bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma Membrane and Chromaffin Granule Characteristics in Digitonin‐Treated Chromaffin Cells
Authors: Holz Ronald W. , Senter Ruth A.
Published: October 2006

Abstract: Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 μM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin‐treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin‐treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 μM reserpine, 30 mM NH4+, or 1 μM carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP‐induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules within digitonin‐treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 μM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin‐treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane‐bound dopamine‐β‐hydroxylase. The studies demonstrate that 20 μM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.
Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1‐Like Dopamine Receptor
Authors: Schulz David W. , Stanford Edward J. , Wyrick Stephen W. , Mailman Richard B.
Published: October 2006

Abstract: The compound [9‐3H]SCH23390 [R‐(+)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1H‐3‐benzazepine‐7‐ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4‐dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high‐affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and olfactory bulb. Specific binding in caudate‐putamen was found to be both temperature‐ and pH‐dependent, with optima at 25–30°C and pH 7.8–8.0. Scatchard or Woolf analyses of binding in caudate‐putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD= 0.7 ± 0.1 nM; Bmax= 347 ± 35 fmol/mg of protein). Both dopamine and cis‐flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis‐flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.
Calcium/Ganglioside‐Dependent Protein Kinase Activity in Rat Brain Membrane
Authors: Goldenring James R. , Otis Laura C. , Yu Robert K. , DeLorenzo Robert J.
Published: October 2006

Abstract: The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high‐molecular‐weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low‐molecular‐weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+ ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1. GD1a was the most potent activator tested. Asialo‐GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+ ‐dependent, ganglioside‐stimulated phosphorylation was qualitatively similar to the pattern for calmodulin‐dependent phosphorylation. However, while calmodulin‐dependent kinase activity was inhibited with an IC50 of 10 μM trifluoperazine, ganglioside‐stimulated kinase was inhibited with an IC50 of 200 μM trifluoperazine. These results indicate that gangliosides have complex effects on membrane‐associated kinase activities and suggest that Ca2+ ‐ganglioside complexes are potent stimulators of membrane kinase activity.
Difference in the Pattern of Soluble Proteins from Rat Brain Regions
Authors: Kler A. , Rosen A.
Published: October 2006

Abstract: The electrophoretic pattern of soluble proteins from seven rat brain regions (amygdala, cerebellum, corpus striatum, cortex, hypothalamus, medulla, and midbrain) was examined by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Although the number of protein bands (36) was identical in all brain regions studied, there were differences in their relative densities, the greatest variation occurring in the low‐molecular‐weight region of the electrophoretogram. The bulk of the soluble proteins had molecular weights between 23,000 and 90,000 daltons. The medulla and amygdala showed the greatest range of protein band concentration. A large number of protein bands in the midbrain and corpus striatum showed a greater concentration of protein compared to the same bands in the other regions. A protein band that migrated with the same characteristic as albumin was found. It was consistently high in all regions, the midbrains showing a 1.5‐fold greater concentration compared to other regions. Linear regression analysis of wet weight of regional brain tissue against protein concentration yielded a regression coefficient (r2) of 0.77. Midbrain and corpus striatum showed a relatively higher protein concentration:weight ratio than other regions.
A Search for Receptors Modulating the Release of γ‐[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices
Authors: Limberger N. , Späth L. , Starke K.
Published: October 2006

Abstract: Various putative striatal transmitters and related compounds were studied for their effects on the release of γ‐aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4‐dihydroxyphenylethylamine (dopamine), an effect not blocked by cis‐flupentixol or domperidone and not mimicked by apomorphine and D1‐selective agonists. The electrically evoked overflow was diminished by 5‐hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal out‐flow whereas oxotremorine caused no consistent change; the effects of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, α2‐adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.
Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling
Authors: Dickinson Kenneth E. J. , Leeb‐Lundberg L. M. Fredrik , Strasser Ruth H. , Caron Marc G. , Lefkowitz Robert J.
Published: October 2006

