bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary
Authors: Memo M. , Castelletti L. , Valeric A. , Missale C. , Spano P. F.
Published: December 2006

Abstract: Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 ± 11 fmol/mg protein and a KD value of 1.4 ± 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L‐Phe‐NT, and 3,000 nM for the pharmacologically inactive form D‐Phe‐NT. NT, up to a concentration of 1 μM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time‐ and dose‐dependent. It appeared to be associated with neurotransmitter‐operated calcium channels since: (1) preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; (2) concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and (3) NT lost its capacity to release PRL in the absence of extracellular calcium. The significant correlation of the effective concentrations of NT and its analogs for binding and biological activities indicates that occupancy of NT receptors in anterior pituitary modifies calcium channel permeability of the cell membranes, resulting in an accumulation of intracellular calcium that participates in the cascade of events that are ultimately expressed as an enhanced release of PRL.
α‐Bungarotoxin Binds to Low‐Affinity Nicotine Binding Sites in Rat Brain
Authors: Wonnacott Susan
Published: December 2006

Abstract: Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]α‐bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an α‐bungar‐otoxin binding protein from rat brain failed to copurify the high‐affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatogra‐phy step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]α‐bungaro‐toxin to P2 membranes was inhibited by (‐)‐nicotine (Ai= 9 × 10‐6 M), and this sensitivity was preserved after affinity purification. It is proposed that α‐bungarotoxin binds to a population of low‐affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80‐fold preference for the (‐) form at the high‐affinity [3H]nicotine binding site, whereas the site labelled by [125I]α‐bungarotoxin displayed little stereoselectivity. In this respect, the brain α‐bungarotoxin binding site resembles the nicotinic acetyl‐choline receptor from Torpedo electric organ.
Evidence for Different States of the Dopamine Dl Receptor: Clozapine and Fluperlapine May Preferentially Label an Adenylate Cyclase‐Coupled State of the Dl Receptor
Authors: Andersen Peter H. , Braestrup Claus
Published: December 2006

Abstract: It has been shown previously that typical neuroleptics have higher affinities for 3,4‐dihydroxyphenyl‐ethylamine (dopamine) Dl receptors as labeled by(R)‐ (+)‐ 8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1 ‐N‐3‐benzazepine‐7‐ol ([3H]SCH 23390) than for inhibiting dopamine‐stimulated adenylate cyclase. We now report that the atypical neuroleptics, clozapine and fluperlapine, exhibit characteristics opposite to typical neuroleptics, i.e., they have higher affinity for inhibiting dopamine‐stimulated adenylate cyclase than [3H]SCH 23390 binding. A variety of compounds, i.e., clozapine, fluperlapine, and dopamine, were tested for their capacity to affect the rate constants of [3H]SCH 23390 binding; these experiments revealed no effect of any tested compound on on‐rate or off‐rate of [3H]SCH 23390 binding. Treatment of striatal membranes with phospholipase A2 (PLA2) caused a rapid decrease in the Bmax value of the [3H]SCH 23390 binding with no effect on the Kd value. The adenylate cyclase, both the unstimulated, the dopamine‐, fluoride‐, and forskolin‐stimulated activity, was far less sensitive than [3H]SCH 23390 binding to PLA2. Treatment of striatal membranes with filipine and (NH4SO4 produced, as did PLA2 treatment, a rapid decline in [3H]SCH 23390 binding. However, opposite to PLA2 treatment, these agents stimulated the adenylate cyclase. In conclusion, a comparison of the pharmacological characteristics of [3H]SCH 23390 binding and dopamine‐stimulated adenylate cyclase suggests the existence of two different Dl binding sites. The rate experiments exclude the possibility of allosterically coupled sites. Instead our results favor that the Dl receptor exists in different states/conformations, i.e., both adenylate cyclase‐coupled and uncoupled, and further, that the atypical neuroleptics clozapine and fluperlapine may have adenylate cyclase‐coupled dopamine Dl receptors as target.
Comparison of a 52‐kDa Phosphoprotein from Synaptic Plasma Membranes Related to Long‐Term Potentiation and the Major Coated Vesicle Phosphoprotein
Authors: Schrama Loes H. , de Graan Pierre N. E. , Zwiers Henk , Gispen Willem Hendrik
Published: December 2006

