bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoprecipitation of an Affinity‐Alkylated Fragment of the Muscarinic Acetylcholine Receptor with an Anti‐Ligand Monoclonal Antibody
Authors: Norman Marjorie W. Gainer , Nathanson Neil M.
Published: October 2006

Abstract: A monoclonal antibody raised against the muscarinic acetylcholine affinity‐alkylating antagonist propylben‐zilylcholine mustard was tested for its ability to recognize affinity‐alkylated muscarinic receptors. We demonstrate here that although the antibody will not recognize the mustard when it is covalently linked to the native muscarinic receptor, trypsinization of affinity‐labeled membranes releases a proteolytic labeled fragment that can be specifically immunoprecipitated by the antibody. Electrophoretic analysis of the immunoprecipitate indicates that the ligand was associated with a polypeptide of molecular weight 5,000. The recognition of this fragment by the antibody provides a means to immunopurify a portion of the muscarinic receptor that is at or near the ligand binding site.
Equilibration of Halothane with Brain Tissue In Vitro: Comparison to Brain Concentrations During Anesthesia
Authors: Bazil Carl W. , Raux Mary Ellen , Yudell Sherry , Minneman Kenneth P.
Published: October 2006

Abstract: A method was devised for reproducing anesthetic concentrations of halothane in slice and membrane preparations of rat brain in vitro. Rats were anesthetized with varying concentrations of halothane, responsiveness was tested, and brain halothane content was determined by heptane extraction and gas chromatography. The inspire concentration of halothane at which half of all animals were unresponsive was 1.05%. At 1.25% halothane, all animals were unresponsive and brain halothane was determined to be 41 × 1.3 nmol/mg lipid. No significant differences in halothane concentration between whole brain and a variety of brain regions were detected. To obtain similar concentrations in vitro, membranes or slices of cerebral cortex were incubated in Krebs‐Ringer bicarbonate buffer (KRB) that had been preequilibrated with anesthetic. Halothane equilibrated rapidly with the buffer and the tissues. The partition coefficient between gas and KRB was found to be 0.78, and between brain slices and KRB approximately 12. Slightly lower gas concentrations were necessary in vitro than in vivo to obtain the same tissue levels of anesthetic. Using this method, it was shown that there was no effect of anesthetic concentrations of halothane on the uptake of [3H]‐norepinephrine or [3H]choline into slices of rat cerebral cortex.
Formation and Utilization of the Active Sulfate Donor [35S]3′‐Phosphoadenosine 5′‐Phosphosulfate in Brain Slices: Effects of Depolarizing Agents
Authors: Gulat‐Marnay Christiane , Lafitte Andrée , Vargas Froylan , Schwartz Jean‐Charles
Published: October 2006

Abstract: The accumulation and utilization of [35S]3′‐phos‐phoadenosine 5′‐phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [35S]sulfate. [35S]PAPS levels were directly evaluated after either isolation by ion‐exchange chromatography or quantitative enzymatic transfer of its active [35S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [35S]PAPS formation was also indirectly followed by incubating slices in the presence of β‐naphthol and measuring the levels of [35S]β‐naphthyl sulfate ([35S]β‐NS). Whereas [35S]PAPS levels rapidly reached a plateau, [35S]β‐NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [35S]PAPS formation in ices was completely and rather potently blocked by 2,6‐dichloro‐4‐nitrophenol (IC50= .10 μM), an inhibitor of the PAPS‐synthesizing enzyme system in a cytosolic preparation. [35S]PAPS accumulation and [35S]β‐NS‘formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N‐methyl‐D‐aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.
Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood‐Brain Transfer of Large Neutral Amino Acids
Authors: Reith J. , Ermisch A. , Diemer N. H. , Gjedde A.
Published: October 2006

Abstract: Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood‐brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood‐brain barrier to AVP relative to mannitol. In brain regions protected by the blood‐brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood‐brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.
Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs
Authors: Mehta Ashok K. , Ticku Maharaj K.
Published: October 2006

Abstract: The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]‐flunitrazepam binding was rapid and saturable. The benzo‐diazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 × 1.6 nM and Bmax of 822 × 194 fmol/mg protein. The association and dissociation cf [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration‐dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15–1788, and β‐carbolines like methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3′‐carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration‐dependent manner by ‐γ‐aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)‐etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor‐ionophore complex. Since these cells can also be studied in parallel for characterizing GABA‐induced 36Cl‐influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.
Blockade of Receptor‐Mediated Cyclic GMP Formation by Hydroxyeicosatetraenoic Acid
Authors: McKinney Michael
Published: October 2006