Abstract: The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I‐2‐[β‐(4‐hydroxyphenyl)ethylaminomethyl]tetralone (125I‐HEAT) and 125I‐cyanopindolol (125I‐CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (±5) pM and 30 (±5) pM, respectively. Binding site maxima were 481 (±63) and 1,020 (±85) fmol/mg protein. The sites labeled by 125I‐HEAT had the pharmacological characteristics of α1‐adrenergic receptors. 125I‐CYP‐labeled β‐adrenergic receptors were characterized as a homogeneous population of β1‐adrenergic receptors. The α1‐ and β1‐adrenergic receptors were covalently labeled with the specific photoaffinity probes 4‐amino‐6,7‐dimethoxy‐2‐{4‐[5‐(4‐azido‐3‐[125I]iodo‐phenyl) pentanoyl]‐1‐piperazinyl}quinazoline (125I‐APDQ) and 125I‐p‐azidobenzylcarazolol (125I‐pABC). 125I‐APDQ labeled an α1‐adrenergic receptor peptide of Mr= 74,000 (±4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I‐pABC labeled a single β1‐adrenergic receptor peptide with a Mr= 42,000 (±1,500), which differed from the 60–65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.
Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina
Authors: Ho Anthony K. , Somers Robert L. , Klein David C.
Published: October 2006

Abstract: Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light‐induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.
UDPgalactose:Ceramide Galactosyltransferase of Rat Brain: A New Method of Purification and Production of Specific Antibodies
Authors: Neskovic N. M. , Roussel G. , Nussbaum J. L.
Published: October 2006

Abstract A new method for purification of UDPgalactose: ceramide galactosyltransferase (EC 2.4.1.45) is described. The principal steps involved solvent extraction at—70°, Triton X‐100 extraction, and DEAE‐Sephadex and Blue Sepharose chromatography. The active configuration of the enzyme was stabilized by phospholipids and a rapid loss of enzymatic activity was observed after removal of these lipids. The inactive enzyme could be fully reactivated in the presence of brain phospholipids dispersed in a Triton X‐100‐containing buffer. The purified enzyme preparation showed two major components by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with apparent molecular weights of 50–70,000. The 53,000‐dalton protein was isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate and used to produce antibodies against UDPgalactosexeramide galactosyltransferase.
Lesions to the Corticostriatal Pathways Ameliorate Hypoglycemia‐Induced Arachidonic Acid Release
Authors: Westerberg Eva , Wieloch Tadeusz
Published: October 2006

Abstract The concentrations of free fatty acids (FFAs) in the neostriatum of control rats and rats subjected to unilateral cortical ablation were measured during and following severe insulin‐induced hypoglycemia. The total FFA concentration in the caudate nucleus contralateral to the lesion increased to approximately 1.5 and 3 times the control level after 5 and 30 min of isoelectricity, respectively, and was similar to the control value following 1 h of recovery. After 5 min of isoelectricity, the total FFA pool was significantly smaller in the decorticated striatum. No difference between hemispheres was noted after 30 min of isoelectricity. After 5 min of isoelectricity the levels of stearic and arachidonic acid were selectively increased whereas palmitic acid and oleic acid remained at control levels. In the decorticated striatum of lesioned animals the arachidonic acid concentration was significantly lower, whereas the level of stearic acid was not significantly different from the control value. After 30 min of isoelectricity the levels of all four FFA species were increased. Apart from a significantly lower level of oleic acid on the decorticated side, there were no interhemi‐spheric differences in the FFA levels. Since the early inter‐hemispheric differences in the levels of arachidonic and stearic acids coincide with a selective decrease in the levels of glutamate and a decreased energy utilization on the decorticated side, the results suggest that glutamate release during hypoglycemia induces an early receptor‐mediated degradation of phospholipids, presumably via the phospha‐tidylinositol cycle.
Regional Distributions of Thiobarbituric Acid‐Reactive Products, Activities of Enzymes Regulating the Metabolism of Oxygen Free Radicals, and Some of the Related Enzymes in Adult and Aged Rat Brains
Authors: Mizuno Yoshikuni , Ohta Keiko
Published: October 2006