Abstract: In the in vitro hippocampal slice preparation a short tetanus induces long‐term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52‐kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52‐kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52‐kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: (1) relative molecular weight on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (2) peptide mapping, (3) phospho‐amino acid content, and (4) isoelectric point. Since coated vesicles are thought to be involved in receptor‐mediated endocytosis and membrane recycling, our data suggest that LTP‐correlated changes in 52‐kDa phosphorylation may reflect increased coated vesicle activity.
Elevation of Striatal Dopamine Receptors by Estrogen: Dose and Time Studies
Authors: Hruska Robert E.
Published: December 2006

Abstract: Administration to male rats of a single dose of 17β‐estradiol valerate (8‐500 μg/rat) or implantation of a pellet containing 17β‐estradiol (0.5‐50 mg/rat) increased serum 17β‐estradiol levels in a dose‐dependent relationship when measured on the sixth day after adminstration. At the same time, after these doses, the serum rat prolactin (rPRL) levels were doubled and the striatal 3,4‐dihydroxyphenyl‐ethylamine (DA, dopamine) receptor densities were increased 20%. A single dose of 17β‐estradiol valerate of 4 μg/ rat or less did not alter serum 17β‐estradiol or rPRL levels or the striatal DA receptor density. After the single injection of 17β‐estradiol valerate (125 μg/rat) the serum 17β‐estra‐diol levels peaked at 1 day, the serum rPRL levels peaked at 2 days, and the striatal DA receptor density elevation peaked from 4 to 8 days. Implantation of a pellet containing 17β‐estradiol (25 mg/rat) produced a constant elevation of serum 17β‐estradiol levels from 1 to 10 days. Whereas the serum rPRL levels were continuously elevated about twofold, the densities of the striatal DA receptors were increased significantly by 20‐25% only from 4 to 8 days after pellet implantation. These results indicate that striatal DA receptor density rises and returns to control levels during the constant elevation of serum 17β‐estradiol and rPRL levels. These results, in comparison with those from the literature, suggest that in male rats the elevation of rPRL levels leads to the increase in striatal DA receptor density and that a substantial sex difference exists in rats in the regulation of striatal DA receptor density after 17β‐estradiol administration.
γ‐Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site
Authors: Robinson Timothy , MacAllan David , Lunt George , Battersby Martin
Published: December 2006

Abstract: The specific binding of [N‐methyl‐3H] flunita‐zepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+‐dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzo‐diazepine receptors in mammals. Thus binding is enhanced by γ‐aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5‐4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photo‐affnity‐labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.
Cellular Origins of Cyclic GMP Responses to Excitatory Amino Acid Receptor Agonists in Rat Cerebellum In Vitro
Authors: Garthwaite J. , Garthwaite G.
Published: December 2006

Abstract: Incubated slices and freshly dissociated cells from 8‐day‐old rat cerebellum were used to try to identify the cells that participate in the large increases in cyclic GMP levels that follow activation of excitatory amino acid receptors in this tissue. In the slices, cyclic GMP responses to L‐gluta‐mate and related excitants were unaffected by tetrodotoxin and could be replicated by the guanylate cyclase activator nitroprusside. Nitroprusside and the receptor agonists appeared to activate the same pool of the enzyme. Prior destruction of neuroblasts, deep nuclei, or Golgi neurones did not cause loss of responses to L‐glutamate. If granule cells were rendered necrotic, however, the cyclic GMP responses to all excitants tested were reduced by ≧ 90%. Substantial losses of responses to veratridine and high K+ levels also occurred, but the nitroprusside‐induced elevations were unaffected. In dissociated cell suspensions, the magnitude of responses to receptor agonists, but not those to nitroprusside, was markedly dependent on cell concentration. Responses to L‐glutamate were the same in cell suspensions that were Purkinje cell depleted and Purkinje cell enriched. It is concluded that granule cells are primarily involved in the cyclic GMP responses to excitatory amino acids but that the cyclic GMP accumulations occur elsewhere, probably in glial cells.
Low‐Level Microwave Irradiations Affect Central Cholinergic Activity in the Rat
Authors: Lai Henry , Horita Akira , Chou Chung‐Kwong , Guy Arthur W.
Published: December 2006