Receptor‐mediated cyclic GMP formation in N1E‐115 murine neuroblastoma cells appears to involve oxidative metabolism of arachidonic acid. Evidence in support of this includes the blockade of this response by lipoxygenase inhibitors, e.g., eicosatetraynoic acid (ETYA) or other metabolic perturbants, e.g., methylene blue. It was recently discovered that the lipoxygenase products 15‐hydroxyeicosatetraenoic (15‐HETE) acid and 12‐HETE, like ETYA, were inhibitors of M1 muscarinic receptor‐mediated cyclic GMP formation. In the present report, the effects of monoHETEs are explored in more detail, particularly with regard to the function of the muscarinic receptor. Like 12‐HETE and 15‐HETE (IC50= 13 and 11 μM, respectively), 5‐HETE inhibited the cyclic GMP response to the muscarinic receptor (IC50= 10 μM). All three of these monoHETEs were shown also to be inhibitors of the cyclic GMP responses to receptors stimulated by carbachol, histamine, thrombin, neurotensin, and bradykinin. 15‐HETE was shown to inhibit the muscarinic receptor‐mediated response in a complex manner (apparent noncompetitive and uncompetitive components; IC50= 18 and 2 μM, respectively). 15‐HETE did not inhibit either the M1 muscarinic receptor‐stimulated release of [3H]inositol phosphates from cellular phospholipids or the M2 muscarinic receptor‐mediated inhibition of hormone (prostaglandin E1)‐induced AMP formation. It seemed possible that the monoHETEs could enter into biochemical pathways for arachidonate in N1E‐115 cells. [3H]Arachidonate and the three [3H]‐monoHETEs all rapidly labeled the membrane lipids of intact NIE‐115 cells, with each [3H]eicosanoid producing a unique labeling profile. [3H] 15‐HETE labeling was noteworthy in that 85% of the label found in the phospholipids was in phosphatidylinositol (PI; t1/2 to steady state = 3 min). Exogenous 15‐HETE inhibited the labeling of PI by [3H]‐arachidonate (IC50= 28 μM) and elevated unesterified [3H]‐arachidonate levels. Thus, the mechanism of blockade of receptor‐mediated cyclic GMP responses by monoHETEs is likely to be more complex than the simple inhibition of cytosolic mechanisms, e.g., generation of a putative second messenger by lipoxygenase, and may involve also alterations of membrane function accompanying the redistribution of esterified arachidonate.
Ion Dependence of Neurotransmitter Uptake: Inhibitory Effects of Ion Substitutes
Authors: Shank Richard P. , Schneider Craig R. , Tighe Joseph J.
Published: October 2006

Several ions commonly used as substitutes for Na+ or Cl‐ were found to inhibit directly the high‐affinity uptake of norepinephrine, dopamine, serotonin, and γ‐aminobutyric acid, but not glutamate or glutamine. When Na+ was partially replaced by any of several different cations or sucrose the uptake of all neurotransmitters studied except that of serotonin was reduced more than could be accounted for by just the inhibitory effect of the cation substitute. In contrast, when Cl was partially replaced by any of several anions only the uptake of dopamine was reduced more than could be accounted for by the inhibitory effect of the anion substitute. These results suggest that for most neurotransmitters the electrochemical potential for Na+, but not for Cl, contributes to the uptake driving force. When either Na+ or Cl was totally replaced by an ion substitute or by sucrose the high‐affinity uptake was virtually abolished, an exception being that glutamate uptake was not affected when isethionate was substituted for Cl. The lack of uptake in the absence of either Na+ or Cl may reflect a specific role for these ions in either increasing the affinity between the substrate and the carrier, or facilitating the translocation process. Alternatively, the transport carriers may undergo a nonspecific conformational change to an inactive form in the absence of Na+ or Cl. A partial substitution of Na+ with Li+ or sucrose differentially affected the kinetics of uptake in that replacement with Li+, but not sucrose, usually resulted in a marked increase in the Km values. The results of this study emphasize the importance of taking into consideration the effects of ions used as substitutes in experiments undertaken to elucidate the roles of Na+ and Cl in the high‐affinity uptake of monoamine and amino acid neurotransmitters.
Enhanced Potassium‐Stimulated γ‐Aminobutyric Acid Release by Astrocytes Derived from Rats with Early Hepatogenic Encephalopathy
Authors: Albrecht Jan , Rafaowska Urszula
Published: October 2006