Abstract: Regional distributions of thiobarbituric acid‐reactive products, activities of enzymes regulating metabolism of oxygen free radicals, and some of the related enzymes were studied in 10 areas of adult and aged rat brains. Thiobarbituric acid‐reactive products were lower in cerebral cortex, septal area, hippocampus, caudateputamen, and substantia nigra compared with other areas studied in adult rats; however, they increased significantly in the former areas with aging. A slight but significant reduction in superoxide dismutase activity was noted in frontal cortex, septal area, caudate‐putamen, and substantia nigra with aging. Glutathione peroxidase and reductase activities were highest in caudate‐putamen and in substantia nigra. Glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase activities were lowest in cortical areas. Phosphofructokinase activity was lowest in septal area and hippocampus in aged rats. Glyceraldehyde‐3‐phosphate dehydrogenase activity showed only small regional and evolutional changes. Lactate dehydrogenase activity declined with age in most of the areas studied. sn‐Glycerol‐3‐phosphate dehydrogenase activity showed small changes with aging except in hippocampus, where 40% reduction was noted. Generally, cerebral cortical areas, hippocampus, and septal areas were not particularly enriched in enzymes regulating the metabolism of oxygen free radicals. The results were discussed in relation to the role of free radicals in aging.
Regional Cerebral Glucose Phosphorylation and Blood Flow After Insertion of a Microdialysis Fiber Through the Dorsal Hippocampus in the Rat
Authors: Benveniste H. , Drejer J. , Schousboe A. , Diemer N. H.
Published: October 2006

Abstract: Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.
Biogenesis of Presynaptic Terminal Proteins
Authors: Garner Judy A. , Mahler Henry R.
Published: October 2006

Abstract: The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two of the major components of axonal transport, and the subsequent persistence and turnover of those proteins were examined in this study. Ganglion cell proteins were radiolabeled by intravitreal injection of radiolabeled amino acids and radioactive axonally transported proteins were analyzed in synaptosomes prepared from the superior colliculi. This procedure allowed examination of presynaptic components of ganplion cell synapses without having to compensate for postynaptic or other unidentified contaminants. Each of the two major axonal transport components supplies a large number of proteins to the presynaptic terminal, in relative quantities similar although not identical to those seen in the axon. Proteins conveyed by the fast component of axonal transport reached the terminals by 3 h after intraocular injection, peaked by 24 h, and were largely undetectable by 15 days. Slow component b proteins reached the terminals by 12 days, peaked around 21 days, and persisted up to 63 days in the terminals. Proteins in both components demonstrated differential turnover relative to cotransported proteins once they reached the terminals. Differential turnover may account for change in relative concentration of a particular protein required to meet new functional demands on that protein once it enters the terminal.
Turnover of Dopamine and Dopamine Metabolites in Rat Brain: Comparison Between Striatum and Substantia Nigra
Authors: Nissbrandt H. , Carlsson A.
Published: October 2006

Abstract: Measurements of the turnover of dopamine (DA) and DA metabolites have been performed in the striatum and substantia nigra (SN) of the rat. Turnover rates of 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid have been assessed from the disappearance rates after blocking their formation by inhibition of monoamine oxidase by pargyline and of catechol‐O‐methyltransferase by tropolone. DA turnover has been measured as 3‐methoxy‐tyramine (3‐MT) plus DA accumulation rate after MAO inhibition by pargyline and as accumulation rate of 3,4‐dihy‐droxyphenylalanine (DOPA) after inhibition of aromatic amino acid decarboxylase by NSD 1015 or NSD 1034. These measures of DA turnover have been compared with α‐methyl‐p‐tyrosine (α‐MT)‐induced DA disappearance rate. In SN all the different measures of DA turnover are in the same range (55–62 nmol/g protein/h) whereas in striatum DOPA accumulation rate after NSD 1015 and α‐MT‐ induced DA disappearance rate (16–23 nmol/g/h) are much lower than DOPAC disappearance rate after pargyline, 3‐MT plus DA accumulation rate after pargyline, and DOPA accumulation rate after NSD 1034 (39–46 nmol/g/h). The data confirm our previous findings indicating that the fractional turnover rate of DA is more rapid in SN than in striatum and that O‐methylation of DA is relatively more important in SN. In striatum at least two pools of DA with different turnover rates appear to exist, whereas in SN, DA behaves as if located in a single compartment.
Postnatal Development of Cholinergic Enzymes and Receptors in Mouse Brain
Authors: Fiedler Eric P. , Marks Michael J. , Collins Allan C.
Published: October 2006