Abstract: Sodium‐dependent high‐affinity choline uptake was measured in various regions of the brains of rats irradiated for 45 min with either pulsed or continuous‐wave low‐level microwaves (2, 450 MHz; power density, 1 mW/cm2; average whole‐body specific absorption rate, 0.6 W/kg). Pulsed microwave irradiation (2‐μs pulses, 500 pulses/s) decreased choline uptake in the hippocampus and frontal cortex but had no significant effect on the hypothalamus, stria‐turn, and inferior colliculus. Pretreatment with a narcotic antagonist (naloxone or naltrexone; 1 mg/kg i.p.) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not significantly alter the effect on the frontal cortex. Irradiation with continuous‐wave microwaves did not significantly affect choline uptake in the hippocampus, striatum, and hypothalamus but decreased the uptake in the frontal cortex. The effect on the frontal cortex was not altered by pretreatment with narcotic antagonist. These data suggest that exposure to low‐level pulsed or continuous‐wave microwaves leads to changes in cholinergic functions in the brain.
myo‐Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels
Authors: Yorek Mark A. , Dunlap Joyce A. , Ginsberg Barry H.
Published: December 2006

Abstract: Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo‐inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V1max for inositol uptake was significantly decreased with little change in the K1max. These data suggest that glucose decreases myo‐inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo‐inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo‐inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.
Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate
Authors: Porter Terence G. , Martin David L.
Published: December 2006

Abstract: In the absence of its cofactor, pyridoxal 5′‐phosphate (pyridoxal‐P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first‐order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the β‐form of the enzyme, the concentration of aspartate giving the half‐maximal rate of inactivation is 6.1 ± 1.3 mM and the maximal apparent rate constant is 1.02 ± 0.09 min−1, which corresponds to a half‐time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or γ‐aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal‐P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5′‐phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal‐P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal‐P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.
Effects of Hyperprolactinemia on Plasma Prolactin and Glucose and on Local Cerebral Glucose Utilization
Authors: Selmanoff Michael , Walovitch Richard C. , Walker Gregory E. , London Edythe D.
Published: December 2006

Abstract: Elevated blood levels of prolactin increase the synthesis, turnover, and release of 3, 4‐dihydroxyphenylethylamine (dopamine) from the tuberoinfundibular dopaminergic neurons, which project to the median eminence. The present study examined whether hyperprolactinemia also increases local cerebral glucose utilization, as determined by the 2‐deoxy‐D‐[I‐14C]glucose method, in the median eminence and other brain structures. Adult male rats were given ovine prolactin (4 mg/kg) subcutaneously every 8 h for 48 h. This treatment exerted an autoregulatory feedback effect on endogenous rat prolactin secretion, as evidenced by decreased circulating levels of rat prolactin. Ovine prolactin treatment also decreased plasma glucose concentrations. However, in both partially immobilized and free‐ranging rats, glucose utilization in brain structures containing tuberoinfundibular dopaminergic cell bodies (the arcuate nucleus) and terminals (the median eminence) was not affected by ovine prolactin treatment. Hyperprolactinemia was, however, associated with decreased glucose utilization in the medial forebrain bundle and the CA subfield of the dorsal hippocampus. The lack of a significant effect of prolactin treatment on glucose utilization in the median eminence indicates (a) that the resolution of the deoxyglucose technique, as used here, is not adequate to detect the ovine prolactin‐induced increase in tuberoinfundibular dopaminergic neuronal activity, (b) that the median eminence does not utilize glucose as its primary energy substrate, or (c) that ovine prolactin treatment causes a counterbalancing decrease in the activity of other neurons projecting to the median eminence.
Translocation of Protein Kinase C in Anterior Pituitary Tumor Cells
Authors: Zatz Martin , Mahan Lawrence C. , Reisine Terry
Published: December 2006