Abstract: Bulk‐isolated astrocytes from rats with early hepatogenic encephalopathy (HE) induced with thioacetamide responded to the increase of potassium in the incubation medium from 5 mM to 75 mM with a markedly enhanced release of previously taken up [14C]γ‐aminobutyric acid ([14C]GABA). The process was not affected by omission of calcium and/or addition of EGTA to the incubation medium. Only a slight stimulation of GABA release by high potassium was observed in astrocytes from control rats. In contrast, histamine and histidine were vigorously released from control astrocytes in high‐potassium medium, and their release was not enhanced by HE, indicating that the observed phenomenon is specific for GABA.
Effect of Repeated Convulsive Seizures on Brain γ‐Aminobutyric Acid Metabolism in Three Sublines of Mice Differing by Their Response to Acoustic Stimulations
Authors: Ciesielski L. , Simler S. , Clement J. , Mandel P.
Published: October 2006

Abstract: The turnover rates and steady‐state levels of γ‐aminobutyric acid (GABA) have been determined in 15 brain areas of three sublines of inbred mice differing in their susceptibility to audiogenic seizures: Rb3, which is seizure resistant; Rb2, which develops clonic seizures; and Rbl, which develops tonic‐clonic seizures. In the Rbl subline, GABA steady‐state levels are lower than in the Rb3 subline in three of the 15 areas examined (cerebellum, anterior colliculus, and amygdala), whereas in the Rb2 subline, steady‐state levels are either higher (posterior colliculus and hippocampus) or lower (amygdala) than in the Rb3 subline. GABA turnover rates differ in three brain areas in Rbl (amygdala, raphe, and hypothalamus) and in a single area (amygdala) in Rb2 when compared with Rb3. Only one area has similar variations of GABA turnover rate and steady‐state levels in the two susceptible sublines: the amygdala. After 2 weeks of repeated auditory stimulations (two times a day, 8,000 Hz, 100 dB), additional alterations in GABA metabolism are observed: mainly large increases in GABA turnover rates (from 40% to three‐to fourfold). The Rb2 subline displays a greater number of alterations (increases of turnover rates in pons, cerebellum, anterior and posterior colliculus, amygdala, olfactory bulbs and tubercles, striaturn, and frontal cortex) than the Rb1 subline (increases of turnover rates in cerebellum, posterior colliculus, olfactory tubercles, raphe, and frontal cortex and a decrease in hypothalamus). In the Rb3 subline, increases of the turnover rate in amygdala and olfactory tubercles and decreases in olfactory bulbs and hippocampus are observed. After repeated auditory stimulations, some significant changes of the GABAergic system are specific and common to the seizure‐susceptible sublines: an increase of turnover rate in the cerebellum, posterior colliculus, and frontal cortex and a decrease of steady‐state level in the hippocampus.
Lack of Usefulness of DN‐1417 for Characterization of a CNS Receptor for Thyrotropin‐Releasing Hormone
Authors: Hawkins E. F. , Wade R. , Engel W. K.
Published: October 2006

Abstract: CNS receptors for thyrotropin‐releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog γ‐butyrolactone‐γ‐carbonyl‐L‐histidyl‐L‐proli neamide (DN‐1417), Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3‐methyl‐histidyl2]‐TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN‐1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4–100 μM) resolved only a single, saturable receptor with high affinity (KD= 12–14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN‐1417 was substituted as the assay ligand, no high‐affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN‐1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low‐affinity binding of DN‐1417 in these regions (Ki values = 2–3 μM; in agreement with values obtained in similar assays with [3H]MeTRH). We found that bacitracin in the assay buffer was useful in preventing the deamidation of [3H]TRH otherwise seen during incubation with limbic forebrain membranes. Our data do not support the existence of TRH receptor subtypes in the CNS. Rather, they favor the view that the neurotransmitter‐like effects of high‐and low‐affinity TRH agonists are mediated by a single type of TRH receptor.
Subsets of Axonally Transported and Periaxonal Polypeptides Are Released from Regenerating Nerve
Authors: Tedeschi Bruce , Wilson David L.
Published: October 2006