Abstract: The developmental profiles for the cholinergic enzymes acetylcholinesterase and choline acetyltransferase, and the muscarinic and nicotinic receptors were determined in whole mouse brain. The enzyme activities (per milligram of protein) increased steadily from birth, reaching adult levels at 20 days of age. These increases were primarily due to increases in Vmax. Muscarinic receptor numbers, measured by [3H]quinuclidinyl benzilate binding, also increased from birth to 25 days of age. Brain nicotinic receptors were measured with the ligands L‐[3H]nicotine and α‐[125I]‐bungarotoxin. Neonatal mouse brain had approximately twice the number of a‐bungarotoxin binding sites found in adult mouse brain. Binding site numbers rose slightly until 10 days of age, after which they decreased to adult values, which were reached at 25 days of age. The nicotine binding site was found in neonatal brain at concentrations comparable to those at the a‐bungarotoxin site followed by a steady decline in nicotine binding until adult values were reached. Thus, brain nicotinic and muscarinic systems develop in totally different fashions; the quantity of muscarinic receptors increases with age, while the quantity of nicotinic receptors decreases. It is conceivable that nicotinic receptors play an important role in directing the development of the cholinergic system.
Amyloid Angiopathy of Alzheimer's Disease: Amino Acid Composition and Partial Sequence of a 4,200‐Dalton Peptide Isolated from Cortical Microvessels
Authors: Pardridge William M. , Vinters Harry V. , Yang Jing , Eisenberg Jody , Choi Thomas B. , Tourtellotte Wallace W. , Huebner Verena , Shively John E.
Published: October 2006

Abstract: The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid‐like, β‐pleated sheet fibrils. Recently, a 4,200‐dalton pep‐tide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N‐terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical micro‐vessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a mi‐crovessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The arteriolc fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200‐dalton peptide. where as the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200‐dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N‐terminal heterogeneity. The N‐terminal sequence was: Asp‐Ala‐Glu‐Phe‐Arg‐His‐Asp‐Ser‐Gly‐Tyr, which is identical to the N‐terminal sequence of the 4,200‐dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques. These studies report the first isolation of intracortical microvessels from Alzheimer's disease brain and the finding of N‐terminal heterogeneity of the 4,200‐dalton peptide comprising the vascular amyloid.
Polypeptides of the Golgi Apparatus of Neurons from Rat Brain
Authors: Gonatas J. O. , Gonatas N. K. , Stieber A. , Louvard D.
Published: October 2006

Abstract: An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100–110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity‐purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200‐, 150‐, 100–110‐, and 64‐kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40‐kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100–110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.
Benzodiazepine Receptor Binding of Triazolobenzodiazepines In Vivo: Increased Receptor Number with Low‐Dose Alprazolam
Authors: Miller Lawrence G. , Greenblatt David J. , Barnhill Jamie G. , Deutsch Stephen I. , Shader Richard I. , Paul Steven M.
Published: October 2006