Abstract: Previous studies have shown that phorbol esters and lithium each stimulate the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary tumor cell line AtT20/D16‐16. Pretreatment with either lithium or phorbol ester desensitizes the cells to subsequent stimulation by phorbol ester. An early consequence of phorbol ester action in other systems is the translocation of protein kinase C from cytosol to membranes. We have assayed protein kinase C activity in cytosol and membranes of AtT20 cells after treatment with phorbol dibutyrate, lithium, or other agents that stimulate secretion of ACTH in these cells. Phorbol dibutyrate clearly induced translocation of protein kinase C, but lithium treatment did not cause translocation itself, nor did pretreatment with lithium affect the translocation induced by phorbol dibutyrate. These results are consistent with a role for translocation of protein kinase C in the stimulatory and desensitizing effects of phorbol esters but fail to implicate translocation in the actions of lithium on AtT20 cells.
Abundance in the Embryonic Brainstem of Adrenaline During the Absence of Detectable Tyrosine Hydroxylase Activity
Authors: Foster George A. , Sundström Erik , Helmer‐Matyjek Elizabeth , Goldstein Menek , Hökfelt Tomas
Published: December 2006

Abstract: The activities of the catecholamine synthetic enzymes tyrosine hydroxylase and phenylethanolamine N‐methyltransferase, and the concentrations of the catecholamines and their respective metabolites, have been measured in the dorsal and ventral halves of the brainstem at various ages in the embryonic and adult rat. The activity of phenylethanolamine N‐methyltransferase in both parts of the brainstem at day 14 of gestation is at or greater than adult levels and thereafter displays relatively small variations during ontogeny. Tyrosine hydroxylase activity, in contrast, is undetectable at day 14 and increases slowly, achieving only 20–25% of adult values by day 18 of gestation. Adrenaline concentrations correlate well with the activity of phenylethanolamine N‐methyltransferase, showing a precocious development, whereas noradrenaline and 3, 4‐dihydroxyphenylethylamine (dopamine) concentrations are more closely related to the enhancement of tyrosine hydroxylase activity; at day 18 of gestation, for example, they are only 5 and 10%, respectively, of the adult values. The concentrations of the metabolites of noradrenaline and dopamine are suggestive of a high rate of turnover. These results confirm previous immunocytochemical evidence of a tardy appearance of tyrosine hydroxylase‐like immunoreactivity in the phenylethanolamine N‐methyltransferase‐positive perikarya of the embryonic medulla oblongata. In addition, the abundance of adrenaline in this area at early gestational stages strongly suggests that, despite the paucity of tyrosine hydroxylase, phenylethanolamine N‐methyltransferase is active in vivo and is utilizing a substrate other than noradrenaline. It is likely, however, that at later stages of gestation, when tyrosine hydroxylase is present at sufficient activity to supply noradrenaline, the conventional synthetic pathway for adrenaline formation comes into being.
Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid‐Turnover Pool Determine Methyl Accepting Capacity
Authors: Meydan Naomi , Egozi Yaakov , Kloog Yoel
Published: December 2006

Abstract: In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70–80%) in their carboxyl methylation as measured either with L‐[methyl‐3H]methionme in the intact lobes after stimulation or in their homogenates with [methyl‐3H]S‐adenosyl‐L‐methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (<7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.
Purification and Amino Terminal Sequencing of Human Melanoma Nerve Growth Factor Receptor
Authors: Marano Nadia , Dietzschold Bernhard , Barley James J. , Schatteman Gina , Thompson Susan , Grob Peter , Ross Alonzo H. , Bothwell Mark , Atkinson Barbara F. , Koprowski Hilary
Published: December 2006

Abstract: The nerve growth factor (NGF) receptor, solubilized with Triton X‐100 detergent, has been purified from human melanoma cell line A875. Purification to near‐homogeneity was achieved by chromatography on wheat germ agglutinin‐agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti‐NGF receptor monoclonal antibody (MAb). The purified receptor, a 75, 000‐dalton protein, retains the capacity to bind NGF as well as anti‐receptor MAbs. Final purification was achieved by sodium dodecyl sulfate‐polyacryiamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.
Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells
Authors: Ananth Uma S. , Leli Ubaldo , Hauser George
Published: December 2006

