bluuebunny/crossref_metadata_2025 huggingface dataset:
The Epithelial Phenotype of Human Neuroblastoma Cells Expresses Bradykinin, Endothelin, and Angiotensin II Receptors That Stimulate Phosphoinositide Hydrolysis
Authors: Ogino Yoshio , Costa Tommaso
Published: October 2006

Abstract: The neuroblastoma line SK‐N‐SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH‐SY5Y), an epithelial phenotype (SH‐EP), and an intermediate cell type (SH‐IN). In SH‐SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH‐EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of brady‐kinin > endothelin > angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH‐EP cells but not in SH‐SY5Y cells. In the intermediate cell clone, SH‐IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides—bradykinin, endothelin, and angiotensin II—on phosphoinositide hydrolysis in SH‐EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH‐EP cells with pertussis toxin under conditions sufficient to ADP‐ribosylate 90–95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short‐term exposure to the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (1 μM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH‐EP cells and partially inhibited that by muscarinic receptors in SH‐SY5Y cells. Prolonged incubation of SH‐EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH‐SY5Y cells. The pattern of phorbol ester‐mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH‐IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH‐EP than to muscarinic receptors in SH‐SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.
Activation and Multiple‐Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands
Authors: Haycock John W. , Wakade Arun R.
Published: October 2006

Abstract: Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 μM tetrahydrobiopterin. Nicotine (3 μM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 μM, 3 min) increased Ser40 phosphorylation, and muscarine (100 μM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.
Quantitative Immunoprecipitation Studies with Anti‐γ‐Aminobutyric AcidA Receptor γ2 1–15 Cys Antibodies
Authors: Duggan Michael J. , Pollard Simon , Stephenson F. Anne
Published: October 2006

Abstract: Antibodies raised against the synthetic peptide NH2‐QKSDDDYEDYASNKTC‐COOH (γ2 1–15 Cys), which corresponds to the N‐terminal amino acid sequence with a C‐terminal cysteine of the human γ2 subunit of the γ‐aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti‐γ2 1–15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam‐ and [3H]muscimol‐reversible binding sites in a dose‐dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti‐α1 324–341 antibodies carried out in parallel with anti‐γ2 1–15 Cys antibodies provided evidence for the promiscuity of the γ2 subunit within native GABAA receptors. These results substantiate the association of the γ2 polypeptide with native GABAA receptors.
Characterization of the Serpin, α1‐Antichymotrypsin, in Normal Human Cerebrospinal Fluid
Authors: Rayford Alan , Rao Jasti S. , Festoff Barry W.
Published: October 2006

Abstract: Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 ± 2.8 SEM) was analyzed for the presence of the serpin α1‐antichymotrypsin (α1‐ACT). A chymotrypsin‐specific chromogenic substrate (succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68‐kDa protein migrating identical to authentic human plasma α1‐ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma α1‐ACT. These studies showed the formation of complexes at 37°C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma α1‐ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human α1‐ACT under these same conditions. α1‐ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma. Recently, the β‐amyloid precursor protein itself has been identified as another serine protease inhibitor, protease nexin II, known to form complexes with bovine chymotrypsin as well as with the epidermal growth factor‐binding protein. Protease nexin II does circulate in plasma; however, complexes of this inhibitor with chymotrypsin are twice as large as the complexes this protease forms with plasma α1‐ACT or with the CSF samples. Using antisera to the β‐amyloid precursor protein, other workers have detected both 125‐kDa and 105‐kDa proteins in human CSF, the 125 kDa reportedly detected by antisera against the Kunitz serine protease inhibitor‐containing domain. The studies with α1‐ACT in CSF should now allow us to evaluate this serpin and its function in CSF of patients with Alzheimer's and other neurodegenerative diseases.
Developmental and Age‐Dependent Changes of 28‐kDa Calbindin‐D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method
Authors: Kurobe Naomi , Inaguma Yutaka , Shinohara Haruo , Semba Reiji , Inagaki Toshiaki , Kato Kanefusa
Published: October 2006