Abstract: Triazolobenzodiazepines are in clinical use as hypnotics and anxiolytics. We analyzed in vivo receptor binding and brain concentrations of alprazolam, triazolam, and estazolam. Drug concentrations measured in the cerebral cortex 1 h after administration were directly proportional to dose for all three compounds. In vivo receptor binding, as defined by the specific uptake of [3H]Ro 15–1788, decreased with increasing doses of estazolam and triazolam, a finding indicating dose‐related increases in receptor occupancy due to these compounds. Triazolam was substantially more potent, with an IC50 value of 16 ng/g, compared with 117 ng/g for estazolam. At higher doses of alprazolam (>0.2 mg/kg), receptor binding by [3H]Ro 15–1788 likewise decreased with increasing dose of the former drug. However, at lower doses of alprazolam (0.02–0.05 mg/kg), which resulted in cortex concentrations of 2–7 ng/g, receptor binding was increased above control values in cortex, hypothalamus, and hippocampus but not in several other brain regions. Binding returned to control values at doses of ≤0.01 mg/kg. Similar results were obtained in time course studies. At 8 and 10 h after a dose of 1 mg/kg i.p., corresponding to cortex concentrations of 2.7–7 ng/g, receptor binding was increased compared with controls. Similarly, at 1, 2, and 3 h after a single dose of 0.05 mg/kg, corresponding to cortex concentrations of 3.7–5.8 ng/g, receptor binding was also increased. The apparent affinity of benzodiazepine receptors for clonazepam in mice receiving alprazolam (0.05 mg/kg) was unchanged from that in untreated control mice, an observation suggesting that low doses of alprazolam increased receptor number. The brain concentration vs. receptor occupancy relationships for triazolam and estazolam resemble those for other benzodiaze‐pines, but alprazolam appears to be anomalous in that low brain concentrations increase benzodiazepine receptor number.
Kinetics of Neutral Amino Acid Transport Across the Blood‐Brain Barrier
Authors: Smith Quentin R. , Momma Seiji , Aoyagi Masaki , Rapoport Stanley I.
Published: October 2006

Abstract: Neutral amino acid (NAA) transport across the blood‐brain barrier was examined in pentobarbital‐anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Kmvalues for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied ∼700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r= 0.910) to the octanol/water partition coefficient, a measure of NAA side‐chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the Mood‐brain barrier. Furthermore, they suggest that affinity of a l‐α‐amino acid for the transport system is determined primarily by side‐chain hydrophobicity.
Agonist‐Induced Regulation of Adrenoceptor Subtypes in Cerebral Cortical Slices
Authors: Nukina Itaru , LaBella Frank S.
Published: October 2006

Cerebral cortical slices from rat brain were incubated at 37°C for 2 h in the presence of isoproterenol, noradrenaline, or adrenaline, and binding affinities and densities of adrenoceptor subtypes were subsequently examined in homogenized tissue. The density of α2‐and total β‐adre‐noceptors was estimated using the radioligands [3H]rauwolscine and [3H]dihydroalprenolol (DHA), respectively. The percentages of β1‐and β2‐adrenoceptors were denned by inhibiting the binding of [3H]DHA with the β1‐selective antagonist metoprolol. Exposure of slices to noradrenaline and adrenaline significantly decreased the maximal number of binding sites (Bmax) of α2‐adrenoceptors (48 and 37% respectively) without significantly affecting affinity; isoproterenol had no effect. Exposure to isoproterenol, noradrena line, and adrenaline significantly decreased the Bmax of β‐adrenoceptors (by 60, 34, and 24%, respectively) but did not affect the affinity. Isoproterenol and adrenaline significantly decreased the density of β1‐adrenoceptors by 75 and 24% and β2‐radrenoceptors by 23 and 28%, respectively. Noradrenaline significantly decreased the density of β1‐adreno‐ceptors by 42% without affecting the number of β2‐adreno‐ceptors. These findings indicate that subtypes of adrenoceptors in rat cerebral cortex are differentially regulated by adrenergic agonists.
Angiotensin III: A Potent Inhibitor of Enkephalin‐Degrading Enzymes and an Analgesic Agent
Authors: Shimamura Mariko , Kawamuki Kosei , Hazato Tadahiko
Published: October 2006

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin‐degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane‐bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N‐terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin‐converting enzyme, which degraded enkephalin, were 0.66 × 10‐6, 1.03 × 10‐6, 2.3 × 10‐4, and 1.65 × 10‐6M, respectively. Angiotensin III potentiated the analgesic activity of Met‐enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.
Cetiedil, a Drug That Inhibits Acetylcholine Release in Torpedo Electric Organ
Authors: Gaudry‐Talarmain Y. Morot , Israël M. , Lesbats B. , Morel N.
Published: October 2006