Abstract: 5‐Hydroxytryptamine (serotonin or 5‐HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 ± 10‐7M. The phosphoinositide response was blocked by the 5‐HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by ˜30%. The 5‐HT1A agonist 8‐hydroxy‐2 (di‐n‐propylamino)tetralin did not cause stimulation. Incubation with 5‐HT (1 μM) for 1 h increased the incorporation of [2‐3H]myo‐inositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5‐HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI‐4‐phosphate, lyso‐PI, glycerophosphoinositol, and IP, but the presence of 5‐HT again did not cause further stimulation. 5‐HT also stimulated the release of IPs in cells prelabeled with [2‐3H]myo‐inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5‐HT (1 μM). Radioactivity in IP2 and IP3 was very low, was stimulated ˜50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was ˜40 times that in IP3. The EC50 value of 1.8 ± 10‐7M was comparable with that obtained from 32Pi studies measuring labeled PI. Ketanserin and spiperone inhibited 5‐HT‐stimulated [2‐3H]IP release with IC50 values of 3.1 ± 10‐9 and 1.8 ± 10, ‐8M, respectively. This study demonstrates that phosphoinositide hydrolysis is enhanced by 5‐HT in C6 glioma cells and that this phenomenon is linked to 5‐HT2‐like binding sites.
GABAergic Inhibition on Dopamine Cells of the Fish Retina: A [3H]Dopamine Release Study with Isolated Cell Fractions
Authors: Ishita S. , Negishi K. , Teranishi T. , Shimada Y. , Kato S.
Published: December 2006

Abstract: Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When γ‐aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 μM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.
Cholinergic Vesicle Specific Proteoglycan: Stability in Isolated Vesicles and in Synaptosomes During Induced Transmitter Release
Authors: Kuhn D. M. , Volknandt W. , Stadler H. , Zimmermann H.
Published: December 2006

Abstract: Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co‐release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P‐40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co‐release with acetylcholine or ATP.
Characterization and Localization of Glutathione‐S‐Transferases in Rat Brain and Binding of Hormones, Neurotransmitters, and Drugs
Authors: Abramovitz Mark , Homma Hisato , Ishigaki Seishi , Tansey Francine , Cammer Wendy , Listowsky Irving
Published: December 2006

Abstract: Rat brain glutathione‐S‐transferases are rich in Yb type subunits with major RNA transcripts coding for a relatively uncommon Yb3 form. The Yb‐containing isoenzymes of brain cytosol bind glucocorticoids and are covalently labeled with dexamethasone 21‐methanesulfonate. Certain neurotransmitters, hormones, and drugs, such as serotonin, dopamine, glucocorticoids, thyroxine, apomorphine, and benzodiazepine derivatives, are effective inhibitors of brain glutathione transferase activity. Immunocytochemical studies show that Yb forms are localized in ependymal cells, subventricular zone cells, astrocytes, tanycytes, and astrocyte foot processes on blood vessels, but Yb was not detected in oligodendrocytes or neurons. Based on their localization and binding properties, brain glutathione‐S‐transferases have the potential to function in intracellular binding of a variety of compounds and thereby govern their uptake and release in brain, transport to neurons, as well as in their detoxification.
D‐Aspartate Uptake and Release in the Guinea Pig Spinal Cord After Partial Ablation of the Cerebral Cortex
Authors: Potashner S. J. , Dymczyk L. , Deangelis M. M.
Published: December 2006