Abstract: For the quantitative analysis of vitamin D‐dependent 28‐kDa calcium‐binding protein (calbindin‐D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin‐D from rat kidneys, and the antibodies were purified with a calbindin‐D‐coupled Sepharose column. The purified antibodies were specific for calbindin‐D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich‐type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin‐D, which was sufficiently sensitive for the measurement of calbindin‐D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin‐D in a single Purkinje cell was 0.05 pg. Calbindin‐D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin‐D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4–5 weeks of postnatal age. In contrast, kidney calbindin‐D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin‐D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain. However, the concentrations in various regions of cerebral cortex from patients with Alzheimer's disease showed values similar to those in the relevant regions of the age‐matched control patients.
Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals
Authors: Wang James K. T. , Andrews Helene , Thukral Vijay
Published: October 2006

Abstract: The wide‐ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non‐NMDA agonists, in a concentration‐dependent manner, resulted in Ca2+‐dependent release of noradrenaline from vesicular transmitter stores. The NMDA‐stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non‐NMDA agonists was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non‐NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.
Expression of Functional Bradykinin Receptors in Xenopus Oocytes
Authors: Phillips Elsa , Conder Michael J. , Bevan Stuart , McIntyre Peter , Webb Michael
Published: October 2006

Abstract: mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108‐15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 μM), with an initial inward current (10–20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 μM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 μM). mRNA from both NG108–15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately fivefold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108‐15 cells.
Relationship of Intracellular Calcium to Dependence on Nerve Growth Factor in Dorsal Root Ganglion Neurons in Cell Culture
Authors: Eichler Marc E. , Dubinsky Janet M. , Rich Keith M.
Published: October 2006

Abstract: During development, neural crest‐derived sensory neurons require nerve growth factor (NGF) for survival, but lose this dependency postnatally. Similarly, dissociated embryonic sensory neurons lose their NGF dependence during the first 3 weeks in cell culture. It has been hypothesized that, in sympathetic neurons, intracellular levels of calcium are related to trophic factor dependence. In vitro during the period in which embryonic‐day‐15 sensory neurons become independent of NGF, intracellular calcium concentrations progressively increased in parallel to the decline in NGF dependence. This elevation of intracellular calcium was directly related to the absolute age of the neurons, not to the length of time in culture. Without NGF, immature sensory, i.e., dependent, neurons survived in the presence of high extracellular potassium, a condition that produces elevated intracellular calcium. In another paradigm, measurements of intracellular calcium were determined in NGF‐dependent neurons “committed to die'’ after NGF withdrawal. These measurements were determined prior to the time that extensive morphological changes, consistent with cell death, were noted by phase‐contrast microscopy. No elevation in intracellular calcium was found in these dying neurons, but rather, a small decrease was observed prior to the disintegration of the neurons. These findings support the hypothesis that trophic factor dependence of neurons may be inversely related to levels of intracellular calcium.
Transcription of the Rat Cholecystokinin Gene Is Initiated at Multiple Sites: Verification by an In Vitro Transcription System
Authors: Kuwano Ryozo , Araki Kazuaki , Usui Hiroshi , Suzuki Yoshiaki , Takahashi Yasuo
Published: October 2006

Abstract: The cDNA that accommodates the most distal 5′‐end region of cholecystokinin mRNA was isolated from an internally primed cDNA library. Using primer extension and S1 nuclease protection analyses, we demonstrated multiple RNA molecules generated from the rat cholecystokinin gene, a single‐copy sequence. The longest RNA is transcribed at position –225 upstream relative to the translation start site. The major transcription, more than 95% of the total cholecystokinin mRNA in rat brain, occurred at —59 and its promoter activity was determined by in vitro RNA synthesis in a HeLa cell extract. Deletion to –105 demonstrated an approximately 60% decrease in transcriptional level compared with the full promoter activity. At least the upstream region between –254 and –105 is necessary for transcription initiated at –59 of the cholecystokinin gene by the cell‐free system.
Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal‐Associated Intermediate Filament Protein Kinase Activity in Astrocytes
Authors: Harrison Beth C. , Mobley Philip L.
Published: October 2006