The effects of cetiedil, a vasodilatator substance with reported anticholinergic properties, were examined on cholinergic presynaptic functions at the nerve electroplaque junction of Torpedo marmorata using either synaptosomes or slices of intact tissue. Cetiedil abolished the calcium‐dependent release of acetylcholine (ACh) triggered by depolarization or by addition of A23187 ionophore, a finding localizing the site of action downstream from the calcium entry step. In addition, a direct effect on the release process itself was indicated by the observation that cetiedil blocks the release of ACh mediated by a recently isolated presynaptic membrane protein, the mediatophore, reconstituted into ACh‐containing proteoliposomes. In all three preparations, ACh release was inhibited by cetiedil with a Ki of 5–8 μM. Under the conditions used in these release experiments, the synthesis of ACh and its compartmentation within the nerve terminals were not modified. However, the drug was able to reduce high‐affinity choline uptake and vesicular ACh incorporation when it was given together with the radioactive precursor, a result showing that cetiedil has a broad inhibitory action on cholinergic uptake processes.
Cerebral Phosphoinositide and Energy Metabolism During and After Insulin‐Induced Hypoglycemia
Authors: Ikeda Masuhiro , Yoshida Shinichi , Busto Raul , Santiso Mercedes , Martinez Elena , Ginsberg Myron D.
Published: October 2006

Abstract: During and after insulin‐induced hypoglycemia, changes in levels of cerebral phosphatidylinositol (PI), phosphatidylinositol 4‐phosphate (PIP), phosphatidylinositol 4,5‐bisphosphate (PIP2), phosphatidic acid (PA), triacylglycerol (TAG), diacylglycerol (DAG), and free fatty acids (FFAs) as well as the cerebral energy state were studied in relation to the EEG. In hypoglycemic rats with an EEG pattern of quasiperiodic sharp or slow sharp waves, which preceded the development of an isoelectric EEG, PIP2 levels increased significantly, together with a slight decrease in PI content. Levels of the other lipids did not change during this period. The cerebral energy state was affected only slightly in spite of profound decreases in plasma and tissue glucose levels. With 30 min of an isoelectric EEG, levels of all phos‐phoinositides and PA decreased significantly; total FFA and DAG contents increased seven‐and twofold, respectively; the TAG‐palmitate level decreased, and that of TAG‐arachidonate increased. Plasma and tissue glucose were nearly depleted, and the cerebral energy state deteriorated severely. The increment in fatty acids in the DAG and FFA pools was less than their loss from phosphoinositides and PA, an observation suggesting vascular washout or oxidation of a portion of the FFAs produced. Following 90 min of glucose infusion, PIP and PA levels recovered to control values; however, the PIP2 content exceeded control levels, and that of PI remained below control levels. DAG and FFA contents returned to normal. The data suggest that PIP2 synthesis is increased or PIP2 degradation is blocked because of disturbed neurotransmission during the preisoelectric and recovery periods, at a time when the tissue energy state is minimally affected, and that all phosphoinositides are degraded during the period of isoelectric EEG, probably owing to energy shortage.
Sialosyllactotetraosylceramide, 3′‐isoLM1, a Ganglioside of the Lactotetraose Series Isolated from Normal Human Infant Brain
Authors: Molin Kent , Mansson Jan‐Eric , Fredman Pam , Svennerholm Lars
Published: October 2006

Abstract: A ganglioside antigen was detected in infant human brains by the monoclonal antibody C‐50. Structural analysis of the isolated ganglioside antigen showed it to be 3′‐isoLM1, Sialosyllactotetraosylceramide. The concentration of this ganglioside in human infant brain was 0.5 nmol/g.
Transport of Leucine‐Enkephalin Across the Blood‐Brain Barrier in the Perfused Guinea Pig Brain
Authors: Zloković Berislav V. , Lipovac Milo N. , Begley David J. , Davson Hugh , Rakić Ljubiša
Published: October 2006