Abstract: This study attempts to determine if L‐glutamate and L‐aspartate may be transmitters of the guinea pig corticospinal tract. Unilateral ablations were made of the frontal and parietal neocortex which destroyed most of the motor and somatosensory areas in the right cerebral hemisphere. In lesioned animals, transverse sections of the cervical enlargement of the spinal cord (segments C6‐T1) were stained to reveal degenerating fibers. Degeneration of axons first appeared 4 days after surgery, reached a maximum on the seventh day, and began to wane by the ninth day. The most prominent loss of axons appeared deep in the dorsal funiculus and in laminae IV–IX of the gray matter contralateral to the cortical lesion. Ipsilaterally, there was very sparse degeneration of fibers in the dorsal and ventral funiculi and in the spinal gray matter. The uptake and release of D‐[3H]‐aspartate, a putative nonmetabolizable marker for L‐glutamate and L‐aspartate, were measured in dissected quadrants of the cervical enlargement taken from intact and lesioned animals. The uptake and the electrically evoked, Ca2+‐dependent release of D[3H]aspartate were depressed by 29–35% at 4 and 7 days after surgery, but only in tissue that was contralateral to the cortical ablation. The lesion had no effect on the uptake and release of exogenous γ‐[14C]aminobutyric acid, which were measured as indices of the postlesion integrity of neurons in the spinal gray matter. These findings suggest that the synaptic endings of corticospinal fibers probably mediate the uptake and release of D‐[3H]aspartate and, therefore, may use L‐glutamate and/or L‐aspartate as a transmitter.
Acetylcholine Mobilization in a Sympathetic Ganglion in the Presence and Absence of 2‐(4‐Phenylpiperidino)Cyclohexanol (AH5183)
Authors: Cabeza R. , Collier B.
Published: December 2006

Abstract: The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2‐(4‐phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13–14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recoveiry from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183‐treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh. and thus we conclude that ACh synthesized in the presence of AH5183 is as releasable as preexisting ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]‐choline): the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183‐induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183‐blocked ganglia, even in the drug's continued presence. Venom‐induced release of ACh from AH5183‐treated ganglia was not less than the venom‐induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, α‐latrotoxin, because an anti‐α‐latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethyl‐homocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of (3H]acetyldiethylhomo‐choline efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.
A Comparison of the Effects of Acrylamide and Experimental Diabetes on the Retrograde Axonal Transport of Proteins in the Rat Sciatic Nerve: Analysis by Two‐Dimensional Polyacrylamide Gel Electrophoresis
Authors: Logan Matthew J. , McLean W. Graham
Published: December 2006

Abstract: Retrogradely transported proteins that accumulated for 18 h distal to a ligature on the sciatic nerve of rats were separated by two‐dimensional polyacrylamide gel electrophoresis and visualised with silver stain. A total of 14 proteins were detectable in this way as being retrogradely transported. In rats rendered diabetic 14 days previously by a single intravenous dose of streptozotocin, the accumulation of four of those proteins was noticeably decreased. Administration of acrylamide to a cumulative dose of 150 mg·kg−1 or 350 mg·kg−1 decreased the accumulation of four and eight proteins, respectively. Three of the four protein changes were common to the diabetic and acrylamide‐treated animals, and were present before signs of neuropathy could be detected. The results suggest that those alterations may play a causal role in the development of neuropathy.
Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character
Authors: Toutant Jean‐Pierre , Arpagaus Martine , Fournier Didier
Published: December 2006

Abstract: The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100. 10‐oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt‐soluble fraction contained the same molecular forms as the low salt‐soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.
Regulation of Norepinephrine Uptake by Adenine Nucleotides and Divalent Cations: Role for Extracellular Protein Phosphorylation
Authors: Hendley Edith D. , Whittemore Scott R. , Chaffee Jean E. , Ehrlich Yigal H.
Published: December 2006

Abstract: This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high‐affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5′‐adenylyl‐imidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+‐stimulated uptake 1 was blocked by App(NH)p; Mg2+‐stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca‐omission, and inhibition of the phosphorylation of one specific, 39,000‐dalton, Ca2+‐dependent, protein component in synaptosomes. This 39K protein was distinct from the α subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.
Effects of Amphetamine on Protein Synthesis and Energy Metabolism in Mouse Brain: Role of Drug‐Induced Hyperthermia
Authors: Nowak Thaddeus S.
Published: December 2006