Abstract: These studies describe a cytoskeletal‐associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK‐C) and the cyclic AMP‐dependent protein kinase (PK‐A). The cytoskeletal‐associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mMMgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK‐C and PK‐A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium‐dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin‐dependent kinase activity acts mainly at serine residues. PK‐A and PK‐C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N‐and the C‐terminal domains of vimentin and the N‐terminal domain of GFAP. In contrast, both PK‐C and PK‐A are known to phosphorylate the N‐terminal domains of both proteins. The intermediate filament protein kinase activity appeared to be distinct from that of PK‐C and PK‐A and was able to phosphorylate unique sites of intermediate filament proteins. Also, it appeared that the intermediate filament protein kinase activity represents two kinases: a magnesium‐dependent protein kinase and a calcium/calmodulin‐dependent protein kinase.
Kinetic Analysis of the Chloride Dependence of the Neuronal Uptake of Dopamine and Effect of Anions on the Ability of Substrates to Compete with the Binding of the Dopamine Uptake Inhibitor GBR 12783
Authors: Amejdki‐Chab N. , Costentin J. , Bonnet J.‐J.
Published: October 2006

Abstract: The specific binding of [3H]l‐[2‐(diphenyl‐methoxy)ethyl]‐4‐(3‐phenyl‐2‐propenyl)piperazine([3H]GBR 12783) to the dopamine (DA) neuronal carrier present in membranes prepared from rat striatum was not affected when Cl– was replaced by either Br– or NO3–. In media containing Cl–, Br–, or NO3–, d‐amphetamine and DA competed with the radioligand in a monophasic manner with Hill coefficients of close to 1 (0.94–1.12). Replacement of CT by Br– impaired the ability of some substrates (d‐amphetamine, DA, p‐hydroxyamphetamine, and m‐tyramine) to compete with [3H]GBR 12783. The potency of Br– to decrease the affinity of substrates for the specific binding site was significantly correlated (t = 7.07, p < 0.001) with their affinity for this binding site. These results suggest that the various substrates tested could bind to recognition sites in which Cl– is differently involved; as a consequence, substrates could bind to the neuronal carrier by means of partly different links. In experiments dealing with the specific uptake of [3H]DA, F–, NO3–, is‐ethionate–, or acetate– was unable to substitute for Cl–, whereas Br– was quite a total substitute. Replacement of Cl– by equimolar concentrations of either NO3 or isethionate– resulted in inhibition curves of DA specific uptake with Hill coefficients of close to 1 (0.77 and 1.04, respectively); this indicates that both NO3– and isethionate” are devoid of inhibitory effects on neuronal uptake and are quite ineffective substitutes for Cl–. Kinetic analyses performed in different experimental conditions provide consistent results revealing that decreasing the CT concentration induced a raise in apparent Km of DA without modification of the Fmax. On the other hand, the apparent Km values of Cl– for the stimulation of the neuronal uptake of DA decreased when the DA concentration increased. These data are consistent with a co‐transport of Cl– and DA by the neuronal uptake system, with CT being the driving force.
Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene
Authors: Kauppinen Risto A. , Halmekyto Maria , Alhonen Leena , Jaime Juhani
Published: October 2006

Abstract: We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was >60 μmol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) Spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate a and ft peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high‐energy metabolites. 1H NMR showed no differences in brain choline/N‐acetylaspartate and total creatine/N‐acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.
In Vivo Neurochemical Evaluation of Striatal Serotonergic Hyperinnervation in Rats Depleted of Dopamine at Infancy
Authors: Jackson Denise , Abercrombie Elizabeth D.
Published: October 2006

