the rat pheochromocytoma line PC 12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of β and iso‐forms of nonmuscle actin in extracts of these differentiating cells showed that the β: ratio decreased from 1.30 ± 0.05 to 0.99 ± 0.05 after 6 days of NGF treatment. Cells treated with. N6, O2‐dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of β: isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the β: actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension‐grown PC 12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a “second messenger” for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC 12 cells.
Production and Characterization of Monoclonal Antibodies Against Myelin‐Associated Glycoprotein
Authors: Nishizawa Masatoyo , Tanaka Masami , Inuzuka Takashi , Tanaka Keiko , Baba Hiroko , Miyatani Nobuyuki , Sato Shuzo , Miyatake Tadashi
Published: December 2006

Abstract: Monoclonal antibodies against myelin‐associated glycoprotein were generated by fusing mouse myeloma cells with spleen lymphocytes from BALB/c mice immunized with human myelin‐associated glycoprotein purified from CNS myelin. Three groups of antibodies were identified: IgG antibodies recognizing the polypeptide moiety and IgG and IgM antibodies recognizing the carbohydrate moiety of the intact molecule. Properties of these antibodies were examined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and the immunostaining technique using human CNS and peripheral nerve myelin, and ganglioside fractions isolated from human brain and peripheral nerve, and with immunohistochemical staining of human peripheral nerves. Part of human peripheral blood mononuclear cells was stained with the antibodies against the carbohydrate moiety, but not with IgG antibodies recognizing the polypeptide moiety. Natural killer activity was partially reduced after treatment of human peripheral blood lymphocytes with an IgM antibody and complement in vitro. The possibility that anti‐myelin‐associated glycoprotein antibodies might play a role in the pathogenesis of demyelinating diseases through modification of natural killer activity is discussed.
Myelin‐Deficient Rat: Analysis of Myelin Proteins
Authors: Yanagisawa Katsuhiko , Duncan Ian D. , Hammang Joseph P. , Quarles Richard H.
Published: December 2006

Abstract: Myelin basic protein (BP), proteolipid protein (PLP), myelin‐associated glycoprotein (MAG), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin‐deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25‐day‐old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.
Insulin Reverses Enhanced Incorporation of 32P into Polyphosphoinositides in Peripheral Nerve of the Streptozotocin Diabetic Rat
Authors: Berti‐Mattera Liliana N. , Peterson Richard G. , Eichberg Joseph
Published: December 2006

Abstract: The ability of insulin treatment to reverse altered phosphoinositide metabolism in sciatic nerve from Streptozotocin diabetic rats was studied. Diabetes was induced in rats by means of a single injection of Streptozotocin. Enhanced incorporation of 32P into phosphatidylinositol‐4,5‐bisphosphate (PIP2) was detectable as early as 8 days following intravenous injection of Streptozotocin and was maximal after 4 weeks. Hormone treatment was initiated at this time by daily injections of protamine zinc insulin followed by the implantation of long‐acting insulin osmotic minipumps, and 4 weeks later sciatic nerves were removed and incubated in the presence of [32P]orthophosphate. The increased labeling of PIP2 was completely reversed by hormone administration. In contrast, insulin (0.1 and 1.0 mU/ ml) added to the incubation medium failed to reverse the altered pattern of 32P incorporation into PIP2. The uptake of 32P into PIP2 was 80% higher into the proximal than into the distal portion of normal sciatic nerve when these were incubated separately. This metabolic difference was abolished in diabetic rats, although the incorporation into both segments was still significantly higher than in controls. These results strengthen the association of altered nerve PIP2 metabolism with the diabetic state and are consistent with the concept that experimental diabetic neuropathy is a distal axonopathy.
N‐Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure
Authors: Braun Axel , Barde Yves‐Alain , Lottspeich Friedrich , Mewes Werner , Thoenen Hans
Published: December 2006

Abstract: A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme‐antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro‐IIe‐Leu‐Glu‐Lys‐Thr‐Pro‐Pro‐Lys‐Met‐Ala.
Human γγ‐Enolase: Two‐Site Immunoradiometric Assay with a Single Monoclonal Antibody
Authors: Federsppiel B. S. Soler , Cras P. , Gheuens J. , Andries D. , Lowenthal A.
Published: December 2006

Abstract: A monoclonal antibody (mAb), termed BBS/NC/VI‐H14 (H14), that reacts with the human enzyme γγ‐enolase was prepared. It was directed against the γ‐subunit and did not cross‐react with the α‐ or β‐subunit. The mAb H14 can be used for quantitative determination of γγ‐enolase in a two‐site immunoradiometric assay (two‐site IRMA). It is also suitable for immunostaining formalin‐fixed tissues. The specific identification of γγ‐enolase provided by the two‐site IRMA with H14 is discussed in relation to the cellular distribution of this protein.
Brain Galactolipid Content in a Patient with Pseudo‐Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno‐, and Other Leukodystrophies
Authors: Harzer K. , Kustermann‐Kuhn B.
Published: December 2006