Abstract: Transport of [tyrosyl‐3, 5−3H]enkephaIin‐(5‐L‐leucine) ([3H]Leu‐enkephalin) across the blood‐brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kim) estimated from the multiple‐time uptake data for [3H]Leu‐enkephalin ranged from 3.62 ± 10−3 to 3.63 ± 10−3 ml min−1 g−1in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu‐enkephalin was not inhibited by unlabelled L‐tyrosine (the N‐terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood‐brain barrier. By contrast, 2 mM unlabelled Leu‐enkephalin strongly inhibited the unidirectional blood‐to‐brain transport of [3H]Leu‐enkephalin by 74‐78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl‐glycyl‐glycyl‐phenylalanine (without the C‐terminal leucine of Leu‐enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl‐glycyl‐phenylalanyl‐leucine (without the N‐terminal tyrosine) at 5 mM was without effect on Leu‐enkephalin transport. Unidirectional brain uptake of Leu‐enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM(I mg/ml), suggesting that there is no binding of Leu‐enkephalin to opioid receptors at the blood‐brain barrier. It is concluded that there is a specific transport mechanism for Leu‐enkephalin at the blood‐brain barrier in the guinea pig.
Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A‐2 Selectivity
Authors: Williams Michael , Abreu Mary , Jarvis Michael F. , Noronha‐Blob Lalita
Published: October 2006

Abstract: Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6‐cyclohexyladeno‐sine ([3H]CHA), [3H]cyclopentyladenosine ([3H]CPA), and [3H]5′‐N‐ethylcarboxamido adenosine ([3H]NECA) to membranes prepared from PC 12 cells showed that the A‐1‐selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve‐fitting programs. However, [3H]NECA, a nonselective A‐l/A‐2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD= 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 μM), a finding suggesting involvement of an N‐protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC 12 cell membranes was stereo‐selective, with the R isomer of N6‐phenylisopropyladenosine (PIA) being ≥ 12 times more active than S‐PIA. The A‐1‐selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki= 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA ≥ 2‐chloroadenosine > CHA; > 5′‐./V‐methylcarbox‐amido adenosine ≥R‐PIA≥ CPA > S‐PIA. Binding was also displaced by the marine adenosine agonist 1‐methyliso‐guanosine and by a series of xanthine antagonists with the activity order being 1,3‐dipropyl–8‐(2‐amino‐4‐chloro)phe‐nylxanthine > 8‐phenyltheophylline > 8‐Psulfophenyl‐theophylline. These findings are consistent with the selective labeling by [3H]NECA of an A‐2 type adenosine receptor in the PC 12 pheochromocytoma cell line.
Distribution of the Two Forms of Monoamine Oxidase Within Monoaminergic Neurons of the Guinea Pig Brain
Authors: Ross Svante B.
Published: October 2006

Abstract: The relative distribution of type A and type B monoamine oxidase (MAO) inside and outside the monoaminergic synaptosomes in preparations from hypothalamus and striatum of the guinea pig was determined by incubation of synaptosomal preparations of these regions with low concentrations of [14C]5‐hydroxytryptamine (5‐HT), noradrenaline, and dopamine. The deamination within the monoaminergic synaptosomes was hindered by selective amine uptake inhibitors. In the absence of these inhibitors, both intra‐ and extraneuronal deamination was measured. The two forms of the enzyme were differentiated with the irreversible and selective MAO‐A and MAO‐B inhibitors clorgyline and selegiline (1‐deprenyl), respectively. [14C]5‐HT was deaminated >90% by MAO‐A both inside and outside the 5‐hydroxytryptaminergic synaptosomes prepared from the guinea pig hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes of the hypothalamic preparation was in the ratio 75:25% for MAO‐A:MAO‐B; the corresponding ratio outside these synaptosomes was 45:55%. The deamination of [14C]dopamine within dopaminergic synaptosomes in the striatal preparation was 65% type A:35% type B, whereas outside these synaptosomes the ratio was 35:65%. Because the relative amounts and the distribution of the two forms of MAO in the guinea pig brain seem to be similar to those previously detected for the human brain, the MAO in the guinea pig brain may be a good model for the MAO in the human brain.
γ‐Aminobutyric Acid Receptor Ionophore Complexes: Differential Effects of Deltamethrin, Dichlorodiphenyltrichloroethane, and Some Novel Insecticides in a Rat Brain Membrane Preparation
Authors: Lummis Sarah C. R. , Chow Shirley Chen , Holan George , Johnston Graham A. R.
Published: October 2006