Abstract: Destruction of nigrostriatal dopamine (DA) neurons with 6‐hydroxydopamine (6‐OHDA) early in development results in hyperinnervation of striatum by the serotonergic afferents deriving from the dorsal raphe nucleus. We have used in vivo microdialysis to investigate the degree to which serotonergic neurotransmission in striatum is altered by this increase in the density of serotonin (5‐HT) terminals. The effects of several manipulations known to influence 5‐HT function on extracellular 5‐HT and 5‐hydroxyindoleacetic acid in striatum were compared in adult rats treated neonatally with 6‐OHDA and in intact adult rats. Basal levels of 5‐HT in extracellular fluid (ECF) of striatum were similar in neonatally DA‐depleted rats and in intact rats. Perfusion with the 5‐HT reuptake blocker, fluoxetine (100 μM), increased 5‐HT in striatal ECF of neonatally DA‐depleted rats to levels that were threefold greater than those achieved in intact rats. Likewise, K+‐depolarization of the 5‐HT terminals (100 mM in perfusate) or systemic administration of the 5‐HT releaser, (±)‐fenfluramine (10 mg/kg i.p.), increased the concentration of 5‐HT in striatal ECF of neonatally DA‐depleted rats to levels approximately threefold greater than those observed in striatum of intact rats. These findings indicate that the 5‐HT hyperinnervation of striatum that takes place in rats depleted of DA at infancy is associated with an increased capacity for neurotransmitter release in this system. Concomitant increases in high‐affinity 5‐HT uptake may prevent the occurrence of any measurable changes in the resting concentration of 5‐HT in striatal ECF.
Dissimilar Aluminum and Gallium Permeation of the Blood‐Brain Barrier Demonstrated by In Vivo Microdialysis
Authors: Allen David D. , Yokel Robert A.
Published: October 2006

Abstract: Aluminum (Al) and gallium (Ga) permeations of the blood‐brain barrier (BBB) were assessed in rats. Unbound extracellular Al and Ga concentrations were ascertained at the two potential sites of BBB permeation, cerebral capillaries and choroid plexuses, by implantation of microdialysis probes in the frontal cortex and lateral ventricle, respectively. A microdialysis probe implanted in the jugular vein revealed unbound blood Al or Ga concentrations. Al or 67Ga citrate was administered via the femoral vein. Peak Al and Ga concentrations were seen within the first 10 min at all three sites. Area under the curve (concentration vs. time to final sample) values were calculated using RSTRIP. Within‐rat overall frontal cortical/blood and lateral ventricular/blood ratios [brain/blood ratios (oBBRs)] were calculated from area under the curve values. Aluminum frontal cortical oBBRs were significantly higher than those for the lateral ventricle. Ga oBBRs were not significantly different between the two sites. Al and Ga oBBRs were significantly different in the lateral ventricle. These results suggest that the primary site of Al permeation across the BBB is at cerebral capillaries, whereas Ga permeation across the BBB does not significantly differ between cerebral capillaries and choroid plexuses. The use of Ga as a model to study Al pharmacokinetics may not be appropriate in the elucidation of the site or mechanism of Al entry into the brain.
Quantitative Examination of Tissue Concentration Profiles Associated with Microdialysis
Authors: Dykstra Kevin H. , Hsiao John K. , Morrison Paul F. , Bungay Peter M. , MefFord Ivan N. , Scully Melinda M. , Dedrick Robert L.
Published: October 2006

Abstract: Spatial solute concentration profiles resulting from in vivo microdialysis were measured in rat caudate‐putamen by quantitative autoradiography. Radiolabeled sucrose was included in the dialysate, and the tissue concentration profile measured after infusions of 14 min and 61.5 min in an acute preparation. In addition, the changes in sucrose extraction fraction over time were followed in vivo and in a simple in vitro system consisting of 0.5% agarose. These experimental results were then compared with mathematical simulations of microdialysis in vitro and in vivo. Simulations of in vitro microdialysis agreed well with experimental results. In vivo, the autoradiograms of the tissue concentration profiles showed clear evidence of substantial differences between 14 and 61.5 min, even though the change in extraction fraction was relatively small over that period. Comparison with simulated results showed that the model substantially underpre‐dicted the observed extraction fraction and overall amount of sucrose in the tissue. A sensitivity analysis of the various model parameters suggested a tissue extracellular volume fraction of approximately 40% following probe implantation. We conclude that the injury from probe insertion initially causes disruption of the blood‐brain barrier in the vicinity of the probe, and this disruption leads to an influx of water and plasma constituents, causing a vasogenic edema.
Biochemical and Pharmacological Characterization of Serotonin‐O‐Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5‐HT1B and 5‐HT1D Binding Sites
Authors: Boulenguez Pascale , Segu Louis , Chauveau Jacques , Morel Anne , Lanoir Jeanne , Delaage Michel
Published: October 2006