Abstract: Using two‐dimensional polyacrylamide gel elec‐trophoresis to analyze proteins, we have found subsets of periaxonal and fast‐transported axoplasmic proteins that are released in vitro from regenerating sciatic nerve into a surrounding bath. Of the fast‐transported proteins that are released from nerve, there is a subset of at least five polypeptides that appears in greater relative abundance in the bath than in the nerve. Some of these released, fast‐transported proteins are glycosylated. Several periaxonally synthesized polypeptides are released in significantly greater amounts from regenerating nerve, and of these polypeptides, two are released in greater amounts from nerve only at regions of regeneration or distal to regeneration. These released polypeptides do not represent the most abundant of the locally synthesized proteins. The released, fast‐transported and periaxonal proteins may play a role in intercellular signaling or in modulation of the extracellular environment during nerve regeneration.
Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes
Authors: Meyer E. M. , Otero D. H. , Morgan E. , Marchand S. , Baker S. P.
Published: October 2006

Abstract: The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37°C, AMMA reduced [3H]QNB binding with an IC50 of 9 μM. Following incubation for 5 min with 50 μM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irrevers‐ibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+‐induced release of newly synthesized [3H]ACh in dose‐dependent manners over a 2.5‐min period. At identical 50 μM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+‐induced [3H]ACh release by AMMA (10–250 μM) was blocked by 2 μM atropine during a 2.5‐min release incubation, but not during a 30‐min release in cubation. After synaptosomes were treated with 50 μM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+‐induced) was reduced. AMMA (50 μM) reduced high‐affinity choline uptake and ACh synthesis by >90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because (a) they were not atropine sensitive and (b) hemi‐cholinium‐3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at pre‐synaptic muscarinic autoreceptors.
Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes
Authors: Nichols Robert A. , Haycock John W. , Wang James K. T. , Greengard Paul
Published: October 2006

Abstract: Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+‐dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87‐kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87‐kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, γ‐aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+‐dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus‐secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.
Purification and Properties of Rubrophilin: A Novel Brain Specific Membrane Polypeptide
Authors: Rosenthal Harold L. , Moore Blake W.
Published: October 2006

Abstract: Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R‐250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatoma‐ceous earth, gel filtration on polyacrylamide (Biogel P‐200). gradient elution chromatography from hydroxylapatite, and reverse‐phase chromatography from phenyl‐Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/ mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryp‐tophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals > birds > reptiles > fishes. It is present in mouse retina and human neu‐roblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C‐6 rat glioma cells.
Isolation of Membrane Fractions with Sodium Channels Sensitive to Veratridine and tetrodotoxin from the Electric Organ of the Eel Electrophorus electricus
Authors: Barnola Flor V. , Sevcik Carlos , Delgado Daniel
Published: October 2006

Abstract: The veratridine/tetrodotoxin‐sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre‐ and Postsynaptic. membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside‐out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.
Purification and Characterization of a Peptidyl Dipeptidase Resembling Angiotensin Converting Enzyme from the Electric Organ of Torpedo marmorata
Authors: Turner Anthony J. , Hryszko John , Hooper Nigel M. , Dowdall Michael J.
Published: October 2006

Abstract: The electric organ of Torpedo marmorata contains a membrane‐bound, captopril‐sensitive metallopepti‐dase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chro‐matography using the selective inhibitor lisinopril immobilised to Sepharose via a 28‐Å spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C‐terminus of a variety ofpeptide substrates, including angiotensin I, brady‐kinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)‐His‐Leu. The hydrolysis of BzGly‐His‐Leu was activated by Cl−. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuro‐peptide substance P. Hydrolysis of substance P occurred primarily at the Phe8‐Gly9 bond with release of the C‐terminal tripeptide, Gly‐Leu‐MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr= 190,000) than the pig kidney enzyme (Mr= 180,000) on sodium dodecyl sulphate‐poly‐acrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.
Myelin Galactolipid Synthesis in Different Strains of Mice
Authors: Sato Carmen , Yu Robert K.
Published: October 2006

Abstract: Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17–21. However, the amount of myelin in the brains of F1 (B6 × D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelino‐genesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14‐, 17‐, and 21‐day‐old D2, B6, and F., mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP‐galactose:ceramide galactosyltransferase and 3′‐phosphoadenylyl sulfate:galactosylceramide 3′‐sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.
Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells
Authors: White Thomas D. , Bourke Jane E. , Livett Bruce G.
Published: October 2006