Abstract: A 4‐year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudo‐arylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachro‐matic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodys‐trophy. A general mechanism of this anomaly in severe de‐myelination is considered.
Characterization and Localization of Phospholipase A2Activity in Sympathetic Ganglia
Authors: Quik M.
Published: December 2006

Abstract:Superior cervical ganglion phospholipase A2activity was characterized using l‐palmitoyl‐2‐[l‐14C]arachidonoyl‐sn‐glycero‐3‐phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver‐Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Kmof 0.53 mMand aVmaxof 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+dependence; in the absence of Ca2+and presence of EGTA, activity was reduced by 40%. The phospholipase A2activity was heat sensitive and was completely inactivated after treatment at 100°C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was l‐palmitoyl‐2‐[l‐14C]arachidonoyl‐sn‐glycero‐3‐phosphocholine = l‐palmitoyl‐2‐[1‐14C]linoleoyl‐sn‐glycero‐3‐phosphocholine > l‐palmitoyl‐2‐[l‐14C]palmitoyl‐sn‐gly‐cero‐3‐phosphocholine. For determination of the localization of the phospholipase A2enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an ˜50% decline in phospholipase A2activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2activity was seen, although after 4 weeks of denervation, a small but non‐significant decrease in enzyme activity was observed. These results suggest that the enzyme has a predominantly post‐synaptic localization. Extrinsic phospholipase A2enzymes have previously been shown to affect postsynaptic function in rat sympathetic ganglia. The presence of a phospholipase A2in the tissue itself could suggest that endogenous phospholipase A2enzymes can regulate postsynaptic neuronal function.
Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells
Authors: Torres Magdalena , France Bader M. , Aunis Dominique , Miras‐Portugal M. Teresa
Published: December 2006

Abstract: Chromaffin cells both recently isolated or in culture present a high‐affinity adenosine transporter with a Km value of 1 μM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3μM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (˜200–260%) in NGF‐cultured cells throughout the period studied.
Release of Leukotriene B4 from Sublethally Injured Oligodendrocytes by Terminal Complement Complexes
Authors: Shirazi Yasaman , Imagawa David K. , Shin Moon L.
Published: December 2006

Abstract: In the present study, the interaction of the terminal complement complexes with Oligodendrocytes was investigated for observation of its effect on membrane lipid hydrolysis. [14C]Arachidonic acid was incorporated into the membrane lipids of cultured Oligodendrocytes before sensitization with anti‐galactocerebroside antiserum. Cells were then exposed to excess C6‐deficient rabbit serum reconstituted with limiting doses of C6 to form various numbers of C5b‐9 complexes. Qualitative analysis of the supernatants by HPLC revealed the presence of compounds that coeluted with arachidonic acid and its oxygenated derivatives, prostaglandin E2, leukotrienes E4 and B4, and 15‐hydroxyeicosatetraenoic acid. The kinetics of leukotriene B4 release by excess C5b‐8 was quantitated by radioimmunoassay. Leukotriene B4 release approached a maximum around 30 min, and C6 dose‐response studies performed at l h showed that maximal levels of leukotriene B4 were detected over a range of sublytic C5b‐9 attack. Maximal release of leukotriene B4 was also achieved by C5b‐8 without further enhancement by addition of lytic doses of C9. Results indicate that sublytic attack of Oligodendrocytes by complement induces release of lipid‐derived inflammatory mediators.
A Similar Calmodulin‐Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles
Authors: Fournier S. , Trifaró J.‐M.
Published: December 2006

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.
Vasoactive Intestinal Peptide Increases Acetylcholine Synthesis by Rat Hippocampal Slices
Authors: Lapchak P. A. , Collier B.
Published: December 2006

Abstract: The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) might have a presynaptic modulatory effect at cholinergic terminals in the rat hippocampal formation. The exposure of rat hippocampal slices to VIP increased [3H]acetylcholine ([3H]ACh) synthesis from the precursor [3H]choline when tissue was incubated in normal or in high K+ medium; the maximal effect was apparent at 10−8M VIP and 10−7M VIP, respectively. Also, 10−7M VIP increased the activity of choline acetyltransferase (ChAT) in a hippocampal homogenate system. The increased synthesis by hippocampal slices was not the result of a VIP‐induced alteration in either the basal release of ACh or the uptake of choline via the high‐affinity uptake system. The increase in ACh synthesis induced by VIP in hippocampal slices was not associated with either adenylate cyclase or protein kinase C second messenger systems. There was no correlation between the effect of VIP on cyclic AMP production with that on ACh synthesis; also, forskolin, an activator of adenylate cyclase that increased cyclic AMP production 3.5‐fold, did not mimic the effect of VIP on ACh synthesis. Similarly, there was no effect of the protein kinase C activator, phorbol myristate acetate, on ACh synthesis in hippocampal slices. However, the effect of VIP to increase ACh synthesis was not evident in the absence of extracellular calcium, suggesting that the effect of VIP is mediated by a calcium‐requiring mechanism. The results suggest that, in the rat hippocampus, VIP has a presynaptic action at cholinergic terminals that results in enhanced synthesis of ACh, possibly by an action that alters ChAT activity.