Abstract: There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin‐1B (5‐HT1B) and serotonin‐ID (5‐HT1D) binding sites. Serotonin‐0‐carboxymethylglycyltyrosinamide (S‐CM‐GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S‐CM‐G[125I]TNH2‐binding sites were studied by quantitative au‐toradiography on rat and guinea pig brain sections. S‐CM‐G[I25I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5‐HT1B sites in the rat and of 5‐HTD sites in the guinea pig. The binding of 0.02 nM S‐CM‐G[125I]TNH2 was inhibited by low concentrations of 5‐HT, S‐CM‐GTNH2, CGS 12066 B, 5‐methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin inhibited S‐CM‐G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5‐HT,c, 5‐HT2, 5‐HT3, and adrenergic receptors, inhibited S‐CM‐G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S‐CM‐G[I25I]TNH2 is then a useful new probe for the direct study of 5‐HT1B and 5‐HT1D binding sites.
Characterization of Neuronal Protein Phosphatases in Aplysia californica
Authors: Endo Shogo , Shenolikar Shirish , Eskin Arnold , Zwartjes Ray E. , Byrne John H.
Published: October 2006

Abstract: Biochemical properties of neuronal protein phos‐phatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase a by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type‐1 protein phosphatase (PrP‐1) was identified in these extracts by the dephosphorylation of the β‐subunit of phosphorylase kinase and its inhibition by the protein, inhibitor‐2. Type‐2A protein phosphatase (PrP‐2A) was demonstrated by the dephosphorylation of the α‐subunit of phosphorylase kinase, which was insensitive to inhibitor‐2. As in vertebrate tissues, only four enzymes, PrP‐1 (47%), PrP‐2A (42%), PrP‐2B (11%), and PrP‐2C (<1%), accounted for all the cellular protein phosphatase activity dephosphor‐ylating phosphorylase kinase. Aplysia PrP‐1 and PrP‐2A were potently inhibited by okadaic acid, with PrP‐1 being approximately 20‐fold more sensitive than PrP‐2A. By comparison, purified PrP‐2A from rabbit skeletal muscle was 15‐ to 20‐fold more sensitive to okadaic acid than PrP‐1 from the same source. Only PrP‐1 was associated with the particulate fractions from Aplysia neurons, whereas PrP‐1 and PrP‐2A, ‐2B, and ‐2C were all present in the cytosol. Extraction of the particulate PrP‐1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factors) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.
Temporal Expression of HNK‐1‐Reactive Sulfoglucuronyl Glycolipid in Cultured Quail Trunk Neural Crest Cells: Comparison with Other Developmentally Regulated Glycolipids
Authors: Jungalwala Firoze B. , Chou Denise K. H. , Suzuki Yasuo , Maxwell Gerald D.
Published: October 2006

Abstract: Monoclonal antibody HNK‐1 is an important marker for embryonic neural crest cells and some of their differentiated derivatives. We have identified 3‐sulfoglucuro‐nylneolactotetraosylceramide (SGGL‐1) as one of the HNK‐1 antigens present in cultures of trunk neural crest cells. This lipid was present at 2 days in vitro and increased in amount with time in culture. Other major HNK‐1‐reactive antigens present in the culture were glycoproteins of apparent molecular masses of 120, 180, and 200 kDa. The 180‐ and 200‐kDa bands were present at 2, 7, and 17 days in vitro, whereas the 120‐kDa band was present only at 17 days in vitro. Gan‐gliosides GD3, LD1, and LM1 were also found in the cultures and exhibited distinct temporal patterns of expression. Gan‐glioside GD3 was present at all stages examined and its expression peaked at 7 days in vitro. In contrast, LD1 was present only at 2 days in vitro and was not detectable at later times. Ganglioside LM1 increased in amount with time in culture in a pattern similar to that seen for SGGL‐1. Taken together, these results indicate that several HNK‐1‐reactive molecules are expressed in neural crest cultures in a temporally regulated manner along with several glycolipids that do not bear this epitope.
1‐Methyl‐4‐(2′‐Ethylphenyl)‐1,2,3,6‐Tetrahydropyridine‐ Induced Toxicity in PC12 Cells Is Enhanced by Preventing Glycolysis
Authors: Basma Alie N. , Heikkila Richard E. , Saporito Michael S. , Philbert Martin , Geller Herbert M. , Nicklas William J.
Published: October 2006