Abstract: A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin‐luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KC1 induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10‐7M and a maximum at 10‐4M. The dose‐response curve for nicotine was bell shaped, with a threshold at 10‐7M, a maximum at 10‐5M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10‐4M nicotine actually induced release of ATP at a faster rate than 10‐5M nicotine. However, the rate of ATP release evoked by 10‐4M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10‐5M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5–1.5 min and a second phase of release beginning at 1.5–2 min, reaching a maximum by 2–3 min. Comparison of the time course for ATP release evoked by 10‐4M nicotine with the time course for secretion of adrenaline and noradrenaline revealed that adrenaline was the predominant catecholamine released during the initial rapid phase of ATP release. A slight plateau for adrenaline release coincided with the plateau for ATP release. Finally, the second phase of ATP release coincided with the time at which release of noradrenaline exceeded the release of adrenaline. The initial phase of ATP release could originate primarily from adrenaline‐containing cells, whereas the second phase could occur primarily from noradrenaline‐containing cells in the monolayer culture.
Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus After Axotomy of Afferent or Centrifugal Fibers
Authors: Staatz‐Benson C. , Potashner S. J.
Published: October 2006

Abstract: Glycine may be an inhibitory transmitter in the mammalian cochlear nucleus (CN). This study attempts to determine if cochlear and/or centrifugal projections to the CN use glycine as a transmitter. The high‐affinity uptake and electrically evoked release of exogenous [14C]glycine were measured in vitro in the three major subdivisions of the guinea pig CN: the anteroventral, posteroventral, and dorsal cochlear nuclei (AVCN, PVCN, and DCN, respectively). [14C]Glycine (3.4 μM) was taken up by each subdivision, reaching tissue concentrations six to seven times that in the medium. Subsequent electrical stimulation evoked a Ca2+‐dependent release of [14C]glycine from each subdivision. These activities were compared in subdivisions from unlesioned animals, and from animals with lesions of centrifugal or cochlear projections to the CN. Two knife‐cut lesions were made to interrupt centrifugal projections to the CN lying in the right acoustic striae and trapezoid body. In one group of animals, centrifugal fibers projecting mainly to the right AVCN and PVCN were severed, which reduced [14C]glycine uptake and release by 44–53% in these subdivisions, but not in the right DCN. In another group of animals, fibers projecting mainly to the right PVCN and DCN were severed, which reduced [14C]glycine uptake and release by 33–47% in these subdivisions, but not in the right AVCN. In CN subdivisions contralateral to either lesion there was no significant change in [14C]glycine uptake or release. Neither of these lesions altered the uptake or release of D‐[3H]aspartate in the right or the left CN. Ablation of the left cochlea, which presumably destroyed cochlear nerve fibers unilaterally, had no effect on [14C]glycine uptake and release. These observations suggest that centrifugal projections contribute a proportion of the glycinergic syn‐aptic endings in the CN. In addition, some glycinergic endings probably arise from neurons intrinsic to the CN. The cochlear nerve contains very few, if any, glycinergic fibers.
411B: A Monoclonal Postsynaptic Marker for Modulations of Synaptic Connectivity in the Rat Brain
Authors: Loessner Bernd , Bullock Sarah , Rose Steven P. R.
Published: October 2006

Abstract: Using 411B, a monoclonal marker raised to chick forebrain postsynaptic densities (PSDs), we have been able to demonstrate by enzyme‐linked immunosorbent assay that the antigen recognised by this monoclonal exists in brain tissue from adult Wistar rats but not in liver, heart, or lung. Moreover, 411B immunoreactivity estimated in various cortical and subcortical brain structures exhibited remarkable differences. The pattern of subcellular distribution of 411B antiserum titre in rats was found to be qualita‐tively similar to that in day‐old chicks, indicating an enrichment of the antigen concentration in the PSD fraction by about 60 times over that observed in the lysed homogenates. One aim of this study was to investigate whether 411B is a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. Antigen was assayed in lysed homogenates from various brain regions dissected from dopaminergically supersensitive rats. Dopaminergic supersensitivity induced by treating animals with haloperidol (1 mg/kg i.p.) for 21 consecutive days resulted in a significant increase in the titre of 411B in corpus striatum (+21%) and hippocampus (+45%) whereas the titre of Q155, a monoclonal marker for an integral synaptic vesicle protein, was unchanged. Our results support the hypothesis that drug‐induced dopaminergic supersensitivity is based on plastic changes at the postsynaptic site. In addition, monoclonal antibody 411B does appear to be a useful tool for further investigation of plastic changes occurring in postsynaptic brain components.
Formation of γ‐Amino‐β‐Hydroxybutyric Acid from 2‐Hydroxyputrescine in Rat Brain
Authors: Noto Tadashi , Hasegawa Takeshi , Nakao Junji , Kamimura Hiroshi , Harada Hiroyuki , Nakajima Teruo
Published: October 2006