Abstract: Changes in brain protein synthesis activity, and in brain levels of glucose, glycogen, and several high‐energy phosphate metabolites, were evaluated under conditions of amphetamine‐induced hyperthermia in mice. Protein synthesis showed a striking dependence on rectal temperature (TR), falling abruptly at TR above 40°C. A similar result was obtained following direct heating of the animals. Protein synthesis activity in liver showed the same temperature dependence observed for brain. Increased synthesis of a protein with characteristics of the major mammalian stress protein, hsp 70, was demonstrated in both brain and liver following amphetamine administration. Brain protein synthesis showed significant recovery within 2 h after amphetamine administration whereas that of liver remained below 30% of control activity, suggesting significant temporal and quantitative differences in the response of individual tissues to elevated temperatures. Brain glycogen levels after amphetamine administration were significantly lower under conditions of ambient temperature which resulted in more severe drug‐induced hyperthermia but did not correlate as strikingly as protein synthesis with the temperatures of individual animals. Brain glycogen also fell in animals whose temperatures were increased by brief exposure at high ambient temperature. Brain glucose levels did not consistently change with hyperthermia. Slight decreases in high‐energy phosphates with increasing TR were likely the result of fixation artifact. These results demonstrate the fundamental role of hyperthermia in the reduction of protein synthesis in brain and other tissues by amphetamine, and suggest that temperature also constitutes a significant source of variability in the effects of this drug on brain energy metabolism, in particular glycogenolysis.
Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex
Authors: Cammer Wendy , Tansey Francine A.
Published: December 2006

Abstract: Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.
Book Review
Authors: Eichberg Joseph
Published: December 2006

Book Reviewin this Article:

Diabetic Neuropathy edited by P. J. Dyck, P. K. Thomas, A. K. Asbury, A. I. Winegrad, and D. Porte, Jr.
Evidence That Receptor‐Linked G Protein Inhibits Exocytosis by a Post‐Second‐Messenger Mechanism in AtT‐20 Cells
Authors: Luini Alberto , De Matteis Maria Antonietta
Published: December 2006

Abstract: In AtT‐20 cells somatostatin inhibits the secretion of adrenocorticotropic hormone (ACTH) through the activation of GTP binding proteins (G proteins) linked to second messengers such as calcium and cyclic AMP (cAMP). Recently, it has been proposed that there may be G proteins that regulate directly the exocytotic machinery. We have investigated whether somatostatin could inhibit secretion at a step distal to second messengers through a GTP binding protein. For these studies two experimental paradigms were used: (1) intact cells stimulated by calcium ionophores and (2) digitonin‐permeabilized cells exposed to buffers of increasing Ca2+ concentrations. Somatostatin inhibited by 70% the ACTH release caused by the calcium ionophore ionomycin without modifying the ionophore‐induced elevation in cytosolic [Ca2+]. This effect was cAMP independent because (1) it was observed in the presence of high concentrations of membrane‐permeant cAMP analogues, and (2) it was not accompanied by a change in cAMP levels. The effect was also independent of the levels of activators of protein kinase C because it could be produced in the presence of high concentrations of phorbol esters. The action of somatostatin was prevented by pertussis toxin. In digitonin‐permeabilized AtT‐20 cells somatostatin inhibited release induced by calcium buffers in a GTP‐dependent manner. These two observations indicate the involvement of a G protein. It is proposed that a G protein coupled to somatostatin receptors inhibits the intracellular machinery of secretion at a step distal to second messengers, perhaps at the exocytotic site.
Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides
Authors: Tzartos Socrates J. , Loutrari Heleni V. , Tang Fen , Kokla Anna , Walgrave Susan L. , Milius Robert P. , Conti‐Tronconi Bianca M.
Published: December 2006

Abstract: Most anti‐nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR α‐subunit. Thirty‐two synthetic peptides, corresponding to the complete Torpedoα‐subunit sequence and to a segment of human muscle α‐subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti‐AChR mAbs directed against epitopes on the α‐subunit other than the MIR. A main constituent loop of the MIR was localized within residues α67‐76. Residues 70 and 75, which are different in the Torpedo and human α‐subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop α67‐76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment α67‐76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68‐71. The most stable structure predicted for this segment, in both the Torpedo and human α‐subunits, is a hairpin loop, whose apex is a type I β‐turn and whose arms are β‐strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non‐MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues α331‐351.