Abstract: The effects of l‐methyl‐4‐(2′‐ethylphenyl)‐1,2,3,6‐tetrahydropyridine (2′Et‐MPTP), l‐methyl‐4‐(2′‐ethyl‐phenyl)pyridinium (2′Et‐MPP+), and the classic complex 1 inhibitor, rotenone, on toxicity as well as on rates of glucose use and lactate production were studied using the pheochro‐mocytoma PC12 cell line. PC12 cells are neoplastic in nature and have a high rate of glycolysis accompanied by a large production of lactate and a low use of glucose carbon through the Krebs cycle. l‐Methyl‐4‐phenylpyridinium (MPP+) and analogues such as 2′Et‐MPP+ are actively accumulated by mitochondrial preparations in vitro and block NADH de‐hydrogenase of complex 1. This blockade results in biochemical sequelae that are ultimately cytotoxic. In this study, untreated PC12 cells used glucose and concomitantly accumulated lactate in a time‐dependent manner at all concentrations of glucose studied. Treatment with 50 pM 2′Et‐MPP+ or 50 nAf rotenone increased both rates significantly, indicating a shift toward increased glycolysis. Cell death caused by the neurotoxins was also time and concentration dependent and markedly enhanced by glucose depletion in the medium. The increase in 2′Et‐MPTP‐induced toxicity in low glucose‐supplemented cells was not due to an increase in pyridinium formation from the tetrahydro‐pyridine, but rather to the lack of glucose for glycolysis. Moreover, inhibition of glycolysis with 2‐deoxyglucose or io‐doacetic acid also enhanced the lethality of the neurotoxins to the cells. The data in this study provide additional support to the hypothesis that 2′Et‐MPP+ or related analogues act to kill cells by inhibiting mitochondrial respiration. Furthermore, these findings support the hypothesis that the ability of 2′Et‐MPTP or its analogues to induce toxicity is dependent on the relative contributions of glycolysis and mitochondrial oxidation to the energy needs of a particular cell or model system.
Tumor Necrosis Factor Modulates the Inactivation of Catecholamine Secretion in Cultured Sympathetic Neurons
Authors: Soliven Betty , Albert Julyan
Published: October 2006

Abstract: Cytokines exert multiple effects on cellular functions. We studied the effects of cytokines on the calcium‐dependent release of catecholamines in cultured neurons from neonatal rat superior cervical ganglia. Incubation of sympathetic neurons with recombinant human interleukin‐β (0.14‐0.7 nM) or recombinant human tumor necrosis factor‐α (1 nM) for 24‐48 h had no effect on the baseline spontaneous release and the initial K+‐evoked [3H]norepinephrine release, compared with untreated cells. A repeat K+‐induced depolarization after 6 min resulted in a decrease of [3H]norepinephrine secretion to 69 ± 5.8% (n = 11) of the initial secretion in recombinant human tumor necrosis factor treated cells, but not in control cells. The secretory response was restored when the interval between the two K+ challenges was increased to 10 min. We conclude that the diminished secretory response to a repeat stimulus in recombinant human tumor necrosis factor‐treated superior cervical ganglia neurons is due to a prolonged recovery from inactivation of secretion in these cells.
Involvement of Dopamine D1 and D2 Receptors in the Regulation of Proenkephalin mRNA Abundance in the Striatum and Accumbens of the Rat Brain
Authors: Angulo Jesus A.
Published: October 2006