Abstract: γ‐Amino‐β‐[3H]hydroxybutyric acid ([3H]‐GABOB) was formed in rat brain from 2‐[3H]‐hydroxyputrescine that had been chemically synthesized from 2‐oxoputrescine and [3H]sodium borohydride. After the injection of 2‐[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion‐exchange chromatography, high‐voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.
Plasma Osmolality Predicts Extracellular Fluid Catechol Concentrations in the Lateral Hypothalamus
Authors: Mason P. A. , Durr J. A. , Bhaskaran D. , Freed C. R.
Published: October 2006

Abstract: The lateral hypothalamus has an important role in regulating food and water intake. We have investigated the endogenous release of monoamines from the lateral hypothalamus during manipulations of plasma osmolality and circulating volume. Adult male Sprague‐Dawley rats implanted with carbon paste in vivo electrochemical (EC) electrodes in the lateral hypothalamus were placed on a 72‐h water deprivation schedule. Although the carbon paste EC electrode has an intrinsically ambiguous signal in which changes in ascorbic acid may appear as changes in catechol concentrations, pharmacologic studies in lateral hypothalamus indicated that the electrode most likely measured nor‐epinephrine and possibly epinephrine. On the test day, the EC electrodes were scanned with linear sweep voltammetry from ‐0.2 to +0.4 V at a rate of 5 mV/s. Semiderivative signal processing showed catechol and hydroxyindole peaks at +0.11 and +0.23 V, respectively. Baseline recordings were made prior to rats drinking distilled water, 10% sucrose, 5% dextrose, 0.30% NaCl, 0.90% NaCl, or 10%D‐mannitol. To control for the act of drinking, other implanted dehydrated rats were intraperitoneally injected with 5% dextrose, 0.30% NaCl, or 0.90% NaCl. To dissociate the effects of osmolality and circulating volume on the EC response, hydrated rats implanted with EC electrodes were subcutaneously injected with 12% NaCl or intraperitoneally injected with 35% polyethylene glycol. Other rats subjected to water deprivation and osmotic challenges were decapitated and trunk blood was collected for measurements of plasma osmolality and hematocrit. Similar experiments were conducted using homozygous Brattleboro rats which lack arginine vasopressin (AVP) but which preserve normal plasma osmolality with prodigious drinking. The use of these rats provided a way to dissociate monoamine changes related to AVP release from changes related to plasma osmolality. Results in Sprague‐Dawley and Brattleboro rats showed that the EC catechol signal was lower in rats with high plasma osmolality and rose with drinking or intraperitoneal fluid administration as a direct function of the fall in plasma osmolality. Manipulations of plasma osmolality had a greater effect on the EC catechol signal than did manipulations of circulating volume. Thus, we conclude that changes in extracellular fluid catechol concentrations in the lateral hypothalamus linearly reflect changes in plasma osmolality.
Properties and Ontogenic Development of Membrane‐Bound Histidine Decarboxylase from Rat Brain
Authors: Toledo A. , Sabriá J. , Rodriguez R. , Brandner R. , Rodriguez J. , Palacios J. M. , Blanco I.
Published: October 2006

Abstract: Histidine decarboxylase (HD) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty‐eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane‐bound HD were compared. No significant differences in the values of Km for histidine and pyridoxal 5′‐phosphate were observed. The solubilization of membrane‐bound HD with Triton X‐100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the Km for substrate and cofactor. The proportion of free pyridoxal 5′‐phosphate‐independent activity was identical in both fractions. The soluble and membrane‐bound forms of the enzyme differ slightly in their pH‐activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane‐bound activity became stabilized from day 20.
Effect of a Deficiency of Thiamine on Brain Pyruvate Dehydrogenase: Enzyme Assay by Three Different Methods
Authors: Elnageh K. M. , Gaitonde M. K.
Published: October 2006