Abstract: The binding of [35S]t‐butylbicyclophosphorothionate ([35S]TBPS), a γ‐aminobutyric acid (GABA)‐activated chloride ionophore ligand; [3H]diazepam, a benzodiazepine agonist; and [3H]muscimol, a GABA receptor probe, were used to assess the effects at 100 μM of deltamethrin, dichlorodiphenyltrichloroethane (DDT), and three experimental insecticides—a DDT‐pyrethroid hybrid, GH414 (cycloprothrin), and two DDT‐analogues, GH266 and GH149 (EDO), on GABA receptor ionophore complexes in a rat brain membrane preparation. GH266 and GH149 were found to inhibit a greater percentage of [35S]TBPS binding than the same concentration of deltamethrin or DDT, although GH414 had little effect. GH266 and GH149 enhanced [3H]diazepam binding by nearly 200%, in contrast to the inhibitory effects of deltamethrin, DDT, and GH414. GH266 and GH149 also caused a dramatic enhancement of [3H]muscimol binding, 367 and 236% of control, respectively, whereas DDT and deltamethrin caused only a moderate enhancement. The effects of the insecticides on binding affinity and density were examined for each of the ligands. The results show a differential interaction of the insecticides on the various ligand binding sites.
Tryptamine and Some Related Molecules Block the Accumulation of a Light‐Sensitive Pool of Cyclic AMP in the Dark‐Adapted, Dark‐Incubated Mouse Retina
Authors: Cohen Adolph I. , Blazynski Christine
Published: October 2006

Abstract: Dark‐adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3‐isobutyl‐methylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5‐methoxytryptamine, bufotenine, and 5‐methoxydimethyltryptamine can block the accumulation of the light‐sensitive pool of cAMP, whereas tryptophan, melatonin, N‐acetylserotonin, 5‐methoxytryptophol, and tetrahydro‐β‐carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light‐adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5‐HT‐2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.
Magnesium Ions Inhibit the Stimulation of Inositol Phospholipid Hydrolysis by Endogenous Excitatory Amino Acids in Primary Cultures of Cerebellar Granule Cells
Authors: Nicoletti F. , Wroblewski J. T. , Costa E.
Published: October 2006

Abstract: Omission of Mg2+ from the incubation buffer results in a six‐ to eightfold increase in [3H]inositol‐1‐phosphate ([3H]Ins‐1‐P) accumulation in primary cultures of cerebellar granule cells at 7–9 days in vitro. This increase is reversed by low concentrations of 2‐amino‐5‐phosphonovalerate (APV), a result indicating that the absence of Mg2+ facilitates the activation of a specific receptor by the endogenous excitatory amino acids (presumably l‐glutamate and l‐aspartate) released from the granule cells. The absence of Mg2+ also potentiates the action of exogenously applied N‐methyl‐d‐aspartate (NMDA), l‐glutamate, l‐aspartate, and kainate. In contrast, the action of quisqualate is virtually unaffected by Mg2+ and is resistant to APV inhibition. Addition of the depolarizing agent veratridine enhances the accumulation of [3H]Ins‐1‐P also in Mg2+‐containing buffer. The action of veratridine is antagonized by APV, a result suggesting that, under depolarized conditions, the NMDA receptor can be activated by the endogenously released excitatory amino acids, despite the presence of Mg2+. Accordingly, in the presence of Mg2+, veratridine potentiates the action of exogenously applied NMDA but does not facilitate the action of quisqualate.
Effect of Sorbinil on myo‐Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels
Authors: Yorek Mark A. , Dunlap Joyce A. , Ginsberg Barry H.
Published: October 2006

Abstract: Neuroblastoma cells were used to determine the effect of sorbinil on myo‐inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo‐inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo‐inositol uptake and in the incorporation of extracellular myo‐inositol into phospholipid. The effect of elevated glucose levels on myo‐inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo‐inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo‐inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.