Abstract: The effects of short‐term treatment (6 h) with selective D1 or D2 agonists and antagonists on the mRNA for proenkephalin in the medial and anterior aspects of the cau‐date‐putamen and the nucleus accumbens were assessed by in situ hybridization histochemistry. Proenkephalin mRNA abundance was significantly changed in the striatum and accumbens in response to D2 receptor manipulation. D2 blockade with haloperidol or raclopride increased, whereas D2 stimulation with LY‐171555 (D2 agonist) decreased, striatal and accumbens proenkephalin mRNA abundance. Antagonism of D1, receptor activity with SCH‐23390 significantly decreased proenkephalin mRNA abundance in all brain regions. Concurrent administration of the D1, agonist SKF‐38393 prevented the SCH‐23390 effect in all brain areas. The data demonstrate that acute treatment with dopaminergic D2 agonists and antagonists affects proenkephalin mRNA abundance in the striatum and accumbens via a D2 receptor mechanism, consistent with the concept that D2 receptor function inhibits the synthesis of the mRNA encoding the enkephalin peptides. Moreover, Df receptor activity, directly or indirectly, exerts modulatory effects on proenkephalin mRNA abundance in the striatum and nucleus accumbens.
Biochemical Characterization and Localization of a Non‐N‐Methyl‐D‐Aspartate Glutamate Receptor in Rat Brain
Authors: Blackstone Craig D. , Moss Stephen J. , Martin Lee J. , Levey Allan I. , Price Donald L. , Huganir Richard L.
Published: October 2006

Abstract: The structure and distribution of non‐N‐methyl‐D‐aspartate glutamate receptors in the rat brain were studied using subunit‐specific antibodies that recognize the receptor subunit GluRl. The GluRl protein, a 106‐kDa glycoprotein, appears predominantly in synaptic plasma membranes, where it is highly enriched in the postsynaptic densities. When synaptic plasma membranes are solubilized with the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐l‐propanesul‐fonate, high‐affinity a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) binding and GluRl immunoreactivity comigrate at a native Mr of 610,000. GluRl is enriched in the hippocampus and cerebellar cortex but is present throughout the CNS. It is found on neuronal cell bodies and processes within most regions of the brain; within the cerebellum, however, it is localized to the Bergmann glia. These data suggest that the GluRl protein is a subunit of multimeric AMPA‐preferring glutamate receptors present on neurons and on specialized glia.
Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn‐1 Diacylglycerol Lipase and a Monoacylglycerol Lipase
Authors: Allen Ann C. , Gammon Charles M. , Ousley Andrea H. , McCarthy Ken D. , Morell Pierre
Published: October 2006

Abstract: In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid ([3HJAA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacyl‐glycerols in unstimulated cultures revealed the presence of two primary [3H]AA‐containing species, l‐palmitoyl‐2‐ar‐achidonoyl and l‐stearoyl‐2‐arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of l‐stearoyl‐2‐arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that A A release occurred through the successive actions of an sn‐1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1‐stea‐royl‐2‐arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK‐stimulated AA release in DRG cultures. Under our assay conditions there were no BK‐stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with (3H]inositol, [3H]choline, or [3H]‐ethanolamine, respectively.
Increased Superoxide Dismutase Activity in Aged Human Cerebrospinal Fluid and Rat Brain Determined by Electron Spin Resonance Spectrometry Using the Spin Trap Method
Authors: Hiramatsu Midori , Kohno Masahiro , Edamatsu Rei , Mitsuta Keiichi , Mori Akitane
Published: October 2006

Abstract: Superoxide dismutase (SOD) activity in CSF of patients was determined by electron spin resonance spec‐trometry using the spin trap method. Variation in SOD activity was found among patients. SOD activity in CSF of subjects increased with age and this was identified as Cu,Zn‐SOD activity by electrophoresis. In addition, animal experiments showed that SOD activities were higher in mitochon‐drial and cytosol fractions of aged rats than in those of adult rats. This finding on aged rat brain validates the increase of SOD activity in aged human CSF.
Dynamic Observation of Dopamine Autoreceptor Effects in Rat Striatal Slices
Authors: Kennedy Robert T. , Jones Sara R. , Wightman R. Mark
Published: October 2006

Abstract: Fast‐scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one‐, two‐, and 50‐pulse, 10‐Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 μM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 μM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 μM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 μM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one‐pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half‐time of ∼ 17 s was found. The addition of picrotoxin (100 μM) and glutamate (10 μM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 μM) attenuated overflow for all the trains tested.
Binding Properties of 3‐[125I]Iodophencyclidine, a New Radioligand for N‐Methyl‐D‐Aspartate‐Gated Ionic Channels
Authors: Chicheportiche Robert , Guiramand Janique , Kamenka Jean Marc , Ponchant Michel , Beaucourt Jean Pierre
Published: October 2006