Abstract: A simple and rapid method based on the NADH‐linked reduction of a tetrazolium dye was described for the determination of pyruvate dehydrogenase activity in rat brain homogenates. The method (method 3) gave a value of 36.06 ± 1.24 nmol of pyruvate utilised/min/mg of whole brain protein. This value was higher than that obtained by measurement of the rate of decarboxylation of [l‐14C]pyruvate (15.10 ± 0.88 nmol/min/mg of protein; method 1) and was comparable with the rate of transfer of acetyl groups to an arylamine (39.04 ± 1.32 nmol/min/mg of protein; method 2). A critique of the values reported by others by different methods was given. The pyruvate dehydrogenase activity in the mitochondria isolated from rat brain was in the “active” (nonphosphorylated) form. A deficiency of thiamine in rats was produced by treatment with pyrithiamine, an antagonist of thiamine. This treatment resulted in abnormal neurological signs, such as ataxia and convulsions. The measurement of the total activity of pyruvate dehydrogenase in the brain by all three methods showed no significant change in the enzymic activity in thiamine‐deficient rats after treatment with pyrithiamine. The activities of the enzyme in the brains of pair‐fed animals were similar to those in the controls.
Aromatic l‐Amino Acid Decarboxylase Activity of the Rat Retina Is Modulated In Vivo by Environmental Light Maria Hadjiconstantinou
Authors: Rossetti Zvani , Silvia Christopher , Krajnc Dimitrij , Neff Norton H.
Published: October 2006

Abstract: Aromatic l‐amino acid decarboxylase (AAAD) activity of rat retina is low in animals placed in the dark. When the room lights are turned on, activity rises for almost 3 h and reaches values that are about twice the values found in the dark. A study of the kinetics of the enzyme revealed that the apparent Km values for l‐3,4‐dihydroxyphenylalanine and pyridoxal‐5′‐phosphate were unchanged in light‐ and dark‐exposed animals, whereas the Vmax increased in the light. Treating the animals with cycloheximide before exposure to light prevented the increase of enzyme activity. Immunoti‐tration with antibodies to AAAD suggested that more enzyme molecules are present in the light than in the dark. When the room lights are turned off AAAD activity drops rapidly at first and then more slowly, suggesting that at least two processes are responsible for the fall of enzyme activity. Exposure to short periods of dark followed by light results in a rapid increase of AAAD activity. Mixing homogenates from light‐and dark‐exposed rats results in activity values that are less than expected, suggesting the presence of an endogenous in‐hibitor(s). These studies demonstrate that AAAD activity is modulated in vivo.
Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane
Authors: Sapirstein Victor S. , Nolan Charles , Stern Richard , Ciocci Michael , Masur Sandra K.
Published: October 2006

Abstract: We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S‐1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes from adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1‐2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.
Characterisation of Na+‐Independent L‐[3H]Glutamate Binding Sites in Human Temporal Cortex
Authors: Cowburn R. F. , Hardy J. A. , Roberts P. J.
Published: October 2006

Abstract: The binding of L‐[3H]glutamate to membranes from human temporal cortex was studied in the absence of Na+, Ca2+, and Cl− ions. Pharmacological characterisation revealed that approximately 35% of specific binding at 50 nM L‐[3H]glutamate was sensitive to a combination of kainate and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid. The remaining approximately 65% of specific binding was to a single population of sites with a KD of 844 nM and a Bmax of 0.92 pmol/mg protein. The pharmacological characteristics were consistent with an interaction at the N‐methyl‐D‐aspartate subclass of excitatory amino acid receptor. The inclusion of Cl− ions revealed additional glutamate binding; this was sensitive to quisqualate and DL‐2‐amino‐4‐phosphonobutyrate, but not to kainate, DL‐2‐amino‐7‐phosphonoheptanoate, or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid.
Melatonin Biosynthesis in Drosophila: Its Nature and Its Effects
Authors: Finocchiaro L. , Callebert J. , Launay J. M. , Jallon J. M.
Published: October 2006

Drosophila melanogaster homogenates incubated with tritiated 5‐hydroxytryptophan, 5‐hydroxytryptamine, or N‐acetylserotonin have the ability of converting them into labelled indolamines, including melatonin. All these compounds were characterized by two‐dimensional thinlayer chromatography by comparison with nonradiolabelled standards, and melatonin by mass spectrometry and radioimmunoassay. On the other hand, the injection of pharmacological doses of melatonin into 2‐day‐old female flies diminishes the mating speed and the oviposition rate.
Racemization of Individual Aspartate Residues in Human Myelin Basic Protein
Authors: Shapira Raymond , Wilkinson Keith D. , Shapira Gary
Published: October 2006

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