Abstract: The binding properties of the 125l‐labeled phencyclidine derivative N‐[1‐(3‐[125I] iodophenyl)cyclohexyl] piperidine (3‐[125I]iodo‐PCP), a new ligand of the N‐methyl‐D‐aspartate (NMDA)‐gated ionic channel, were investigated. Association and dissociation kinetic curves of 3‐[125I]iodo‐PCP with rat brain homogenates were well described by two components. About 32% of the binding was of fast association and fast dissociation, and the remaining binding was of slow association and slow dissociation. Saturation curves of 3‐[125I] iodo‐PCP also were well described using two binding sites: one of a high affinity (KDH= 15.8 ± 2.3 nM) and the other of a low affinity (KDL= 250 ± 40 nM). 3‐Iodo‐PCP inhibited the binding of 3‐[125I]iodo‐PCP with inhibition curves that were well fitted by a two‐site model. The binding constants (KiH, BmaxH; KiL, BmaxL) so obtained were close to those obtained in saturation experiments. Ligands of NMDA‐gated ionic channels also inhibited the binding of 3‐ [125I]iodo‐PCP with two constants, KiH and KiL. There was a very good correlation (r = 0.987) between the affinities of these ligands to bind to NMDA‐gated ionic channels and their potencies to inhibit the binding of 3‐[125I]iodo‐PCP with a high affinity. Moreover, the regional distribution of the high‐affinity binding of 3‐[125I]‐iodo‐PCP paralleled that of tritiated N‐[1‐(2‐thienyl)cyclohexyl]piperidine ([3H]TCP). In contrast to that of [3H]TCP, the binding of 3‐[125I]iodo‐PCP to well‐washed rat brain membranes was fast and insensitive to glutamate and glycine. We conclude that 3‐[125I]iodo‐PCP, at low concentrations, is suited for future rapid autoradio‐graphical studies of both open and closed forms of NMDA‐gated ionic channels and that 3‐[123I]iodo‐PCP could be used successfully for in vivo studies by single‐photon emission computed tomography analysis.
Mono‐N‐Methylation of 1,2,3,4‐Tetrahydro‐β‐Carbolines in Brain Cytosol: Absence of Indole Methylation
Authors: Matsubara Kazuo , Collins Michael A. , Neafsey Edward J.
Published: October 2006

Abstract: In an accompanying report we demonstrated enzyme activity in guinea pig brain cell nuclei that catalyzes S‐adenosylmethionine (SAM)‐dependent N‐methylations of heteroaromatic β‐carbolines (BCs) on the 2[β]‐nitrogen and subsequently on the 9[indole]‐nitrogen, ultimately yielding N2,N9‐dimethylated BCs. Presented here are the esults of a parallel study of the N‐methylation of 1,2,3,4‐tetrahydro‐BCs (THBCs), which form endogenously via condensations of tryptophan and its derived indoles with carbonyl compounds or, like their BC oxidation products, are environmental constituents and plant alkaloids. THBCs were enzymatically methylated on the 2[β]‐nitrogen by [3H]‐SAM in undialyzed homogenates of rat or guinea pig brain, but [3H]methyl transfer to the 9[indole]‐nitrogen was not observed. The structure of the 2[β]‐methyl THBC product was verified with capillary gas chromatography‐mass spectrometry. Furthermore, whereas BC N‐methylation was largely particulate and displayed micromolar Km values for BC substrate, THBC 2[β]‐N‐methylation activity was cytosolic and displayed a relatively high (millimolar) Km for THBC substrate. The N‐methylation of THBCs may be due to cytosolic N‐methyltransferases that others have studied using different azaheterocyclics. Our overall studies indicate that N2,N9‐dimethylated BCs could be unique neurotoxic factors that are bioactivated within brain by sequential N‐methylations of BCs. These results suggest the possibility of an additional route to the putative 2,9‐dimethylated toxins involving, as a first step, 2[β]‐N‐methylation of environmental or endogenously derived THBCs in the brain and perhaps other organs.